Galactose-derived Glycopolymers : Synthesis, characterization

and applications

by

Pierre-Olivier Ferko

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Chemistry University of Toronto

© Copyright by Pierre-Olivier Ferko

ii

Galactose-derived Glycopolymers : Synthesis, characterization

and applications

Pierre-Olivier Ferko

Master of Science

Department of Chemistry

University of Toronto

2018

Abstract

This thesis explores the synthesis, characterization and application of galactose-derived

glycopolymers. Glycopolymers are synthetic polymers possessing pendant carbohydrate moeities.

Desirable properties such as variable structural complexity, biological compatibility and tunable

responsiveness to environmental conditions have made glycopolymers prominent in biomedical

engineering applications such as synthetic tissues and targeted drug delivery. Among

carbohydrates, galactose is particularly relevant in drug delivery applications due to binding with

asialoglycoprotein receptors (ASGPR) overexpressed in liver cancer cells. This thesis investigates

the synthesis of polymerizable galactose derivatives via organoboron catalysts to efficiently access

a new class of glycopolymeric biomaterials. Notably, RAFT polymerization is utilized to

synthesize well-defined glycopolymers, and a hydrogel is formed as proof of concept for potential

applications.

iii

Acknowledgments

First and foremost, I offer my deepest gratitude to Professor Mark Taylor, who made the effort to

recruit me to his group and had the patience thereafter to teach me modern chemistry and the

application of the scientific method. These are lessons that I will hold forever dear as I continue in

my career, and for this opportunity to learn, I am grateful.

I extend this gratitude to all the 2017-2018 members of the Taylor group, who supported me over

the course of my degree; Ekaterina, Sanjay, Seung-Joon, Daniel, Grace, Victoria, John, Kashif,

Alvaro, Ali, Jacklyn and Liwah. Specifically, I would like to highlight the support of Daniel

regarding the monomer synthesis.

I also wish to thank the people that I’ve met from the various other groups in the department of

chemistry, who all contributed to my professional development and education over the year.

Particularly, I would like to thank Hyungjun and Rachel from the Winnik group for their help and

knowledge regarding RAFT polymerization and GPC instrumentation respectively. Their

contributions are deeply appreciated, along with that of Professor Mitchell Winnik for accepting

second readership of this thesis.

Finalement, je tiens à remercier les membres de ma famille qui m’ont supporté au cours de mon

éducation : mes parents (Dany et Jim), mon frère Maxime-Alexandre, ma grand-mère (Huguette)

et son partenaire (Guy), mes oncles (Pierre et Sylvain) et mes tantes (Courtney et Sylvie), mes

cousines (Camille et Alice), et ma copine Juliana.

iv

Table of Contents

Acknowledgments.......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Tables ...................................................................................................................................v

List of Figures ................................................................................................................................ vi

List of Appendices ......................................................................................................................... ix

Introduction .................................................................................................................................1

1.1 Background ..........................................................................................................................1

1.2 1.2 Reversible Addition-Fragmentation Chain-Transfer (RAFT) Polymerization ..............1

1.3 RAFT Glycopolymers ..........................................................................................................2

1.4 Glycopolymer Hydrogels .....................................................................................................5

1.5 Scope of Thesis ....................................................................................................................9

Results and Discussion ..............................................................................................................12

2.1 Methacrylate Glycomonomer Synthesis ............................................................................12

2.2 Glycopolymer Synthesis ....................................................................................................15

2.3 Summary and Future Directions ........................................................................................27

Experimental .............................................................................................................................28

3.1 Monomer Synthesis ...........................................................................................................29

3.2 Polymer Synthesis ..............................................................................................................31

References ......................................................................................................................................36

v

List of Tables

Table 1 : Synthesized glycopolymers and their properties ……………………………………21

vi

List of Figures

Figure 1 : Glycomonomers (1.01 to 1.04) investigated by Narain and coworkers. ……...………4

Figure 2 : Simplified visual interpretation of galactose-containing glycopolymers binding to

asialoglycoprotein receptors overexpressed on liver cancer cells. ……………………………….5

Figure 3 : Complexation and relative equilibria of arylboronic acid with 1,2-diols at neutral and

basic pH. ………………………………………………………………………………………….6

Figure 4 : Complexation of benzoboroxole with cis-diol motifs at varying

pH.……………………………………………………………………………………………...….6

Figure 5 : RAFT statistical polymerization of MAAmBO and NIPAAm to form P(NIPAAm-st-

MAAmBO).……………………………………………………………………………………….7

Figure 6 : Boronic-diol interaction to form crosslinks between P(NIPAAm-st-MAAmBO) and

glycoplymers. ……………………………………………………………………………………..8

Figure 7 : Above: Equilibrium of 2-acrylamidophenylboronic acid in water. Below: Structure of

P(2APBA-co-DMA)………………………………………………………………………………9

Figure 8 : Selective monobenzylation of pyranoside substrates catalyzed by oxoboraanthracene

(1.09) ……….……………………………………………………………………………………10

Figure 9 : Selective functionalization of the O-3 position of b-methyl-D-galactopyranoside via

oxoboraanthracene catalysis……………………………………………………………………..11

Figure 10: 1H NMR Spectrum (400 MHz, DMSO-d) of the crude reaction mixture containing

monomer 2.01, showing the full spectrum………………………………………………………13

Figure 11: 1H NMR Spectrum (400 MHz, DMSO-d) of the crude reaction mixture containing

monomer 2.01, magnifying the region containing the signals corresponding to the alkene

moeity……………………………………………………………………………………………14

Figure 12: 1H NMR Spectrum (400 MHz, DMSO-d) of the crude reaction mixture containing

polymer 2.04, circling the regions of interest used for monomer conversion estimation……….16

vii

Figure 13: 1H NMR Spectrum (400 MHz, DMSO-d) of the crude reaction mixture containing

polymer 2.04, before precipitation…………………………………………………………….…16

Figure 14: 1H NMR Spectrum (400 MHz, DMSO-d) of precipitated polymer 2.04……………17

Figure 15: 1H NMR Spectrum (400 MHz, DMSO-d) of polymer 2.05-40 after two

precipitations, magnifying the aromatic region of the

spectrum……………………………………………………………………………………….…19

Figure 16: Representative 1H NMR Spectrum (400 MHz, DMSO-d) of polymer 2.05-40,

highlighting the relevant peaks used for endgroup analysis……………………………………..20

Figure 17: Hydrogel made from 6% 2.05-40 and 6% PVPBA in aqueous 2M NaOH……..…..26

viii

List of Schemes

Scheme 1 : Mechanistic overview of the RAFT polymerization process ……..…………...…….2

Scheme 2: Syntheses and yields of methacrylate glycomonomers 2.01, 2.02, and

2.03…………………………………………………………………………………...……….....12

Scheme 3: Syntheses of poly (methyl methacrylate)-derived glycopolymers 2.04, 2.05 and

2.06……………………………………………………………………………………………....19

Scheme 4: Condensation of polymer 2.05-20 …………………………………...……………...24

Scheme 5: Conceptual hydrogel formation using polymer 2.05 and boric acid …...…………...25

Scheme 6: Synthesis of PVPBA…………………………………...……………………………26

Scheme 7: Hydrogel synthesis using polymer 2.05 and PVPBA………………………………..27

ix

List of Appendices

A1: NMR Spectra of Compounds

A2 : GPC Traces of Compounds

1

Introduction

This thesis investigates the synthesis of polymerizable carbohydrate derivatives using organoboron

catalysts to efficiently access a new class of polymeric biomaterials.

1.1 Background

Carbohydrates, as a class of molecules, perform various essential roles in various biological

systems and have been the subject of intense research in the past century. A key feature of

carbohydrates is their structural complexity, achieved by combining densely functionalized

monosaccharides in their various forms (i.e. pyranose, furanose, open-chain) to perform a wide

range of roles. In recent years, societal and research interest in ‘green’ chemistry has drawn further

attention to carbohydrate valorization, given their emblematic properties as renewable,

biocompatible and biodegradable chemicals.1 Accordingly, materials and polymer chemists have

capitalized on the broad utility of carbohydrates by incorporating them into polymeric

biomaterials, allowing them to interface with biological systems for applications such as tissue

engineering and targeted drug delivery.2 Indeed, these carbohydrate-derived biomaterials have

garnered great interest for therapeutic purposes and are often used in tandem with nanoparticle

chemistry to push the boundaries of medicine,3 among other important fields.

1.2 1.2 Reversible Addition-Fragmentation Chain-Transfer (RAFT) Polymerization

Discovered in the twilight years of the 20th century, reversible addition-fragmentation chain-

transfer polymerization (RAFT) has since made a remarkable impact on the fields of material and

polymer chemistry; with over 3000 citations, the original 1998 report of RAFT is the most cited

article in Macromolecules.4 As the name suggests, RAFT polymerization relies on the presence of

a chain transfer agent (CTA) as a chemical additive in an otherwise conventional free radical

polymerization to control the growth rate and uniformity of the polymer chains.2 Typically, CTAs

tend to possess a thiocarbonate moiety that enables their function, but this is not a strict

requirement and some CTAs are able to function with alternate moieties, such as a diene, present.5

Beyond control of polymer growth, polymer properties can be heavily influenced by the nature of

the CTA chosen, as the endgroups of the polymer will originate mainly from the CTA and

ultimately determine the type of post-synthesis modifications that can be carried out. For example,

2

polymers synthesized via RAFT protocols can potentially serve as macro RAFT agents in the

synthesis of block copolymers, provided that the endgroups of the former are preserved. Access to

complex polymer architectures can thus be achieved via RAFT and is the focus of much research

attention; the record for consecutive RAFT block copolymerizations is 20 as of 2013 by the Perrier

group.6

Scheme 1: Mechanistic overview of the RAFT polymerization process. (1) describes the

initiation step and growing polymer chain as per conventional free radical polymerization.

(2) describes the reversible-deactivation step that allows the RAFT agent to reduce radical

concentration and therefore termination reactions. (3) summarizes the RAFT equilibria

between growing polymer chains that controls their growth.

1.3 RAFT Glycopolymers

Aside from the mechanistic advantages of RAFT polymerizations, the generally non-toxic nature

of the end-products,7 high compatibility with various monomer archetypes5 and utility of the

endgroups8 has allowed for powerful use of RAFT polymers in various biological applications,

particularly RAFT glycopolymers. Glycopolymers comprise a subset of carbohydrate-derived

biomaterials, broadly defined as synthetic polymers possessing pendant carbohydrate groups.

Despite their synthetic origins, glycopolymers are at the forefront of biocompatible systems design

and engineering, serving multiple roles ranging from artificial tissue to drug-carrying vesicles. The

power of the RAFT polymer approach is illustrated in a highly relevant manner to this thesis by

the Narain group, who regularly apply RAFT protocol in the design and ultimate application of

3

their glycopolymeric drug delivery vehicles. A key recurring feature of their research is the

presence of pendant galactose groups on their polymers, allowing the latter to preferentially bind

with liver cancer cells as a form of targeted drug delivery. Specifically, liver cancer cells are known

to overexpress asialoglycoprotein receptors that are competitively inhibited by galactose, therefore

offering the possibility of targeted drug delivery by the incorporation of galactose into the drug

delivery vehicles. The Narain group generally gains access to their desired therapeutic

glycopolymers via the RAFT polymerization of open-chained glycomonomers, LAMA (2-

lactobionamidoethyl methacrylate) and LAEMA (2-lactobionamidoethyl methacrylamide) (Figure

1).9–14 These glycomonomers are necessarily open-chained due to the introduction of methacrylate

or methacrylamide group at the glycosidic position of the sugar monomers. Advantageously, there

is no need for protecting group chemistry to access the glycomonomers, allowing for ease of access

to bulk quantities of glycomonomers. The versatility of RAFT polymerization then allows for

statistical copolymerizations of these glycomonomers with monomers that confer further

functionality to the polymers, such as chelation to therapeutic nanoparticles.15 Moreover, the

thiocarbonate endgroups in the resulting polymers can be easily hydrolyzed to afford thiol

endgroups,16–19 further increasing the potential functionality of the polymers.

4

Figure 1: Glycomonomers (1.01 to 1.04) investigated by Narain and coworkers. The glucose-

derived monomers (1.03 and 1.04) are generally used as controls, to which the performance

of the lactose-derived monomers is compared.

5

Figure 2: Simplified visual interpretation of galactose-containing glycopolymers binding to

asialoglycoprotein receptors overexpressed on liver cancer cells.

1.4 Glycopolymer Hydrogels

A popular application of glycopolymers is the formation of hydrogels. As the name suggests,

hydrogels are macromolecular gels composed of crosslinked, hydrophilic polymer networks in

aqueous media. Owing to their biocompatibility and highly diverse and tunable properties,

hydrogels have received much attention in biological engineering applications such as artificial

tissue20 and wound dressings.21 Crosslinking methods vary with the polymers involved and the

desired properties of the end-product; such methods may involve ion pairing,22 imine formation,23

Diels-Alder cyclization,24,25 boronic acid esterification,26,27 thiol-ene click reactions,28,29 or others.

Given the high density of hydroxyl groups present in many glycopolymers, crosslinking via

boronic acid esterification has arisen as an effective method to afford hydrogels from suitable

glycopolymers.

In the case of boronic acid esterification, the crosslinks are a product of the well-known interaction

between boronic acids and 1,2- and 1,3-diols; the reversible nature of this interaction also allows

for resulting gels to ‘self-heal’ after being torn, a highly sought-after property for biological

engineering applications. The requirement of biological compatibility for such applications also

necessitates that the hydrogel can be formed and preserve function in controlled, pH-neutral

6

environments. Optimal pH for binding between boronic acids and diols can be estimated by the

average pKa of the boronic acid and diol in the system, as per the following equation:30

𝑝𝐻𝑜𝑝𝑡𝑖𝑚𝑎𝑙 =𝑝𝐾𝑎−𝑎𝑐𝑖𝑑+𝑝𝐾𝑎−𝑑𝑖𝑜𝑙

2 (1)

It is important to note that this equation does not consider the contributions of solvent, buffer,

hydrogen bonding, or other intermolecular interactions, and is therefore best seen as a rule of

thumb only.30,31 Typical boronic acids have a pKa significantly above 7, and generally require

basic environments to function as crosslinking agents. Accordingly, this prevents the use of

many boronic acids in biological applications. Recently, however, there have been novel

approaches to boronic acid crosslinker design that have allowed for hydrogels to form at neutral

pH with great efficacy.

Figure 3: Complexation and relative equilibria of arylboronic acid with 1,2-diols at neutral

and basic pH.

One such approach is the use of benzoboroxole crosslinkers to complex with glycopyranosides,

pioneered by the Hall group and applied in hydrogels with the collaboration of the Narain group.

Benzoboroxoles are part of a class of molecules known as ‘improved’ Wulff-type boronic acids,

featuring a stabilizing intramolecular B-O bond as opposed to the weaker B-N bond present in

conventional Wulff-type boronic acid.32 The characteristic stability of Wulff-type boronic acids

generally results in a lower pKa and generally higher binding affinity with diols compared to other

varieties of boronic acids.

7

Figure 4: Complexation of benzoboroxole with diol motifs at varying pH.

In 2008, the Hall group reported that this increased Lewis acidity allowed the complexation of

benzoboroxole (pKa 7.4) with glycopyranosides possessing either 3,4- or 4,6- diols in neutral pH

aqueous environments (Figure 4), augmented by the anionic nature of the complex at neutral pH.33

Subsequently, the groups of Narain and Hall jointly reported a novel class of hydrogels composed

of glycopolymers crosslinked via statistical copolymers containing benzoboroxoles (Figure 5).27

In this system, 5-methacrylamido-1,2-benzoboroxole (MAAmBO) was statistically

copolymerized with N-isopropylacrylamide (NIPAAM) via RAFT to create well-defined polymers

(PNIPAAMx-st-MAAmBOy) with temperature, pH and glucose responsive properties. Here,

NIPAAM was chosen by the authors as polymeric NIPAAM (PNIPAAM) has well-known

temperature responsive properties, including reversible hydrophilic/hydrophobic character around

its lower critical solution temperature (LCST) of 32 oC. Strikingly, the authors were able to form

hydrogels by simply mixing PNIPAAMx-st-MAAmBOy of various composition with PGAEMA

in aqueous phosphate-buffered saline (PBS) (pH 7.4). However, the authors noted that this method

failed when using PLAEMA 21 or PGAEMA30, noting the importance of chain length in this

hydrogel system. Moreover, the authors also noted the temperature responsiveness of their

hydrogel system; when subjected to excess glucose at 4 oC, the gel disintegrated, whereas at 40 oC

the gel remained intact. The authors attributed this difference to the increased lability of the

crosslinking moieties due to higher temperature, favouring transient networks, as opposed to

decreased lability favouring the more stable complexation with the free glucose. Interestingly, the

gel was destroyed at both 0 oC and 40 oC in weakly acidic aqueous media. Accordingly, the authors

identified this novel system to have potential biological application as a drug delivery vehicle that

allows for slow release of encapsulated therapeutic agents via temperature and pH responsive

properties.

8

Figure 5: RAFT statistical polymerization of MAAmBO and NIPAAm to form P(NIPAAm-

st-MAAmBO).

Figure 6: Boronic-diol interaction to form crosslinks between P(NIPAAm-st-MAAmBO) and

glycoplymers.

Another approach for forming similar systems involving ‘improved’ Wulff-type boronic acids has

been pioneered by the Sumerlin group, prominently featuring 2-acrylamidophenylboronic acid26

(2APBA) (Figure 7) in an analogous role to MAAmBO in the Narain-Hall system. In this

9

approach, 2APBA is statistically copolymerized with N,N-dimethylacrylamide (DMA) in various

ratios via free radical polymerization to afford biocompatible and hydrophilic polymeric

crosslinking agents (P(2APBA-co-DMA)). P(2APBA-co-DMA) is then mixed with either pendant

catechol-bearing polymer or poly(vinyl alcohol) (PVOH) in deionized water to yield a hydrogel,

verified qualitatively via the gel inversion test. Notably, the authors note that these systems are

capable of formation and self-healing in both neutral (pH 7) and acidic (pH 4) conditions, offering

the possibility of biological applications in acidic environments such as the gastrointestinal tract.

Figure 7: Above: Equilibrium of 2-acrylamidophenylboronic acid (2APBA) in water. Below:

Structure of P(2APBA-co-DMA)

1.5 Scope of Thesis

In the last decade, Taylor and coworkers have devised and examined organoboron catalysts that

allow for regioselective functionalization of saccharides, by way of 1,2- and 1,3-cis diol

complexation with the organoboron catalysts34–37. Aside from the general non-toxicity of

10

organoboron catalysts, their method is distinguished by the advantage of eschewing protecting

group chemistry altogether in many instances due to the regioselectivity of the catalyzed reactions.

Figure 8: Selective monobenzylation of pyranoside substrates catalyzed by

oxoboraanthracene (1.09)38

Among the organoboron catalysts commonly featured in the Taylor group’s work is

oxoboraanthracene, a borinic acid capable of selectively enhancing the nucleophilicity of a

singular hydroxyl over another in a cis-1,2-diol pair via complexation (Figure 8).38 The selectivity

is dependent on the substrate; notably, unprotected galactopyranosides will tend to be

functionalized at the O-3 position (Figure 9). This thesis was encouraged by the potential access

to glycomonomers offered by this catalyst, as well as the groundbreaking work by the Narain-Hall

group. Specifically, this thesis investigates the synthesis of polymerizable carbohydrate derivatives

via organoboron catalysts to efficiently access a new class of polymeric biomaterials. Novel

methacrylate monomers derived from methyl-D-galactopyranosides will be investigated for

potential applications in RAFT glycopolymers inspired by the Narain group, sharing conserved

cyclic pendant galactose moieties while differing by absence of glucose and closer promixity of

the saccharide pendant groups to the synthetic backbone. Investigation aimed at the formation of

a hydrogel will serve as a proof-of-concept application for these new glycopolymers.

11

Figure 9: Selective functionalization of the O-3 position of methyl-β-D-galactopyranoside via

catalytic action of 1.09

12

Results and Discussion

2.1 Methacrylate Glycomonomer Synthesis

The investigation began by treating methyl-β-D-galactopyranoside with methacryloyl chloride in

the presence of organoboron catalyst to yield monomer 2.01. This synthesis had been tested by

fellow group member Ekaterina Slavko in an exploratory manner prior to this formal investigation,

and the reproduction of her work served as an entry point for the project.

Scheme 2 : Syntheses and yields of methacrylate glycomonomers 2.01, 2.02, and 2.03

Initial reproduction of the reaction resulted in significantly higher yield (50%) than what was

achieved in the test reaction (9%) according to 1H NMR analysis with a quantitative internal

standard. This difference was attributed to comparatively longer cooling period of the reaction

flask in ice prior to dropwise addition of methacryloyl chloride, likely reducing the extent of

decomposition of the latter in solution prior to participating in the intended reaction. The reaction

was not optimized as priority turned towards obtaining purified monomer for analysis. Notably,

increasing the temperature of the reaction to 40 oC (following the ice-cold addition of methacryloyl

chloride) did not alter the yield of the reaction.

13

Figure 10: 1H NMR Spectrum (400 MHz, DMSO-d) of the crude reaction mixture containing

monomer 2.01, showing the full spectrum. The integrated peak on the left side of the

spectrum arises from the presence of 2.01 (see Experimental 3.2 for full structural

assignment), whereas the integrated peak on the right is the peak from the internal standard

(mesitylene, 9H signal). The table in the figure displays the calculations to determine NMR

yield.

Purification of the monomer proceeded at first via standard silica flash column chromatography

using a gradated hexane: ethyl acetate solvent system. However, this system provided a much

lesser isolated yield (13%) than what had been suggested by the NMR yield. Compounding these

difficulties was the presence of a side-product that was difficult to separate from the desired

product. The side product was surmised to be the O-6 methacrylate isomer of the desired product

14

in light of the group’s previous observations when the acrylation of this substrate is catalyzed by

1.09.34

Figure 11: 1H NMR Spectrum (400 MHz, DMSO-d) of the crude reaction mixture containing

monomer 2.01, magnifying the region containing the signals corresponding to the alkene

moeity. The larger peaks, denoted with the blue arrows, correspond to the O-3 isomer, while

the smaller peaks, denoted with the red arrows, are assumed to correspond to the O-6 isomer.

Ultimately, a dichloromethane: methanol system was able to achieve a much higher isolated yield

of 41%. Subsequently, the optimized reaction and purification procedures were then applied to

methyl-α-D-galactopyranoside and isopropyl β- D-1-thiogalactopyranoside with slightly reduced

success to expand the substrate scope to similar galactopyranosides (Scheme 2). 2D COSY NMR

analysis (Figure A02) was used to assign the structure of 2.01 (Experimental 3.1); the other

monomers were assigned based off their 1H NMR spectra (Figures A05 and A07 for 2.02 and 2.03

respectively), comparison with the structural assignment of 2.01 and the Taylor group’s previous

work involving borinic acid-catalyzed reactions of galacto-configured sugars.

______________________________________________________________________________

15

2.2 Glycopolymer Synthesis

With a method of obtaining pure methacrylate glycomonomer 2.01 in acceptable yield, focus

turned towards polymerization. As mentioned earlier in section 1.3 (RAFT Glycopolymers), well-

defined and monodisperse glycopolymers are advantageous for biological applications, and the

methacrylate moiety of the monomer allowed the application of controlled radical polymerization

(CRP) as the method of choice for incorporation of these properties into the polymers. Among the

current methods of CRP, RAFT was chosen due to technical accessibility, ease of thiol

introduction to the chain for future functionalization, and literature precedent for related monomers

by Narain and coworkers.9,10,15 Atom-transfer radical polymerization (ATRP) was also considered,

but ultimately did not fit in the current scope of this project given the possibility of residual

transition metals arising from ATRP catalysts in polymers destined for biological purposes.

RAFT polymerizations of monomer 2.01 were initially conducted on small scale (100.0 mg, 0.381

mmol 2.01) with 2-cyano-2-propyl dodecyl trithiocarbonate (2.07) serving as RAFT CTA due to

known compatibility with methacrylates and ready availability.5 As polymerization of this

monomer is unprecedented, a reaction protocol was decided upon and carried out by adapting

conditions from reports by the Narain group on similar glycopolymer syntheses (Experimental

3.2). Promisingly, qualitative physical properties such as increased viscosity of the reaction

solution along with characteristic broad peaks in the 1H NMR spectrum of the crude product

suggested that polymerization had occurred. Analysis of monomer conversion was attempted by

comparing the integrals of the alkene peaks corresponding to the residual monomer (5.66 and 6.10

ppm) with the integrals of the peaks corresponding to the methyl hydrogen signals that would

necessarily be unique to the polymer (approx. 0.8 to 1.5 ppm) (Figure 11). However, conversion

16

was not able to be confidently assessed due to significant signal overlap with non-polymeric

species in the alkane region of the spectrum.

Figure 12: 1H NMR Spectrum (400 MHz, DMSO-d) of the crude reaction mixture containing

polymer 2.04, circling the regions of interest used for attempted monomer conversion

estimation. The blue circle contains the signals corresponding to the two hydrogens

belonging to the alkene moiety of the monomer. The red circle contains the signal

corresponding to the three hydrogens belonging to the methyl group on the backbone of the

repeat unit. As noted above, the signals of the methyl hydrogens partially overlap multiple

other signals belonging to solvent, residual monomer, initiator and RAFT agent, greatly

limiting the utility of this analysis.

Given the presence of polymeric species in the crude reaction mixture that were likely of structure

2.04, precipitation from dimethylformamide into a small volume of ethyl acetate at room

temperature was attempted to retrieve them selectively. Ethyl acetate was chosen as it was able to

dissolve 2.01 and caused the appearance of a precipitate, thought to be the polymer, when added

to the crude polymerization reaction mixture. Vacuum filtration of the precipitate from the ethyl

acetate solution yielded a colourless solid with a mass corresponding to less than 20% recovery of

monomer mass input, highlighting the need for optimization. Broad peaks in the 1H NMR spectrum

of the solid heavily suggested that the solid was polymeric in nature, but endgroup analysis was

17

not possible as it was discovered that the characteristic signal of the methylene alpha to the

thiocarbonyl at the Ω-end of the polymer, reported to be between 3.0 and 3.5 ppm in DMSO-d,39

partially overlapped with the broad peaks from the repeat carbohydrate pendant groups(Figure 13).

The obfuscation of what was thought to be the endgroup peak was unexpected, particularly as it

was partially visible in the crude reaction mixture prior to precipitation (Figure 12). Quantitation

of the methyl hydrogen signals at the terminal end of the trithiobenzoate was also prevented by

significant overlap in the region of the spectrum where alkanes are expected to produce signals

(Figure 12).

Figure 13: 1H NMR Spectrum (400 MHz, DMSO-d) of the crude reaction mixture containing

polymer 2.04, before precipitation. The blue circle overlaid on the spectrum highlights the

partially resolved methylene peaks belonging to the methylene alpha to the trithiobenzoate

endgroup at 3.36 and 3.37 ppm.

18

Figure 14: 1H NMR Spectrum (400 MHz, DMSO-d) of precipitated polymer 2.04. The blue

circle overlaid on the spectrum highlights the broad peaks near 3.5 ppm and their shoulders,

obfuscating the signal of the endgroup of the trithiobenzoate RAFT CTA 2.07.

Consequently, an accurate degree of polymerization was not obtainable from the 1H NMR, and it

was therefore initially unclear whether the synthesized polymer adhered to the expected RAFT

kinetic parameters. It is possible that a larger scale experiment, superior technical handling or

alternate NMR solvents may have led to improved results and interpretability of the experiment,

particularly given its nature as a preliminary foray into polymerization. Endgroup analysis via UV-

Vis absorbance measurements as per Skrabania and coworkers was also considered as an

alternative to NMR analysis for future polymerizations, but ultimately decided against given the

apparent need of calibration standards consisting of well-defined structural analogues to 2.04 to

achieve accuracy comparable to NMR analysis.40 MALDI-TOF mass spectroscopy as per

Vandenbergh and coworkers also offered a potential method for endgroup analysis of the sample,

but was decided against due to comparative complexity of the data analysis,41 and the decision to

19

use 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid dithiobenzoate (2.08) as RAFT agent

instead of 2.07 for further experiments.

Scheme 3: Syntheses of poly (methyl methacrylate)-derived glycopolymers 2.04 and 2.05

Often used by the Narain group, 2.08 was also known to be compatible with methyl methacrylates,5

was commercially available, and possessed an aromatic ring, a structural feature otherwise absent

in this reaction system, that could serve as an NMR handle for endgroup analysis. Furthermore,

2.08 also introduces a carboxylic acid endgroup to polymers instead of a nitrile group from 2.07,

slightly increasing water solubility of the polymers without need for endgroup modification.

Polymerizations were performed with 2.08 following the same protocol as with 2.07; initial

attempts at determining monomer conversions were also complicated by overlap of non-polymeric

peaks with the methyl hydrogen peaks of the polymer. However, the presence of broad peaks

suggested that polymerization had occurred nonetheless. Solvent screening then took place to

identify superior precipitation conditions that would selectively precipitate the polymer while

leaving the unreacted reagents behind in solution. Among the solvents tested, ethyl acetate was

20

found to be the only solvent that could satisfy both constraints. Ethers precipitated polymer 2.05

as well as the monomer from the crude reaction mixture according to 1H NMR analysis.

Importantly, lowering the temperature of the precipitating solvent to 0 oC and filtering with finer

pore size increased the recovered polymer mass drastically from the initial 13% using 2.07 (Table

1). Polymers of general structure 2.05 were subsequently synthesized and purified using this

method, detailed in Table 1.

Figure 15: 1H NMR Spectrum (400 MHz, DMSO-d) of polymer 2.05-40 after two

precipitations, magnifying the aromatic region of the spectrum. The ratio of the three signals

(2 : 1 : 2, from left to right) are in agreement with the expected endgroup of the polymer.

21

Polymer Mass

Recovery

(% of mass

input)

Expected

DPnb

Observed

DPn (NMR,

GPC)

Polydispersity

(Mn/Mw)

2.05-20 51.0 20 34, 18 1.27

2.05-40 53.5 40 45, 31 1.25

2.05-100 30.7 100 102, 37 1.6

Table 1: Synthesized glycopolymers and their properties. bBased on initiator loading.

Figure 16: Representative 1H NMR Spectrum (400 MHz, DMSO-d) of polymer 2.05-40,

highlighting the relevant peaks used for endgroup analysis. The red box (far left) contains

the aromatic peaks corresponding to the dithiobenzoate endgroup; the integration of the

22

aromatic peak at 7.9 ppm was assigned as 2 hydrogens total, and served as reference for the

other peaks in the spectrum. The blue box (second from the left) contains two broad peaks,

each corresponding to a single proton present on the repeat units (Experimental 3.1). The

purple box (second from the right) contains a broad peak corresponding to the methylene

present on the methacrylate backbone of the polymer. The green box (far right) contains a

broad peak corresponding to the methyl hydrogens present on the methacrylate backbone

of the polymer. Once the methylene and methyl hydrogens are normalized by dividing by

two and three respectively, the average of the integrations of the peaks in the blue, purple

and green boxes is denoted as the DPn of the polymer.

Gel permeation chromatography (GPC) was performed to confirm that the polymerizations had

been controlled by the addition of the RAFT agent; the GPC traces can be found in the appendix

(Figure A17). The degree of polymerization (DPn) determined via 1H NMR endgroup analysis

(Figure 15) agreed with the GPC results for 2.05-40 only , while the low polydispersities of the

polymers were indeed found to be within the range expected from controlled RAFT

polymerizations.42

Polymer 2.05-100 seems to be a clear outlier in mass recovery, observed DPn from GPC and

polydispersity; polymer 2.05-20 also shows a disagreement between GPC and NMR DPn

determination. Increased reaction time for larger polymers and/or increasing the concentration of

monomer in the reaction are factors worth considering for increasing mass recovery in the future.

It is also worth noting that GPC determination of molecular weight is made relative to calibration

standards; in this case, relative to PMMA standards. Thus, it is possible that the GPC-determined

molecular weight is erroneous due to significant structural difference between the sample and the

calibration standards, causing discrepancies at the low and high end of the molecular weight range

shown. Alternatively, it is also possible that the NMR-derived DPn may be erroneous due to the

signal-to-noise ratio (<1) for the signal of the dithiobenzoate endgroups used as reference in 2.05-

100, resulting in significantly inflated integration of the other peaks in the spectrum comparatively.

Increasing the relaxation delay (T1) from 1 to 10 seconds may improve the reliability of the

integrations given the polymeric nature of the compound. From a technical standpoint, the absolute

quantity of initiator required for reaching the expected DPn of 100 may have been too low for

practical purposes at the small scale used (300 mg, 1.114 mmol 2.01). Reproduction of these

experiments for 2.05-20 and 2.05-100 will be required to confirm if the divergence from the

23

expected DPn is a technical or chemical issue. Larger-scale experiments would also serve to reduce

the ambiguity surrounding the technical aspects of the procedure. Based on these current results,

the polymerization method seems to be successful for targeted chain length of approximately 40

repeat units, and this polymer was the focus of the hydrogel investigation as a result.

Given their importance for intended applications, confirming the preservation and functionality of

the hydroxyl groups on the O-4 and O-6 positions of the repeat galactose units then took place. To

investigate this, 2.05-20 was treated with 4-(trifluoromethyl)phenylboronic acid (2.09) in refluxing

toluene to introduce boronate esters on the repeat units via condensation reactions (Scheme 4).

2.05-20 had a well-resolved 1H NMR spectrum, and was thought to be suitable for this experiment

despite discrepancy between NMR- and GPC-determined DPn. 19F NMR analysis of the crude

product 2.10 suggested the presence of multiple boronate esters along the polymeric backbone due

to the appearance of a broad peak overlapping the narrow peak at 63.2 ppm characteristic of the

free acid species (Figure A14). However, the reaction solution changed colour from pink to

colourless during the reaction, suggesting that the dithiobenzoate endgroups of the polymer had

been hydrolyzed as observed in literature when subjected to hydrogen peroxide in hot water.18

The hydrolysis was likely caused by the water produced as a by-product of the polycondensations

and the high temperature of the reaction. Confirmation of the endgroup hydrolysis or conversion

by way of NMR spectroscopy was not straightforward, once again due to broad overlap of peaks

belonging to both the modified and unmodified polymers, in both 1H and 19F NMR spectra (Figures

A12 and A13, respectively). Endgroup conservation is critical for RAFT polymers meant for

incorporation into block copolymers, as polymers with conserved RAFT groups can be considered

as ‘macro RAFT agents’ and used for additional polymerizations themselves. Given that block

copolymerization was considered at the time as a potential future direction for this polymer system,

a short investigation into the preservation of the endgroup while transforming the O-4 and O-6

moieties was carried out.

Thus, the experiment was repeated with molecular sieves and acetonitrile at room temperature

instead of refluxing toluene, to determine if the endgroups could remain while the polymer was

modified in such a way. In these conditions, the polymer retained its pink colour, and 19F NMR

analysis again possessed a broad peak at the same location characteristic of polymeric material

(Figure A14). However, quantitative 1H NMR analysis of the crude reaction mixture was again

24

thwarted due to the broad overlap of peaks belonging to both the dithiobenzoate endgroup and the

aromatic ring of the repeating boronic ester (Figure A12). Although the general peaks known to

belong to the protons on the repeat galactose units were visible, the nature of the analysis remained

qualitative. Consequently, conversion nor endgroup analysis was not able to be determined.

Despite this difficulty, the broad peaks in both the 19F and 1H NMR spectra did suggest that the

boronic acids were now part of a polymeric material and were most likely of structure 2.11.

Accordingly, the polymerizations were assumed to have left the O-4 and O-6 hydroxyl moieties

intact for further transformations. The colour of the crude product also remained pink, as opposed

to 2.10, serving as further qualitative evidence of endgroup preservation. Given that the goal of

confirming the functionality of the O-4 and O-6 hydroxyl moieties had been achieved, the

experiment was not repeated or investigated further. UV-Vis measurements to analyse the presence

of the endgroup and boronic acid moeities could potentially serve to avoid the pitfalls of the NMR

analysis if this experiment were to be revisited. Investigation into purification via precipitation of

2.10 and 2.11 may also serve to afford a simpler polymer spectrum to analyze.

Scheme 4: Condensation of polymer 2.05-20. Structures 2.10 and 2.11 are tentatively

assigned based on 1H NMR spectroscopy, and have not been purified.

2.3 Hydrogel and Crosslinking Agent Synthesis

25

After having confirmed the functionality of the O-4 and O-6 hydroxyls on the pendant groups,

formation of a hydrogel using this new class of glycopolymer was explored. As part of an initial

proof of concept, boric acid was chosen as the crosslinking agent due to its non-toxic nature and

well-known ability to form gels with diol-bearing polymers such as poly (vinyl alcohol) under

basic aqueous conditions. Initial attempts in carbonate-buffered systems ranging from pH 8 to 10.5

did not produce gels that could pass the qualitative gel inversion test, but the viscosities of the

systems did increase, suggesting that crosslinking between the chemical species had occurred. In

this case, it is hypothesized that boric acid failed to adequately crosslink the polymer chains due

to its relatively weak interactions with diol groups and the steric strain per molecule of boric acid

binding the O-4 and O-6 positions of multiple chains.

Scheme 5: Proposed hydrogel formation using polymer 2.05 and boric acid

The success of polymeric crosslinking agents synthesized by the Sumerlin group,26 as discussed

in the introduction (1.4) inspired the use of poly(4-vinylphenylboronic acid) (PVPBA) as an

alternative crosslinking agent. Although not expected to form hydrogels at neutral pH, PVPBA

could still serve as a proof of concept as intended with previous crosslinking agents. Compared to

TRENBASIM, polymeric crosslinking agents such as PVPBA have the advantage of lessened

steric strain (depending on the solvent) and abundance of boronic acid groups per crosslinking

molecule, ideally leading to a higher density of crosslinking events.

26

Scheme 6: Synthesis of PVPBA

PVPBA was synthesized as per literature protocol43 (Scheme 6, Experimental 3.2) and purified by

precipitation of the reaction mixture in chloroform to afford a 47% yield. A drop of the crude

reaction mixture found to be partially miscible with methanol, allowing for a crude 1H NMR

analysis that revealed broad peaks assumed to belong to polymeric material along with unreacted

monomer (Figure A18). Pellon and coworkers noted difficulties associated with chemical analysis

of the polymer in their report, mainly due to low solubility of the polymer in single-solvent

systems, and relied mostly on viscosity measurements, gravimetric analysis and infrared

spectroscopy to support their claims. Importantly, the polymer was noted to require the presence

of aqueous NaOH to dissolve in water,43 preventing use of a pH-controlled system for the intended

hydrogelation experiments. NMR analysis of the precipitated polymer was not conducted due to

the aforementioned difficulty in dissolving the polymer in an appropriate deuterated solvent. Given

the matching physical properties and presence of polymeric material in the crude 1H NMR

spectrum, the precipitated solid was assumed to be PVPBA and used without further

characterization. When this solid was mixed in aqueous NaOH with 2.05-40 and gently heated

(Experimental 3.2), a hydrogel was formed that passed the gel inversion test (Figure 17), providing

further circumstantial evidence for the identity of PVPBA along with proving the possibility of

forming a hydrogel with polymer structure 2.05, as originally intended by this investigation.

27

Scheme 7: Hydrogel synthesis using polymer 2.05 and PVPBA

Figure 17: Hydrogel made from 6% 2.05-40 and 6% PVPBA in aqueous 2M NaOH. (A) is

the gel after 15 seconds in a 40 oC hot water bath. (B) is the same gel, inverted for the gel

inversion test.

2.3 Summary and Future Directions

In summary, a novel class of glycopolymers was designed and synthesized in this project. Notably,

usage of an organoboron catalyst allowed for easily accessible monomers from unprotected

carbohydrates in a singular step, despite unoptimized sub-50% yields (Scheme 1). Glycopolymers

synthesized via RAFT fell within narrow polydispersity ranges expected from RAFT

polymerization methods. More work is needed to assess reproducibility of RAFT polymerizations

and to define reliable protocols to separate polymers with a given DPn. The synthesized

glycopolymers were successfully used to form hydrogels, albeit limited to highly basic and

unbuffered systems.

Future work within the context of this project is directed towards formation of a hydrogel in a

controlled, neutral pH environment, to be followed by characterization and evaluation of the

properties of this new material. Synthesis of the polymeric crosslinking agent used by the Sumerlin

28

group, 2APBA (Figure 8), is underway as well as optimization and expansion of scope with respect

to the carbohydrate monomers and their respective polymers.

Experimental

General

All reactions were stirred with Teflon-coated stir bars dried at 140 oC prior to use and carried out

without effort to exclude air or moisture, unless noted otherwise. Stainless steel needles and gas-

tight syringes were used to transfer air- and moisture-sensitive liquids. Flash silica gel

chromatography was performed using (60 Å, 230-400 mesh) (Silicycle). Aluminium-backed silica

gel 60 F254 plates (EMD Milipore) were used to perform TLC analysis and visualized with a UV

254 lamp and/or aqueous potassium permanganate (KMnO4) solution.

Materials and Methods

Methyl methacrylate was filtered through basic alumina prior to use in polymerizations.

Azobisisobutyronitrile (AIBN) was recrystallized from methanol. Acetonitrile was purified by

passing through two columns of activated alumina under nitrogen (Innovative Technology, Inc.).

Deuterated solvents were purchased from Cambridge Isotope Laboratories or Sigma-Aldrich. All

other starting materials were procured from Sigma-Aldrich.

Nuclear Magnetic Resonance Spectroscopy

Nuclear Magnetic Resonance (NMR) spectra were recorded using the following spectrometers:

Bruker Avance III 400 and Varian Mercury 400. The spectra were processed using MestReNova.

1H NMR spectra are reported in parts per million (ppm) relative to tetramethylsilane and referenced

to residual protium in the solvent. Spectral features are tabulated in the following order : chemical

shift (σ, ppm); multiplicity (s-singlet, d-doublet, t-triplet, q-quartet, quin-quintet, m-complex

multiplet, app-apparent, br-broad); number of protons; coupling constants (J, Hz). 19F NMR

spectra were calibrated to an external standard of 2,2,2-trifluoroethanol (σ -78.222 ppm, C6D6).

Gel Permeation Chromatography

Gel permeation chromatography (GPC) was conducted at 85 oC using N-methylpyrrolidine (NMP)

as eluent, at a flow rate of 1.0 ml/min through a guard column and one Agilent PLgel 5um MIXED-

29

C columns equipped with a refractive index detector. Poly(methyl methacrylate) (PMMA)

standards were used for external calibration.

3.1 Monomer Synthesis

General Procedure A - (2R,3S,4S,5R,6R)-3,5-dihydroxy-2-(hydroxymethyl)-6-

methoxytetrahydro-2H-pyran-4-yl methacrylate

To an oven-dried round-bottomed flask was added methyl-β-D-galactopyranoside (388.0 mg, 2.0

mmol) and 1.09 (39.2 mg, 0.2 mmol, 0.10 equiv). Under an atmosphere of argon, anhydrous

acetonitrile (20 mL) was added, followed by diisopropylethylamine (522.5 μL, 3.0 mmol, 1.5

equiv). The resulting clear mixture was then cooled to 0 oC, at which point methacryloyl chloride

(214.9 μL, 2.2 mmol, 1.1 equiv) was added dropwise. Afterwards, the reaction mixture was left to

warm up to room temperature and proceed overnight, at which point the orange reaction mixture

was quenched by the addition of methanol (20 mL). The resulting orange solution was then

evaporated under vacuum to produce a dark orange oil identified as the crude product. Purification

of the crude product via silica gel column chromatography, using an eluent system of

DCM/MeOH, 4:96 to 8:92) resulted in off-white/white powder weighing 215.1 mg (41 % isolated

yield)

30

1H NMR (400 MHz, Chloroform-d) δ 6.23 (s, 1H, HI), 5.67 (s, 1H, HJ), 4.90 (dd, J = 10.1, 3.2

Hz, 1H, HC), 4.32 (d, J = 7.7 Hz, 1H, HA), 4.20 (app t, J = 3.4 Hz, 1H, HD), 4.03–3.87 (m, 3H, HB

and HF), 3.61 (s, 3H, HG), 2.56 (d, J = 4.0 Hz, 1H, HE),1.99 (s, 3H, HH)

13C NMR (100 MHz, CD3OD) δ 169.35, 138.62, 127.39, 106.86, 78.63, 77.26, 70.83, 68.65,

63.06, 58.18, 19.27.

HRMS (DART+): calcd for C11H22N1O7 [M + NH4]+ = 280.1 m/z ; found 280.140 m/z

(2S,3R,4S,5S,6R)-2,3,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl

methacrylate

Synthesized according to General Procedure A, using methyl-α-D-galactopyranoside (388 mg, 2

mmol). Purification of the crude product via silica gel column chromatography, using an eluent

system of DCM/MeOH, 4:96 to 8:92) resulted in off-white/white powder weighing 162.3 mg (31

% isolated yield)

1H NMR (400 MHz, Methanol-d4) δ 6.11 (s, 1H), 5.54 (s, 1H), 4.91 (dd, J = 10.5, 3.2 Hz, 1H),

4.67 (d, J = 3.9 Hz, 1H), 4.03 – 3.94 (m, 2H), 3.75 (app t, J = 6.1 Hz, 1H), 3.65–3.55 (m, 2H), 3.24

(s, 2H), 1.86 (s, 3H).

13C NMR (100 MHz, Methanol-d4) δ 169.51, 138.68, 127.35, 102.40, 76.03, 72.87, 69.47, 68.54,

63.36, 56.57, 19.28.

HRMS (DART+): calcd for C11H19O7 [M + H]+ = 263.270 m/z ; found 263.1130 m/z

(2R,3S,4S,5R,6S)-3,5-dihydroxy-2-(hydroxymethyl)-6-(isopropylthio)tetrahydro-2H-pyran-

4-yl methacrylate

31

Synthesized according to general procedure A, using isopropyl β-D-1-thiogalactopyranoside

(476.7 mg, 2.0 mmol). Purification of the crude product via silica gel column chromatography,

using an eluent system of DCM/MeOH, 4:96 to 8:92) resulted in off-white/white powder weighing

145.2 mg (24 % isolated yield)

1H NMR (400 MHz, Methanol-d4) δ 6.20 (s, 1H), 5.64 (s, 1H), 4.79 (dd, J = 9.7, 3.3 Hz, 1H),

4.51 (d, J = 9.8 Hz, 1H), 4.09 (d, J = 4.0 Hz, 1H), 3.75–3.63 (m, 2H), 3.31–3.27 (m, 1H), 3.25 (p,

J = 6.8 Hz, 1H), 1.96 (s, 3H), 1.31 (app t, J = 6.5 Hz, 6H).

13C NMR (100 MHz, Methanol-d4) δ 169.30, 138.66, 127.38, 88.18, 81.08, 79.86, 69.93, 68.93,

63.18, 36.79, 25.27, 19.28.

HRMS (DART+): calcd for C13H26N1O7S1 [M + NH4]+ = 324.41 m/z ; found 324.148 m/z

3.2 Polymer Synthesis

General procedure B - Compound #2.05-20

To an oven-dried 25 mL Schlenk tube was added a small stir bar, monomer 2.01 (300.0 mg, 1.1

mmol) and 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (15.9 mg, 0.06 mmol, 0.05 equiv).

A portion of freshly made stock solution of DMF in AIBN (1.0 mL, 1.88 mg, 0.01 mmol, 0.001

equiv) was added to the Schlenk tube, followed by DMF to reach a total reaction volume of 3 mL.

32

The reaction mixture was then degassed via 5 freeze-pump-thaw cycles, backfilled with argon gas,

sealed and left to react while stirring in a pre-heated oil bath at 80 oC for 15 hours. The reaction

was terminated by opening the Schlenk tube to air and removing from heat. Analysis of an aliquot

of crude reaction mixture by 1H NMR indicated broad peaks across the spectrum, heavily

suggesting the presence of a polymeric species in the reaction mixture. The polymer was

precipitated by dropwise addition of the crude reaction mixture into ice-cold EtOAc, followed by

vacuum filtration through a Hirsch funnel to yield a light pink powder. Analysis of the polymer

product via 1H NMR spectroscopy indicated presence of alkene groups attributed to residual

monomer. The polymer was thus reprecipitated by dissolution in minimal MeOH, followed again

by dropwise addition into ice-cold EtOAc and vacuum filtration to yield a light pink powder (161.2

mg, 51.0% mass recovery). This colour is consistent with the polymer possessing a dithiobenzoate

endgroup originating from the chain transfer agent. 1H NMR spectroscopy of the twice-

precipitated polymer indicated no residual monomer, as well as a degree of polymerization of 34

(Mn = 9200 Da) based on integration of the aromatic protons belonging to the dithiobenzoate

endgroup compared to that of the protons belonging to the repeat units (normalized) at 4.29,4.13,

2.5–1.8 and 1.5–0.8 ppm. GPC (N-methylpyrrolidone, 85 oC, calibrated to PMMA standards)

indicated a Đ of 1.27 and Mn = 5000 Da relative to PMMA.

1H NMR (399 MHz, Methanol-d4) δ 7.85 (br s, 2H), 7.55 (br s, 1H), 7.39 (br s, 2H), 4.84 (br s,

212H), 4.56 (br s, 46H), 4.27 (br s, 36H), 4.11 (br s, 36H), 3.74 (br s, 102H), 3.58 (br s, 141H),

2.34–1.80 (m, 65H), 1.47 – 0.94 (m, 98H).

Compound #2.05-40

33

Synthesized and precipitated according to General Procedure B, altering the [RAFT CTA] :

[Monomer] ratio to 1 : 0.025 to target a DPn of 40. Vacuum filtration yielded a light pink powder

(164.8 mg, 53.5% mass recovery). This colour is consistent with the polymer possessing a

dithiobenzoate endgroup originating from the chain transfer agent. 1H NMR spectroscopy of the

twice-precipitated polymer indicated no residual monomer, as well as a degree of polymerization

of 45 (Mn = 1.2 x 104 Da) based on integration of the aromatic protons belonging to the

dithiobenzoate endgroup compared to the average of the protons belonging to the repeat units

(normalized) at 4.29,4.13, 2.5–1.8 and 1.5–0.8 ppm. GPC (N-methylpyrrolidone, 85 oC, calibrated

to PMMA standards) indicated a Đ of 1.25 and Mn = 8600 Da relative to PMMA.

1H NMR (399 MHz, Methanol-d4) δ 7.87 (br s, 2H), 7.56 (br s, 1H), 7.40 (br s, 2H), 4.56 (br s,

48H), 4.28 (br s, 47H), 4.13 (br s, 45H), 3.94 – 3.67 (m, 146H), 3.58 (br s, 196H), 2.51 – 1.88 (m,

83H), 1.61 (m, J = 61.0 Hz, 19H), 1.17 (m, J = 64.0 Hz, 139H)

Compound # 2.05-100

Synthesized and precipitated according to General Procedure B, altering the [RAFT CTA] :

[Monomer] ratio to 1 : 0.01 to target a DPn of 100. Vacuum filtration yielded a light pink powder

(90.2 mg, 30.7% mass recovery). This colour is consistent with the polymer possessing a

dithiobenzoate endgroup originating from the chain transfer agent. 1H NMR spectroscopy of the

twice-precipitated polymer indicated no residual monomer, as well as a degree of polymerization

of 102 (Mn = 2.7 x 104 Da) based on integration of the aromatic protons belonging to the

dithiobenzoate endgroup compared to that of the protons belonging to the repeat units at 4.29,4.13,

2.5–1.8 and 1.5–0.8 ppm. GPC (N-methylpyrrolidone, 85 oC, calibrated to PMMA standards)

indicated a Đ of 1.6 and Mn = 10 090 Da relative to PMMA.

34

1H NMR (399 MHz, Methanol-d4) δ 7.88 (br s, 2H), 7.56 (br s, 1H), 7.40 (br s, 2H), 4.83 (br s,

937H), 4.56 (br s, 117H), 4.28 (br s, 108H), 4.13 (br s, 107H), 3.75 (br s, 328H), 3.59 (br s, 444H),

2.47–1.89 (m, 178H), 1.69 (br s, 36H), 1.58 – 0.88 (m, 317H).

Compound # 2.10

To a 20 mL screwcap vial was added a small stir bar, followed by 2.05-20 (11.6 mg, 0.04 mmols),

4-(trifluoromethyl)phenylboronic acid (7.6 mg, 0.04 mmols, 1 equiv), 3Å molecular sieves (40.0

mg) and acetonitrile (2.0 mL). The resulting light pink solution was capped and left to stir

overnight. After 24 hours, the solution was filtered through cotton and evaporated under vacuum

to afford a light pink solid (18 mg, 93.8% crude yield). Further purification was not attempted. 1H

and 19F NMR spectra are provided in the appendix (Figures A14, A15 and A16), and show

presence of both polymeric and monomeric species, as well as what appear to be solvent peaks.

Poly (4-vinylphenylboronic acid) (PVPBA)

Synthesized as per previously reported procedure.43

To an oven dried, 25 mL Schlenk tube was added a small stir bar, 4-vinylphenylboronic acid (200

mg, 1.35 mmol), AIBN (5.0 mg, 0.03 mmol, 0.023 equiv) and water : tert-butyl alcohol solution

35

(1 : 4, 1.0 mL). The Schlenk tube was then sealed with a rubber septum, subjected to five freeze-

pump-thaw cycles, backfilled with argon and left to stir in a pre-heated oil bath at 70 oC for 100

minutes, at which point the reaction was quenched by opening to air and removing from the oil

bath. The clear, viscous solution was then added dropwise to chloroform (200 mL), affording

translucent, insoluble strings identified as the polymer. Vacuum filtration yielded a white solid

(94.0 mg, 47% mass recovery), used without further purification. NMR spectroscopy was only

possible for a small drop retrieved from crude reaction mixture that was found to be partially

miscible with methanol; the insolubility of the polymer in the vast majority of solvents as per the

investigation of Pellon and coworkers prevented NMR analysis of the precipitated product.43 1H

NMR spectroscopic analysis of the crude sample indicated the presence of polymeric species, as

well as unreacted monomer (Figure A17).

Hydrogel Formation from 2.05-40 and PVPBA

To a ½ dram vial was added 2.05-40 (0.0120 g) and distilled water (0.100 mL) to make a 12 wt%

polymer solution. To another ½ dram vial was added crosslinking agent PVPBA (12.0 mg) and

aqueous NaOH solution (2 M, 0.100 mL) to make a 12 wt% crosslinker solution. The polymer

solution was added to the crosslinker solution dropwise, and then heated in a 35 oC water bath for

15 seconds, resulting in a colourless, opaque gel. The gel inversion test was then conducted by

inverting the vial and observing whether the gel would flow due to gravity. The gel did not flow

during this test.

36

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Appendices

A1. NMR Spectra of Compounds

Figure A01 : 1H NMR Spectrum (399 MHz, CDCl3 ) of 2.01

Figure A02 : 2D COSY NMR Spectrum (399 MHz, CDCl3 ) of 2.01

Figure A03 : 1H NMR Spectrum (399 MHz, DMSO-d ) of 2.01

Figure A04: 13C NMR Spectrum (100 MHz, MeOD) of 2.01

Figure A05: 1H NMR Spectrum (399 MHz, MeOD) of 2.02

Figure A06: 13C NMR Spectrum (100 MHz, MeOD) of 2.02

Figure A07: 1H NMR Spectrum (399 MHz, MeOD ) of 2.03

Figure A08: 13C NMR Spectrum (100 MHz, MeOD ) of 2.03

Figure A09 : 1H NMR Spectrum (399 MHz, MeOD ) of 2.05-20

Figure A10: 1H NMR Spectrum (399 MHz, MeOD ) of 2.05-40

Figure A11 : 1H NMR Spectrum (399 MHz, MeOD ) of 2.05-100

Figure A12: 1H NMR Spectrum (399 MHz, MeOD ) of 2.10

Figure A13: 19F NMR Spectrum (375 MHz, MeOD ) of 2.10

Figure A14: 1H NMR Spectrum (399 MHz, CDCl3 ) of 2.11

Figure A15: 19F NMR Spectrum (375 MHz, CDCl3 ) of 2.11; full spectrum shown.

Figure A16: 19F NMR Spectrum (375 MHz, CDCl3 ) of 2.11; full spectrum shown.

Figure A17: 1H NMR Spectrum (399 MHz, MeOD ) of crude PVPBA reaction mixture; full spectrum shown.

A2. GPC Traces of Compounds

Figure A18 : GPC Traces (N-methylpyrrolidone, 85 oC) of 2.05-20, 2.05-40 and 2.05-100, overlaid. Measurements were made relative to poly (methyl methacrylate) (PMMA) standards.

160

165

170

175

180

185

3 4 5 6 7 8

Inte

nsi

ty (

AU

)

Retention volume (mL)

GPC Traces of Polymers with General Structure 2.05

2.05-20

2.05-40

2.05-100