Anti-Malarial Polymer-Peptide Conjugates

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By Paul Reader

Dissertation presented in partial fulfilment of the requirements for the degree of PhD

(Polymer Science)

Promoter: Prof. Bert Klumperman

University of Stellenbosch

Department of Chemistry and Polymer Science

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Declaration

By submitting this dissertation electronically, I declare the entirety of the work contained therein is my own, original work, that I am the owner of the copyright thereof (unless to the extent explicitly otherwise stated) and that I have not previously in its entirety or in part submitted it for obtaining any qualification.

Paul Reader September 2014

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Abstract

The primary aim of this study was to investigate an amphiphilic polyvinylpyrrolidone (PVP)-based drug delivery system for the treatment of the Plasmodium falciparum strain of malaria, using a known anti-malarial cylic decapeptide, tyrothricin.

A triazole-based reversible addition-fragmentation chain-transfer (RAFT) agent, comprising an acetal-based R-group and a xanthate-based Z-group was synthesised. α-Acetal, ω-xanthate heterotelechelic PVP was synthesised via RAFT polymerisation and it was shown that the polymerisation was adequately controlled. A one-pot orthogonal chain-end functionality deprotection strategy was developed and conditions were established to conjugate a model targeting ligand and a model drug linker to the α- and ω-chain-ends, respectively.

A micellar drug delivery system was developed by conjugating the aggregation-prone tyrothricin to PVP, via its ω-chain-end functionality through an acid-labile linker. The α-chain-end functionality of the PVP was sparsely conjugated to a targeting ligand and to a fluorescent marker. In aqueous media, the conjugate exhibited self-assembly into micelles. The tyrothricin formed the core of the micelle, stabilised via the hydrophilic PVP, decorated with the targeting ligands and fluorescent marker. The conjugate was shown to inhibit chloroquine-resistant P. falciparum strains of malaria in picomolar concentrations with virtually no haemolysis observed; a 700-fold improvement of the IC50 over tyrothricin alone was observed. In addition, the conjugates were found to vaccinate the erythrocytes against re-infection. The drug delivery system appears to be a promising candidate for further investigation as a treatment against drug-resistant strains of malaria.

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Opsomming

Die primêre doel van hierdie studie was om 'n amfifiliese polivinielpirolidoon (PVP)-gebaseerde medisyne-toedieningstelsel vir die behandeling van die Plasmodium falciparum stam van malaria te ondersoek deur gebruik te maak van 'n bekende anti-malaria sikliese dekapeptied, genaamd tyrotrisien.

'n Triasool-gebaseerde omkeerbare byvoeging-fragmentasie kettingoordrag (BFKO) agent is gesintetiseer wat bestaan het uit 'n asetal-gebaseerde R-groep en 'n xantaat-gebaseerde Z-groep. α-Asetal, ω-xantaat heterotelesheliese PVP is gesintetiseer deur BFKO gemedieerde-polimerisasie en daar is bevind dat die gemedieerde-polimerisasie voldoende beheer is. 'n Een-pot ortogonale endketting funksionaliteitsontskerming strategie is ontwikkel en kondisies is bepaal om 'n model teiken ligand en 'n model medisyne aanhegter aan die α- en ω-endkettings, onderskeidelik, te voeg.

‘n Missel-gebaseerde medisyne-toedieningstelsel is ontwikkel deur konjugasie van die tyrotrisien, wat geneig is om te aggregeer, aan die PVP. Dit het geskied deur die ω-endketting funksionaliteit deur middel van 'n suur-sensitiewe aanhegter. Die α-endketting funksionaliteit van die PVP is yl gekonjugeer met 'n teiken ligand en fluoreserende merker. In ‘n watermedium het die gekonjugeerde molekules selfsamevoeging-eienskappe getoon om miselle te vorm. Die tyrotrisien het die kern van die miselle gevorm en is gestabiliseer deur die hidrofiliese PVP, versier met die teiken ligande en fluoreserende merker. Daar is bevind dat die gekonjugeerde molekules chloroquine-weerstandige P. falciparum stamme van malaria geïnhibeer het in picomolare konsentrasies met feitlik geen hemoliese waargeneem; 'n 700-voudige verbetering van die IC50 van tyrotrisien alleen is waargeneem. Daarbenewens is bevind dat die gekonjugeerde molekules die rooibloedselle teen herbesmetting ge-ent het. Die medisyne-toedieningstelsel blyk 'n belowende kandidaat te wees vir verdere ondersoek as 'n behandeling teen medisyne-weerstandige stamme van malaria.

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Acknowledgements

Firstly, I would like to thank God for blessing me with the opportunity to study and friends and family that support me.

I would like to acknowledge and thank my promoter, Bert Klumperman, for his invaluable guidance, encouragement and friendship. You have always remained optimistic and that has been a great motivation to me. Thank you for giving me an opportunity to work with you. I would also like to thank Marina Rautenbach, with whom I collaborated in the later stages of this project, who helped bring it to completion. I particularly appreciate the many hours that you spent with me, working through the data.

I would also like to thank a friend and past colleague, Eric van den Dungen, for his mentorship that shaped the rigour with which I work today.

I extend my gratitude to all my colleagues, mentors, assistants and friends from Polymer Science, past and present. There are too many to name but I thank you all for the assistance, discussions and friendships that we made. I would also like to thank the staff and support-staff of Polymer Science. Thank you for the many ways you aided me during my PhD studies and the friendships that we made.

I thank the Biopep Peptide group for welcoming me into their lab. In particular, I would like to thank Arnie, Wikus and Helba for their assistance, guidance and friendship.

I would like to thank Elsa Malherbe and Jaco Brand for NMR spectroscopy assistance, Ben Loos for fluorescence microscopy analysis, Mohammed Jaffer for TEM analysis and Nadine Pretorious for SEC analysis.

I am eternally grateful for the funding I received from the National Research Fund, the Postgraduate Merit Bursary and Stellenbosch University.

On a more personal level, I deeply value the support and encouragement I received from my family. Above all, I cannot thank my wife Tammy enough, for her love, encouragement and support during my studies, especially through the many late nights towards the end of my PhD. Thank you for always believing in me!

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List of Figures

Figure 1.1 Schematic representation of miceller structure of the PVP-tyrothricin drug

delivery system. 2

Figure 2.1 Functionalities achieved through thiocarbonyl thio moiety manipulation. 8

Figure 2.2 Azide- and alkyne-terminal RAFT agents for the polymerisation of vinyl acetate

and styrene, respectively. 10

Figure 2.3 Ringsdorf's designed drug delivery system. 11

Figure 3.1 1H NMR spectrum confirming the deprotection of acetal 9. 29

Figure 3.2 1H NMR spectrum of RAFT agent 10. 30

Figure 3.3 Molar mass as a function of conversion plot for the synthesis of PVP using RAFT

agent 10. Dotted lines represent the theoretical curves. 30

Figure 3.4 Representative 1H NMR spectrum of PVP synthesised using RAFT agent 10. 32

Figure 4.1 1H NMR spectrum of the cleavage of the xanthate, affording the pyridyl

disulphide activated product, 13. 41

Figure 4.2 1H NMR spectrum of the deprotection of the acetal functionality, affording 14. 43

Figure 4.3 1H NMR spectrum of the deprotection of the PVP polymer system. 46

Figure 4.4 DRI and UV SEC chromatogram comparing PVP 11 to PVP 15. 46

Figure 4.5 1H NMR spectrum of Gly-DL-Ser conjugated to 11, i.e. compound 17. 48

Figure 4.6 Thiol-Michael addition 1H NMR spectra of product from 17 and 18, yielding

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Figure 4.7 Phenolate release as a function of time. 52

Figure 5.1 GSRSKGT structure. 62

Figure 5.2 1H NMR spectrum of the incorporation of targeting ligand in PVP 20. 64

Figure 5.3 Results of the Kaiser test performed on PVP 20 and 21, respectively: (1) Control –

DMF and reagents; (2) PVP 16 – control; (3) PVP 21; (4) PVP 15 – control; (5) PVP 20. 65

Figure 5.4 Primary structure for TrcB. The variable positions 3-,4-, 7- and 9-, with the

alternative amino acid residues found in the Trc analogues. 66

Figure 5.5 UPLC-MS chromatogram depicting the separation of tyrothricin and tabulated

assignments of the major peaks. 67

Figure 5.6 UPLC-MS chromatogram depicting the separation of the modified tyrocidines and

tabulated assignments of the major peaks. 69

Figure 5.7 Aggregation of TrcA as observed via mass spectroscopy; (a) mass spectrum for

TrcA; (b) calculated mass spectrum for TrcA. 70

Figure 5.8 Aggregation MS data for the acrylate-modified tyrocidine A; (a) mass spectrum

for Mod-TrcA; (b) calculated mass spectrum for Mod-TrcA. 71

Figure 5.9 1H NMR spectra comparison between acrylate-modifed tyrocdine 22 and

conjugate 23. 73

Figure 5.10 - DLS size distributions of conjugate 23 and 24. 74

Figure 5.11 TEM images of conjugate 23 and 24; (a) conjugate 23; (B) conjugate 24. 74

Figure 5.12 Release of tyrocidine from conjugate 23 over time, in a phosphate buffer (pH =

5.5). 75

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Figure 5.14 IC50 and HC50 results for the dose-response assay of conjugates 23 and 24. 78

Figure 5.15 Giemsa-stained blood smears from P. falciparum cultures (24 hours) treated with

conjugates 23 and 24; (a) growth control; (b) growth control; (c) conjugate 24; (d) conjugate

24; (e) conjugate 23; (f) conjugate 23. Refer to the text for an explanation of the arrows. 79

Figure 5.16 CFM images of the blood smears taken at 48 hours; (a,b,c) growth control; (d,e,f)

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List of Schemes

Scheme 2.1 Main equilibrium of the RAFT process. 6

Scheme 3.1 Xanthate-mediated RAFT polymerisation. 22

Scheme 3.2 Triazole-based RAFT agent. 23

Scheme 3.3 Synthesis of RAFT agent 3; (a) THF, r.t.; (b) NaN3, DMSO, 100 oC; (c)

CuSO4.5H2O, sodium ascorbate, DMF, r.t. 24

Scheme 3.4 Synthesis of RAFT agent 5; (a) Tosyl chloride, KOH, diethyl ether, 0 oC to r.t.;

(b) THF, r.t.; (c) CuSO4.5H2O, sodium ascorbate, DMF, r.t. 25

Scheme 3.5 Synthesis of model cyclic acetal 6; (a) CuSO4.5H2O, sodium ascorbate, DMF, r.t. 26

Scheme 3.6 Acetal deprotection. 27

Scheme 3.7 Synthesis of model acetal 9; (a) Tosyl chloride (TsCl), KOH, 0 oC, imidazole,

HCl; (b) CuSO4.5H2O, sodium ascorbate, DMF, r.t. 28

Scheme 3.8 Synthesis of RAFT agent 10; (a) CuSO4.5H2O, sodium ascorbate, DMF, r.t. 29

Scheme 4.1 Strategy 1: Deprotection of model compound 10; (a) hexyl amine, DTP, THF,

r.t.; (b) 1 M HCl in acetone, r.t. 41

Scheme 4.2 Strategy 2: Deprotection of model compound 10; (a) 1 M HCl in acetone, r.t.; (b)

hexyl amine, DTP, THF, r.t.; 42

Scheme 4.3 One-pot deprotection of model compound 10; (a) hexylamine, acetone, r.t.; (b) 4

M HCl in dioxane, acetone, r.t. 44

Scheme 4.4 One-pot deprotection of PVP system; (a) hexylamine, acetone, r.t.; (b) 4M HCl in

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Scheme 4.5 Schiff base formation and subsequent reductive amination. 47

Scheme 4.6 Conjugation of model targeting ligand (Gly-DL-Ser) to 15; (a) sodium borate

buffer (pH = 9.1), NaBH3CN, r.t. 47

Scheme 4.7 Thiol-Michael-Addition reaction. 49

Scheme 4.8 Synthesis of model drug 18; (a) TEA, DCM, r.t. 49

Scheme 4.9 Thiol-Michael addition between 17 and 18; (a) TCEP, ethylene diamine, DMF,

r.t. 50

Scheme 4.10 Conversion of phenol to the phenolate ion; (a) NaOH, H2O, r.t. 52

Scheme 5.1 Conjugation of targeting ligand to 15 and 16; (a) GSRSKGT,

6-aminofluorescein, n-propylamine, NaBH3CN, sodium borate buffer (pH = 9.7), r.t. 63

Scheme 5.2 Synthesis of acrylate-modified tyrocidines (22); (a) DIPEA, DMF, 4 oC 68

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List of Tables

Table 2.1 PEG alternative hydrophilic polymers. 13

Table 2.2 Acid-labile linkers commonly found in drug delivery systems. 16

Table 3.1 Polymerisation conditions and results for PVP polymerised using RAFT agent 3 24

Table 3.2 Polymerisation conditions and results for PVP polymerised using RAFT agent 5 26

Table 3.3 Polymerisation conditions and results for PVP polymerised using RAFT agent 10

31

Table 5.1 Summary of the tyrocidines and tryptocidines and their analogues. 66

Table 5.2 Dose-response data for conjugates 23 and 24. The concentration values are given in

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List of Acronyms

AIBN 2,2’-Azobis(isobutyronitrile)

ATRP Atom transfer radical polymerisation BHT 2,6-Di-tert-butyl-4-methylphenol BSA Bovine serum albumin

CFM Confocal fluorescence microscope CRP Controlled radical polymerisation CTA Chain-transfer agent

CuAAC Copper-catalysed alkyne azide 1,3-dipolar cycloaddition DLS Dynamic light scattering

DMAc Dimethylacetamide DP Degree of polymerisation DRI Differential refractive index DTP 2,2’-Dithiopyridine

EPR Enhanced permeability and retention FDA Food and drug administration

IC50 50 % inhibitory concentration Hb Haemoglobin HC50 50 % haemolytic concentration L-Lys L-lysine L-Orn L-ornithine L-Tyr L-tyrosine

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xvii MADIX Macromolecular design via the exchange of xanthates

MALDI-TOF Matrix-assisted laser desorption/ionisation time-of-flight MaxEnt Maximum Entropy

MS Mass spectroscopy

MWCO Molecular weight cut-off

NMP Nitroxide mediated polymerisation NMR Nuclear magnetic resonance NVP N-Vinylpyrrolidone

ODN Oligodeoxynucleotide PDI Poly dispersity indices PEG Polyethylenegylcol

pHEMA Poly(2-hydroxyethyl methacrylate)

pHPMA Poly (N-(2-hydroxypropyl) methacrylamide PMMA Poly(methyl methacrylate)

PMOZ Poly(2-methyl-2-oxazalonine) pNIPAAm Poly(N-isopropylacrylamide) POZ Poly(2-ethyl 2-oxazoline) PVP Polyvinylpyrrolidone r.t. Room temperature

RAFT Reversible addition-fragmentation chain-transfer SEC Size exclusion chromatography

TEM Transmission electron microscopy

TCEP Tris(2-carboxyethyl)phosphine hydrochloride TLC Thin layer chromatography

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Trc Tyrocidine

Tpc Tryptocidine

UPLC-MS Ultra performance liquid chromatography linked to electrospray mass spectrometry

ESMS Electrospray mass spectroscopy

UV Ultra-violet

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Chapter 1: Prologue

1.1 Introduction

Malaria is endemic in 97 developing world countries with an estimated 207 million clinical malaria cases reported in 2012. There were an estimated 627 000 deaths reported in 2012, where the highest mortality rate occurred among children living in Africa.1 Malaria is caused by parasites from the genus Plasmodium, of which there are 4 species that infect humans. P.

falciparum, however, is the most concerning, as it contributes to the highest mortality rate.2 In addition, many strains of Plasmodium falciparum are drug resistant, predominantly towards chloroquine.3 Thus, there is a pressing need to develop new effective antimalarial drugs.

Antimicrobial peptides, that have membrane-linked mechanisms of action, have been introduced as potential candidates for antimalarial agents, as their membranolytic activity reduces the chances of parasites developing drug resistance.4 In particular, tyrothricin, a mixture of cyclic decapeptides, produced by Bacillus Brevis, was found to be active against chickens infected with Plasmodium gallinaceum in 1944.5 Sixty years later, the tyrocidines, major components of tyrothricin, were purified and found to be active against P. falciparum with nanomolar 50 % P. falciparum inhibitory concentrations (IC50). However, a micromolar

P. falciparum 50 % haemolytic concentration (HC50) was reported and the tyrocidines were found to not be selective between normal and malaria-infected erythrocytes, thus decreasing their potential for drug use.6

In traditional drug delivery, the therapeutic agent is distributed throughout the entire body, post administration. Consequently, only a low concentration of the drug reaches its target site.7 Targeted drug delivery is a strategy to overcome the concentration deficiency, by delivering the therapeutic agent in a high concentration, to a specific part of the body. In addition, side-effects are minimised as only the target site is exposed to the drug.8 This is accomplished by using polymer therapeutics, an umbrella term for supramolecular drug-delivery systems, polymer-protein conjugates and polymer-drug conjugates.9 These systems have the ability to hide the drug from the bodies physiological environment, until it reaches

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2 the target site, after which a change in the physiological environment can trigger the release of the therapeutic agent.10

It is well known that the pH of erythrocytes is 7.4 while the pH of the food vacuole in the malaria parasite, within the erythrocyte is 5.5.11 This dissertation focusses on the development of a targeted drug delivery system for P. falciparum, comprising a hydrophilic polyvinylpyrrolidine (PVP) segment conjugated to aggregation-prone tyrothricin, through an acid-labile linker, self-assembled in water to form miceller structures, sparsely decorated with malaria-infected erythrocyte-targeting ligands (Figure 1.1).

Figure 1.1 Schematic representation of miceller structure of the PVP-tyrothricin drug delivery system.

1.2 Objectives of dissertation

The objectives of this study can be summarised as follows:

1. To synthesise α-acetal, ω-xanthate heterotelechelic PVP via controlled radical polymerisation (CRP).

2. To develop an orthogonal deprotection strategy for the heterotelechelic PVP and conjugate the α- and ω-chain-end functionalities to a model drug and targeting ligand. 3. To conjugate the heterotelechelic PVP to tyrothricin, a suitable targeting ligand and a

fluorescent marker, and subsequently self-assemble the construct.

4. To study the structure of the construct and test its effectiveness against P. falciparum via malaria assays, in-vitro.

5. To study the selectivity of the drug delivery system against malaria-infected erythrocytes.

Acid-labile linker

PVP

Tyrothricin

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1.3 Outline of dissertation

Chapter 1 – Prologue

Chapter 1 briefly introduces malaria, antimicrobial peptides that have shown excellent results against malaria and the ability to incorporate them into a drug delivery system that targets malaria. In addition, the objectives of the study are presented.

Chapter 2 – Historical and Theoretical Background

A comprehensive literature review is presented, that assesses the critical elements of CRP, the ability to introduce and orthogonally manipulate chain-end functionality. In addition, polymer-protein/polypeptide conjugation is discussed, including aspects such as targeted drug delivery and drug-release mechanisms.

Chapter 3 – Synthesis and Characterisation of Polyvinylpyrrolidone

Chapter 3 addresses the choice of a suitable chain transfer agent for the synthesis of an α-acetal, ω-xanthate heterotelechelic PVP via reversible addition-fragmentation chain-transfer (RAFT) mediated polymerisation. Additionally, characterisation of the polymers is discussed.

Chapter 4 - Orthognal Deprotection of Model Conjugation Studies

Chapter 4 presents the development of an orthogonal deprotection strategy for α-acetal, ω-xanthate heterotelechelic PVP. Conjugation chemistries are developed for the addition of a model targeting ligand and model drug linker.

Chapter 5 - Tyrocidine PVP Anti-Malarial Conjugates

Chapter 5 addresses the conjugation and self-assembly of PVP to tyrothricin and a suitable malarial-infected erythrocyte-targeting ligand. The conjugate is tested against malaria-infected erythrocytes and selectivity is studied via fluorescence microscopy.

Chapter 6 – Epilogue

A summary of the work described in this dissertation is presented. Suggestions are presented for future research for the development of this drug delivery system.

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1.4 References

(1) World Health Organization. Media centre: Malaria. Retrieved 07 September, 2014, from http://www.who.int/mediacentre/factsheets/fs094/en/.

(2) Good, M. F.; Kaslow, D. C.; Miller, L. H. Annu. Rev. Immunol. 1998, 16, 57. (3) Bruce-Chwatt, L. BMJ (Clinical research ed.) 1982, 285, 674.

(4) Brown, E. D.; Wright, G. D. Chem. Rev. 2005, 105, 759.

(5) Taliaferro, L. G.; Coulston, F.; Silverman, M. J. Infect. Dis. 1944, 75, 179.

(6) Rautenbach, M.; Vlok, N. M.; Stander, M.; Hoppe, H. C. Biochim. Biophys. Acta 2007, 1768, 1488.

(7) Keraliya, R. A.; Patel, C.; Patel, P.; Keraliya, V.; Soni, T. G.; Patel, R. C.; Patel, M. M. ISRN Pharmaceutics 2012, 2012, 9.

(8) Mills, J. K.; Needham, D. Expert Opin. Ther. Pat. 1999, 9, 1499. (9) Haag, R.; Kratz, F. Angew. Chem., Int. Ed. 2006, 45, 1198. (10) Schmaljohann, D. Adv. Drug Del. Rev. 2006, 58, 1655.

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Chapter 2: Historical and Theoretical Background

2.1 Introduction

The majority of therapeutic drugs today have a short half-life in the body and are distributed evenly in the tissue. As a result, a low concentration of the drug reaches the target site and many side-effects are experienced. The ability to conjugate polymers to polypeptide or protein therapeutics has greatly improved the impact of many of those drugs. The polymer can improve the drug’s solubility and biocompatibility, while the drug can impart its pharmacological activity to the conjugate. In order to design a conjugate, the polymeric element has to be carefully designed.

2.2 Controlled radical polymerisation

Living polymerisation was first reported by Szwarc in 1956, while studying the anionic polymerisation of styrene.1 Szwarc discovered that the viscosity of the polymerisation mixture increased until all the monomer was consumed; however, after subsequent addition of extra monomer, a further increase of viscosity was observed. He made the conclusion that living polymerisation is a chain growth process, where termination and chain transfer processes are absent. Living radical polymerisation, often coined controlled radical polymerisation (CRP), is a technique derived from conventional radical polymerisation that ensures that polymer chains grow simultaneously, by supressing irreversible termination reactions. This is achieved by establishing a dynamic equilibrium between a high concentration of dormant chains and a very low concentration of propagating radicals, thus minimising the chance of termination.2 As a consequence, polymer chains grow linearly with respect to monomer conversion and well-defined characteristics, such as targeted molar mass and low dispersity, can be attained. The most well-known classes of CRP techniques used today are reversible addition-fragmentation chain-transfer (RAFT)-mediated polymerisation,3 atom transfer radical polymerisation (ATRP)4 and nitroxide mediated polymerisation (NMP).5 Further, emphasis will be placed on RAFT polymerisation as it plays a critical role in this dissertation.

2.2.1 RAFT polymerisation

RAFT-mediated polymerisation was first reported by the CSIRO group in 1998 and is controlled by a reversible chain transfer mechanism.6 Reversible chain transfer agents (CTAs), known as RAFT agents, facilitate this process and have a thiocarbonyl thio motif,

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6 such as trithiocarbonate or dithioesters.3 The important pre-equilibrium and the main equilibrium steps of the RAFT process are illustrated in Scheme 2.1.

Scheme 2.1 Main equilibrium of the RAFT process.

The polymerisation starts when radicals, generated by a free radical source, react with monomer (M) to yield propagating radicals, which may in turn react with additional monomer to yield longer propagating radicals (Pn●). The propagating radical reacts with the RAFT agent to form the intermediate RAFT adduct, which may reversibly fragment in either direction (pre-equilibrium). An additional propagating radical (Pn●) is generated when the released radical (R●) reacts with monomer. All chains have equal probability to grow, as a result of a rapid equilibrium between the dormant chain and propagating radical (main equilibrium). Termination occurs when two radicals react, yielding irreversibly dead chains.

2.2.2 Macromolecular design via the exchange of xanthate (MADIX) polymerisation

During the same time that the CSIRO group reported RAFT-mediated polymerisation, the Zard group reported MADIX polymerisation.7 MADIX works on the same principle as RAFT but employs xanthates as RAFT agents. Less activated monomers such as vinyl acetate and N-vinyl monomers generate highly reactive radicals, due to the electron-donating effect of their pendant groups and the lack of resonance, attributed to their non-conjugated structure. As a consequence, they form very poor leaving groups, resulting in highly reactive propagating radicals that are hard to control. Xanthates have been found to control the polymerisation of non-conjugated N-vinyl monomers particularly well, due to the effect of their

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electron-7 donating O-ethyl Z-group that destabilises the intermediate radical, increasing the rate of fragmentation and decreasing the rate of addition.8

2.2.2.1 Rationale for use of polyvinylpyrrolidone (PVP)

Polyethyleneglycol (PEG) is the universal standard for therapeutic polymer drug conjugates because, among other things, it is non-toxic, water-soluble and FDA-approved.9 PEGylation, a term coined for PEG-drug conjugates, offers increased blood circulation half-life as a result of a reduced renal clearance, if the molar mass of the total construct exceeds 50 kDa.10 PVP offers the same benefits as PEG and in addition, it was shown to have a longer plasma half-life when compared to PEG conjugates and has a very low tissue distribution.11 This makes PVP a very attractive alternative to PEG.

PVP is an attractive water-soluble and biocompatible polymer that is synthesised from N-vinylpyrrolidone (NVP) and has been used in numerous applications such as in the pharmaceutical and cosmetic industries. PVP is also well known for its use as a plasma expander in trauma patients in the 1950s.12 High molar mass PVP can be synthesised by conventional radical polymerisation. Well-defined PVP, on the other hand, can be synthesised by CRP techniques such as ATRP13 and MADIX. MADIX polymerisation of NVP was first reported by Okamoto and co-workers in 2005.14 Since then, many researchers have focussed their efforts on synthesising well-defined PVP for the design of polymer therapeutics.

2.3 Chain-end functionality

RAFT/MADIX polymerisation offers advantages over other CRP techniques as it allows the facile introduction of chain-end functionality by manipulating the RAFT agent. RAFT agents contain two variable substituents, the R- and the Z-group, each performing a different role. The R-group must be a good leaving group, i.e. be a sufficiently stabilised radical in the pre-equilibrium stage, but also be able to reinitiate the growth of a new chain. The Z-group affects the stability of the intermediate radical, leading to reversible-fragmentation, and the reactivity of the S=C bond, affecting the addition to the propagating radical (Pn●).15 Post-polymerisation, the R-group becomes the α-chain-end functionality and the Z-group, the ω-chain-end functionality. Careful consideration of both of these groups is thus necessary when designing chain-end functional polymers.

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2.3.1 Z-group manipulation

Post-polymerisation modification of the labile thiocarbonyl thio moiety, present in all RAFT agents, has been extensively studied.16 End-group removal has been carried out in a number of strategies including:

 thermolysis, achieving an unsaturated chain-end.

 radical-induced reduction, yielding a sulphur-free hydrocarbon end-group.

 reduction in the presence of nucleophiles, attaining thiol chain-end functionality.16 Aminolysis or reduction of the thiocarbonyl thio moiety achieves a thiol-terminal chain-end, which is a highly reactive species capable of reacting with a number of functionalities (Figure 2.1).

Figure 2.1 Functionalities achieved through thiocarbonyl thio moiety manipulation.

Thiol moieties are difficult to handle as they tend towards disulphide formation and as a result, one-pot reductions and subsequent couplings are generally preferred. Le Neindre and Nicolay reported a one-pot deprotection of a dithiobenzoate terminal copolymer, via aminolysis, and subsequent functionalisation, among others, with thiol-epoxy ring opening, thiol-isocyanate reaction, thiol-disulphide exchange and thiol-acrylate Michael additions.17 In

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9 particular, the Michael addition has been widely studied as a means to introduce functionality into RAFT-synthesised polymers.

The Michael addition was discovered by Arthur Michael when he speculated and proved that a sodium malonate ester or a sodium acetoacetate ester could react via an addition mechanism to an α,β-unsaturated acid ester.18,19 Subsequently, a Michael addition has been defined as the nucleophilic addition between a nucleophile, often a carbanion, and an α,β-unsaturated carbonyl compound.20 The thiol-Michael additions, a subset of Michael additions, was first reported by Allen et al. in 1964 and since then it has been widely used in the manipulation of RAFT synthesised polymers.21,22 Today, the thiol-Michael addition is considered a “click” reaction, as it is highly efficient with no side reactions.

The lability of the thiocarbonyl thio moeity, under reducing conditions, combined with the high reactivity of the thiol moiety, makes the thiol-Michael addition an attractive strategy for chain-end manipulation. Lowe and co-workers took advantage of this by synthesising three-armed star polymers, using thiol-ene chemistry. In this work, poly(n-butylacrylate) and poly(N,N-diethylacrylamide) were synthesised using RAFT-mediated polymerisation and were reacted with a triacrylate, through a one-pot reduction of the thiocarbonyl thio end-group and subsequent phosphine-catalysed thiol-acrylate Michael addition, yielding 3-armed polymer stars.23

2.3.2 R-group manipulation

As previously described, the R-group has to serve a certain purpose by stabilising the radical, in the pre-equilibrium state, and reinitiate chain growth. However, these conditions only need to be met adjacent to the thiocarbonyl thio moeity and therefore an array of functionalities can be introduced further away. Stenzel and co-workers developed two RAFT agents for the polymerisation of vinyl acetate and styrene, respectively, that contained azide and protected alkyne R-groups (Figure 2.2). Post-polymerisation, the two homopolymers were coupled using copper-catalysed alkyne azide 1,3-dipolar cycloaddition (CuAAC) to achieve well-defined poly(styrene-b-vinyl acetate).24 However, using similar RAFT agents, a whole array of compounds could be conjugated to such polymers, as long as they contained an alkyne or azide R-group, respectively.

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Figure 2.2 Azide- and alkyne-terminal RAFT agents for the polymerisation of vinyl acetate and styrene, respectively.

In a similar strategy, Akeroyd et al. designed RAFT agents, where the R-group was generated through the CuAAC coupling of an alkyne and azide, achieving a triazole-based R-leaving group.25 Using this methodology, a wide array of functionalities could be introduced to the RAFT agent, before the polymerisation process.

2.3.3 Orthogonal end-group manipulation

In more complex polymer systems, it is necessary to manipulate both the α- and the ω-chain-end functionality, in order to conjugate multiple components to the construct. Often, the chain-end functionalities are protected, such as the thiocarbonyl thio moiety, which under reducing conditions, reveals a thiol. However, in order to effectively employ this strategy, deprotection chemistries have to be chosen carefully in order that they do not affect each other. This is more commonly known as the orthogonal deprotection of a heterotelechelic system.

As an example of this strategy, Maynard and co-workers synthesised well-defined poly(N-isopropylacrylamide) (pNIPAAm) using a RAFT agent comprising an α-biotin disulphide and a ω-trithiocarbonate.26 Post-polymerisation, the trithiocarbonate was converted into a maleimide, via a radical coupling reaction, affording the α-biotin disulphide, ω-maleimide heterotelechelic pNIPAAm. The polymer system was then conjugated to a free cysteine, available on bovine serum albumin (BSA), via the maleimide and to neutravidin-coated 96-well plates via the biotin. The conjugate was cleaved from the 96-96-well plates via reduction of the disulphide, revealing a reversibly immobilised system.

In another example, Roth et al. synthesised α-pentafluorophenyl, ω-dithiobenzoate poly(methyl methacrylate) via RAFT mediated polymerisation.27 A fluorescent dye, Texas Red, was conjugated to the α-chain-end through amide formation and the ω-dithiobenzoate

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11 chain-end functionality was cleaved to form thiols, through aminolysis, and subsequently reacted with methyl methanethiosulfonate to form ω-methyl disulphide (SS-CH3) end-groups, in a one-pot reaction. The SS-CH3 were tethered to metals and semiconductor nano-particles through liquid exchange.

In both of the described examples, careful thought was applied to designing the orthogonal post-polymerisation deprotection and coupling strategies. It should be noted that in drug-delivery systems, it is necessary to apply these techniques to develop novel polymer-drug conjugates.

2.4 Polymer-protein/polypeptide conjugates

In 1975, Helmut Ringsdorf envisioned a strategy for a drug delivery system, stating that it should have a biocompatible, water-soluble macromolecular backbone, a therapeutic drug, a spacer separating the drug from the backbone and a targeting ligand, to selectively find the target site (Figure 2.3).28

Figure 2.3 Ringsdorf’s designed drug delivery system.

There are two common methods of delivering therapeutic agents: encapsulation of the drug within a three-dimensional amphiphilic polymeric construct or conjugation of the drug to the polymeric system, as envisioned by Ringsdorf. In 1989, Kabanov et al. introduced the concept of “micelles as microcontainers” in which a hydrophobic drug was contained within the hydrophobic core of amphiphilic block co-polymer micelle.29,30 This fast became the preferred strategy for drug delivery.31 However, this strategy has several disadvantages such as instability of the constructs over time, the necessity that the drug be hydrophobic and the release of the therapeutic drug occurs over a short period of time.32 Ringsdorf’s idea of

Spacer

Therapeutic drug

Targeting ligand Polymer

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12 conjugation of the therapeutic drug to a polymer has been shown to overcome some of these shortfalls.

During Ringsdorf’s career, CRP had not yet been developed and thus implementing his design was not possible. However, with the advent of CRP and the ability to orthogonally control end-group functionality, the speculated drug delivery system became conceivable. Many drug delivery systems have since used parts of Ringsdorf’s design. PEGylation is a classic example, where hydrophilic PEG chains are attached to low molar mass therapeutics, in order to increase their circulation time in the body, thereby maximising their therapeutic index.33

More recently, proteins and polypeptides have immerged as therapeutic agents, which are typically larger than 1000 Da and delivered intravenously.34 In addition, most of these proteins and polypeptides are sensitive to the physiological environment and thus conjugation with a suitable polymer offers stability, inherent to the polymer.35 PEGylation has been widely studied, probably because it was possible to synthesise well-defined PEG chains, via anionic polymerisation, prior to researchers fully grasping CRP techniques. PEGylation was first reported by Abushowski et al. in 1977 when PEG chains were attached to bovine liver catalase and BSA, respectively, improving the immunological properties compared to the native protein.36,37

2.4.1 Alternative hydrophilic polymers for drug-polymer conjugation

As this dissertation focusses on PVP-polypeptide conjugates, PEGylation alternatives will be discussed. Keeping in line with Ringsdorf’s model, the macromolecular backbone needs to be hydrophilic in nature. Over the past decade, various hydrophilic polymeric backbones have been studied and a few of the major polymers are summarised in Table 2.1.

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13

Table 2.1 PEG alternative hydrophilic polymers.

Name Abbreviation Structure

Poly (N-(2-hydroxypropyl)

methacrylamide38,39 pHPMA

Poly(N-vinylpyrrolidone)40,41 PVP

Poly(2-hydroxyethyl methacrylate39,42 pHEMA

Poly(2-ethyl 2-oxazoline)43,44 POZ

Interestingly, the first polymer-peptide conjugate was reported in 1954 when Jatzkewitz synthesised a PVP-mescalin conjugate which proved to have an extended plasma half-life.45 Furthermore, in 1996, Zalipsky et al. synthesised two hydrophilic polymers, poly(ethyl 2-oxazoline) (POZ) and poly(2-methyl-2-oxazalonine) (PMOZ), respectively, and conjugated them to distearoylphosphatidyl-ethanolamine via their carboxylic acid chain-end functionality.46 In parallel, comparable PEG conjugates were also synthesised. These conjugates associated themselves into liposomes and their tissue distribution and plasma clearance kinetics were studied in mice. It was found that the POZ and PMOZ conjugates had increased plasma half-lifes and low hepatosplenic uptake. In addition, the POZ conjugates were quantitatively comparable to the PEG conjugates, making them a viable alternative.

Various strategies have been designed for the production of polymer-drug conjugates. For example, Maynard and Bontempo introduced a biotinylated ATRP initiator to streptavidin via its four subunits, capable of binding to biotin. Poly(N-isoprppylacrylamide) (pNIPAAm) was

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14 subsequently grown from the protein using ATRP. pNIPAAM has a lower critical solution temperature (LCST) in water at 32 oC and as a result, the conjugate precipitated out of water above that temperature. This confirmed that the streptavidin had been successfully modified with pNIPAAm. The authors reported that this technique as a novel versatile approach to preparing conjugates.47 McDowall et al. reported a similar conjugate design; however, using a grafting-to approach. PVP was synthesised via MADIX, leading to well-defined PVP chains comprising N-succinimidyl ester α-functionalised chain-ends.48 Lysozyme contains seven primary amine attachment anchors and the PVP was conjugated to them, through amide formation, yielding seven-armed stars with a lysozyme core. Both examples offer alternative conjugation strategies to produce polymer-drug conjugates. It should be noted that Gauthier and Klok comprehensively reviewed the different strategies to introduce functionality onto the amino-acids present in proteins and polypeptides, to activate them for conjugation with polymers.49

2.4.2 Site-selective conjugation

Although conjugation of polymers to proteins or polypeptides increases the pharmacological effectiveness of the drug, it can also have an undesirable effect on the pharmacological activity of the drug, by hindering its binding ability to its target.50 As a result, it is beneficial to study the protein or polypeptide’s binding ability and site-selectively introduce polymer chains to a position that does not affect this binding site. Styslinger et al. developed a site-specific glycolysation of haemoglobin (Hb) with various sized carbohydrates. Glycans are far more degradable than PEG, making their use advantageous when conjugates are administered over a long period of time.51

2.4.3 Targeted drug delivery

Many modern drug delivery systems still cling to Ringsdorf’s design; however, only in recent years have active targeting drug conjugate delivery systems been brought to life. These systems fully embrace the use of polymeric orthogonal deprotection strategies. Previously, only passive targeting drug delivery systems existed, for instance, those that relied on an increased circulation time within the body. An example of this methodology involves conjugating a hydrophilic polymer to the drug, increasing the molar mass of the total construct, making it invisible to the renal clearance system of the body.

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15 The majority of targeted drug delivery systems present in literature are targeted encapsulation-based delivery systems. Very few publications can be found that describe a polymer-drug conjugate contains targeting ligands. For example, Vadlapudi et al. increased the bioavailability of acyclovir by conjugating a lipid raft, a plasma membrane-based lipid, to the drug, thereby increasing its lipophilicity.52 In addition, a targeting ligand that can be recognised by a specific receptor in the cell membrane was bound to the other end of the lipid raft. Cellular accumulation studies were performed and it was found that the combination of the lipid moiety and targeting ligand improved cellular uptake significantly. This design can facilitate higher cell membrane permeability of hydrophilic therapeutic agents such as proteins, polypeptides and genes.

In a specific example, Patri et al. synthesised two dendritic drug delivery systems, one in which the drug was conjugated to the dendrimer and another where the drug was complexed within the dendrimer.53 In particular, a targeting moiety of folic acid was attached to the surface of the dendrimers. It was found that the conjugate was stable in both water and phosphate-buffered saline; however, the inclusion complex was only stable in water. It was suggested by the cultures that the conjugate was more suitable for targeted drug delivery than the dendritic inclusion complex.

With the recent increase in the development of α-,ω- heterotelechelic orthogonal deprotection strategies of polymeric systems, there is no doubt that targeted delivery of polymer-drug conjugates will be studied more extensively in years to come.

2.5 Design of drug-release triggering mechanisms

As previously mentioned, attachment of polymer chains to a therapeutic drug may have undesirable effects on the drug’s pharmacological activity. However, if the drug delivery system is able to release the drug, affording its original structure, then the site of conjugation is less important. Stimuli-responsive linkers, that have the ability to cleave under certain physiological stimuli, can facilitate the release of drugs from a conjugate. However, the majority of examples in literature are still associated with encapsulated systems.54 A number of methods used to release drugs from polymers are described in the next section.

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16

2.5.1 pH-responsive linkers

The change in pH is the most commonly used physiological stimulus for cleavage. For example, the pH of blood is 7.4; however the extracellular pH in tumours, is closer to 6.5.55 Polymer-drug conjugates can thus be designed to take advantage of this, releasing the drug under acidic conditions. Table 2.2 summarises common pH-responsive linkers found in drug delivery systems.56

Table 2.2 Acid-labile linkers commonly found in drug delivery systems.

Name Structure Degradation Products

Acetal/ketal

Imine

Hydrazone

Orthoester

In a specific example, Etrych et al. synthesised a pHPMA-doxorubicin conjugate, with a hydrazone acid-labile linker conjugating the drug to the pHPMA and an antibody targeting ligand, for the treatment of cancerous tumours. In this research, it was found that the doxirubicin was released at pH 5, which is relevant upon endosomal uptake in cancer cells.57

2.5.2 Light-responsive triggers

Photochemical drug-release mechanisms can also be incorporated into polymer-drug conjugates by including light-sensitive linkers. Electromagnetic radiation, generally ultra-violet (UV) light, can thus be applied to specific parts of the body to initiate a response, via laser irradiation. Derivatives of ortho-nitrobenzene are commonly used as photo-labile “caging groups” that deactivate a biologically active molecule. Upon UV irradiation, the biological agent is rendered active again.58 In a specific example, Choi et al. synthesised and evaluated a folate receptor-targeted polyamidoamine dendrimer conjugated to doxorubicin,

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17 through an ortho-nitrobenzyl photocleavable group.59 The resulting nanoconjugate targeted cancer cells and released its’ therapeutic agent via a photochemical mechanism.

2.5.3 Temperature-responsive triggers

The use of thermally-responsive molecules for drug-release has been widely reviewed in amphiphilic block copolymer drug encapsulated systems.60,61 In most cases, thermally-responsive polymers, such as pNIPAAm, that have a LCST, are used as the hydrophilic block in the copolymer, and under changes in temperature, micellular structure is lost, resulting in a release of the encapsulated drug. Currently, to the best of our knowledge, no thermoresponsive linker has been introduced into polymer-drug conjugates.

2.6 Conclusion

Polymer-protein/polypeptide conjugates have not yet been fully investigated; however, they offer promising results for the future of drug delivery. Through the development of CRP, strategies for orthogonal chemistry on both chain ends are readily accessible. This makes it possible to conjugate a therapeutic drug through a stimuli-responsive linker, as well as a targeting ligand to such polymer chains. These developments open up the doors for future “smart” drug delivery systems.

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18

2.7 References

(1) Szwarc, M. Nature 1956, 178, 1168.

(2) Braunecker, W. A.; Matyjaszewski, K. Prog. Polym. Sci. 2007, 32, 93. (3) Moad, G.; Rizzardo, E.; Thang, S. H. Polymer 2008, 49, 1079.

(4) Matyjaszewski, K. Macromolecules 2012, 45, 4015. (5) Grubbs, R. B. Polym. Rev. 2011, 51, 104.

(6) Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P. T.; Mayadunne, R. T. A.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S. H. Macromolecules 1998, 31, 5559.

(7) Biadatti, T.; Charmot, D.; Corpart, P.; Michelet, D.; Zard, S.; Google Patents: 1998. (8) Nakabayashi, K.; Mori, H. Eur. Polym. J. 2013, 49, 2808.

(9) Veronese, F. M.; Pasut, G. Drug Discov. Today 2005, 10, 1451. (10) Yamaoka, T.; Tabata, Y.; Ikada, Y. J. Pharm. Sci. 1994, 83, 601.

(11) Kaneda, Y.; Tsutsumi, Y.; Yoshioka, Y.; Kamada, H.; Yamamoto, Y.; Kodaira, H.; Tsunoda, S.-i.; Okamoto, T.; Mukai, Y.; Shibata, H.; Nakagawa, S.; Mayumi, T. Biomaterials 2004, 25, 3259.

(12) Ravin, H. A.; Seligman, A. M.; Fine, J. New Engl. J. Med. 1952, 247, 921. (13) Lu, X.; Gong, S.; Meng, L.; Li, C.; Yang, S.; Zhang, L. Polymer 2007, 48, 2835. (14) Wan, D.; Satoh, K.; Kamigaito, M.; Okamoto, Y. Macromolecules 2005, 38, 10397. (15) Keddie, D. J.; Moad, G.; Rizzardo, E.; Thang, S. H. Macromolecules 2012, 45, 5321. (16) Willcock, H.; O'Reilly, R. K. Polym. Chem. 2010, 1, 149.

(17) Le Neindre, M.; Nicolaÿ, R. Polym. Int. 2014, 63, 887. (18) Michael, A. J. Prakt. Chem. 1887, 35, 349.

(37)

19 (20) Little, R. D.; Masjedizadeh, M. R.; Wallquist, O.; Mcloughlin, J. I. Org. React. 2004,

47, 315.

(21) Allen, C. F. H.; Fournier, J. O.; Humphlett, W. J. Can. J. Chem. 1964, 42, 2616. (22) Nair, D. P.; Podgórski, M.; Chatani, S.; Gong, T.; Xi, W.; Fenoli, C. R.; Bowman, C.

N. Chem. Mater. 2013, 26, 724.

(23) Chan, J. W.; Yu, B.; Hoyle, C. E.; Lowe, A. B. Polymer 2009, 50, 3158.

(24) Quemener, D.; Davis, T. P.; Barner-Kowollik, C.; Stenzel, M. H. Chem. Commun. 2006, 5051.

(25) Akeroyd, N.; Pfukwa, R.; Klumperman, B. Macromolecules 2009, 42, 3014.

(26) Heredia, K. L.; Tao, L.; Grover, G. N.; Maynard, H. D. Polym. Chem. 2010, 1, 168. (27) Roth, P. J.; Kim, K.-S.; Bae, S. H.; Sohn, B.-H.; Theato, P.; Zentel, R. Macromol.

Rapid Commun. 2009, 30, 1274.

(28) Ringsdorf, H. J. Polym. Sci. Sym. 1975, 51, 135.

(29) Kabanov, A. V.; Chekhonin, V. P.; Alakhov, V. Y.; Batrakova, E. V.; Lebedev, A. S.; Melik-Nubarov, N. S.; Arzhakov, S. A.; Levashov, A. V.; Morozov, G. V.; Severin, E. S.; Kabanov, V. A. FEBS Lett. 1989, 258, 343.

(30) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. J. Controlled Release 2002, 82, 189.

(31) Riess, G. Prog. Polym. Sci. 2003, 28, 1107. (32) Torchilin, V. P. Pharm. Res. 2007, 24, 1.

(33) Pasut, G.; Veronese, F. M. J. Controlled Release 2012, 161, 461.

(34) Craik, D. J.; Fairlie, D. P.; Liras, S.; Price, D. Chem. Biol. Drug. Des. 2013, 81, 136. (35) Edwards, C. M. B.; Cohen, M. A.; Bloom, S. R. QJM-Int. J. Med. 1999, 92, 1.

(36) Abuchowski, A.; McCoy, J. R.; Palczuk, N. C.; van Es, T.; Davis, F. F. J. Biol. Chem. 1977, 252, 3582.

(38)

20 (37) Abuchowski, A.; van Es, T.; Palczuk, N. C.; Davis, F. F. J. Biol. Chem. 1977, 252,

3578.

(38) Krakovičová, H.; Etrych, T.; Ulbrich, K. Eur. J. Pharm. Sci. 2009, 37, 405.

(39) Tao, L.; Liu, J.; Xu, J.; Davis, T. P. Organic & Biomolecular Chemistry 2009, 7, 3481. (40) Pound, G.; McKenzie, J. M.; Lange, R. F. M.; Klumperman, B. Chem. Commun.

2008, 3193.

(41) Trellenkamp, T.; Ritter, H. Macromolecules 2010, 43, 5538.

(42) Bontempo, D.; Heredia, K. L.; Fish, B. A.; Maynard, H. D. J. Am. Chem. Soc. 2004, 126, 15372.

(43) Tong, J.; Luxenhofer, R.; Yi, X.; Jordan, R.; Kabanov, A. V. Mol. Pharm. 2010, 7, 984.

(44) Mero, A.; Pasut, G.; Via, L. D.; Fijten, M. W. M.; Schubert, U. S.; Hoogenboom, R.; Veronese, F. M. J. Controlled Release 2008, 125, 87.

(45) Jatzkewitz, H. Hoppe Seylers Z. Physiol. Chem. 1954, 297, 149.

(46) Zalipsky, S.; Hansen, C. B.; Oaks, J. M.; Allen, T. M. J. Pharm. Sci. 1996, 85, 133. (47) Bontempo, D.; Maynard, H. D. J. Am. Chem. Soc. 2005, 127, 6508.

(48) McDowall, L.; Chen, G.; Stenzel, M. H. Macromol. Rapid Commun. 2008, 29, 1666. (49) Gauthier, M. A.; Klok, H.-A. Chem. Commun. 2008, 2591.

(50) Harris, J. M.; Chess, R. B. Nat. Rev. Drug Discov. 2003, 2, 214.

(51) Styslinger, T. J.; Zhang, N.; Bhatt, V. S.; Pettit, N.; Palmer, A. F.; Wang, P. G. J. Am. Chem. Soc. 2012, 134, 7507.

(52) Vadlapudi, A. D.; Vadlapatla, R. K.; Kwatra, D.; Earla, R.; Samanta, S. K.; Pal, D.; Mitra, A. K. Int. J. Pharm. 2012, 434, 315.

(53) Patri, A. K.; Kukowska-Latallo, J. F.; Baker Jr, J. R. Adv. Drug Del. Rev. 2005, 57, 2203.

(39)

21 (55) Vaupel, P.; Kallinowski, F.; Okunieff, P. Cancer Res. 1989, 49, 6449.

(56) Binauld, S.; Stenzel, M. H. Chem. Commun. 2013, 49, 2082.

(57) Etrych, T.; Jelı́nková, M.; Řı́hová, B.; Ulbrich, K. J. Controlled Release 2001, 73, 89. (58) Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006, 45, 4900.

(59) Ki Choi, S.; Thomas, T.; Li, M.-H.; Kotlyar, A.; Desai, A.; Baker, J. J. R. Chem. Commun. 2010, 46, 2632.

(60) Nakayama, M.; Okano, T. J. Drug Deliv. Sci. Technol. 2006, 16, 35.

(61) Ma, X.; Zhou, Z.; Jin, E.; Sun, Q.; Zhang, B.; Tang, J.; Shen, Y. Macromolecules 2012, 46, 37.

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22

Chapter 3: Synthesis and Characterisation of Polyvinylpyrrolidone

3.1 Introduction

Extensive research over the past few decades has shown the great versatility of the CRP process.1 CRP offers control over molar mass and dispersity, as well as the ability to introduce chain-end functionality. There are various different approaches to CRP, one of them being RAFT-mediated polymerisation, which is well known for the synthesis of well-defined polymers.2 In order to achieve this, careful thought has to be given to the choice of RAFT agent and the reaction conditions.

As macromolecular systems increase in complexity, there is a greater need to control chain-end functionality in an orthogonal fashion. In RAFT polymerisation, this can be achieved by synthesising a RAFT agent that contains two protected functionalities, that can be deprotected independently of each other, post-polymerisation.3 For example, Grover et al. synthesised trithiocarbonate-based RAFT agents comprising bis-functional protected-aminoxy and pyridyl disulphide R-groups, respectively. This allowed direct access to aminooxy- and pyridyl disulphide-inherent homotelechelic poly[poly(ethylene glycol) acrylate] polymers in one step, via RAFT-mediated polymerisation. These polymers were easily conjugated to peptides and small molecules without additional post-polymerisation modification.4

It is well known that xanthate-based RAFT agents control the polymerisation of NVP (Scheme 3.1). This was first reported by Kamigaito and co-workers in 2005, when they reported the effective use of benzyl (CH2Ph) and 1-ethylphenyl (CH3CHPh) R-groups in xanthate-mediated polymerisation.5

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23 Since then, numerous studies have shown the success of the xanthate-moiety in combination with a vast array of R-functionalities. This area has recently been reviewed by Nakahayashi and Mori.6

3.1.1 Choice of RAFT agent

With the growth in the design of intricate nano-systems, it is imperative to be able to introduce orthogonal chain-end functionality. As previously mentioned, NVP can be polymerised with xanthate-based RAFT agents. The xanthate moiety allows the introduction of an inherent protected ω-chain-end functionality, which can be manipulated into a variety of moieties, post-polymerisation, such as thiols.7 Roth et al. highlighted the immense array of thiol-reactive species that can be introduced through these reactive chain-ends, such as disulphide formation with cysteine, present in many proteins and polypeptides.8 In addition, PVP is a biocompatible and non-toxic polymer and thus an attractive choice in the design of a protected orthogonal system.

In another example, Akeroyd et al. showed that 1,2,3-triazole-based leaving groups offered good control over molar mass and dispersity for vinyl acetate, styrene, n-butyl acrylate and NVP. This methodology allows the introduction of a vast range of α-chain-end functionalities through copper click chemistry (Scheme 3.2).9

Scheme 3.2 Triazole-based RAFT agent.

It is well known that aldehydes can be conjugated to amines, present in the N-terminus of peptide sequences, through imine formation. Aldehydes can react with a two equivalents of an alcohol or an equimolar amount of a 1,2-/1,3-diol, acid-catalysed, to form linear acetals and

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24 cyclic acetals, respectively. Both linear and cyclic acetals are widely recognised as acid-labile protecting groups for aldehydes in organic synthesis.

3.2 Synthesis and characterisation of RAFT systems

3.2.1 Cyclic acetal RAFT systems

The synthesis of RAFT agent 3 is shown in Scheme 3.3. First, propargyl bromide was reacted with potassium ethyl xanthate, through nucleophilic substitution, to yield compound 1. In parallel, 2-(bromomethyl)-1,3-dioxolane was converted into its azide 2. Products 1 and 2 were coupled via CuAAC to attain the desired RAFT agent 3.

Scheme 3.3 Synthesis of RAFT agent 3; (a) THF, r.t.; (b) NaN3, DMSO, 100 o

C; (c) CuSO4.5H2O, sodium

ascorbate, DMF, r.t.

RAFT agent 3 contained two protected functionalities, a cyclic acetal and a xanthate, that would inherently be incorporated into the polymer chain. PVP was subsequently synthesised using RAFT agent 3, targeting two degrees of polymerisation (DP), 100 and 200, respectively. The RAFT agent/thermal initiator (AIBN) ratio was kept constant at 5:1 and the results are summarised in Table 3.1. It was not possible to determine the molar mass via 1H nuclear magnetic resonance (NMR) spectroscopy, as the chain-end retention was not quantitative.

Table 3.1 Polymerisation conditions and results for PVP polymerised using RAFT agent 3.

DP αa (%) Reaction Time (hr) Reaction Temp (oC) Mn, theo, (g/mol) Mn, SECb (g/mol) Ð 100 60 5 60 6950 6360 1.91 200 62 5 60 14000 12760 1.73 a_conversion b _M

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25 Generally, for a controlled radical polymerisation, a dispersity lower than 1.5 is expected. In both polymerisations, dispersities higher than 1.5 were obtained from size exclusion chromatography (SEC). Many other reaction conditions were attempted but under no circumstance was it possible to improve the control of the polymerisations. Akeroyd et al. attributed the stabilisation of the intermediate radical to the triazole moiety and compared it to the phenyl group in the well-known benzyl leaving group. However, in every case described, R1 was a phenyl moiety (Scheme 3.2).9 RAFT agent 3 contains a methylene group adjacent to the triazole-ring, which cannot offer the same stabilisation that phenyl could, through resonance. This is a speculative reason for the loss of control and high dispersities.

In order to overcome the suspected instability of the radical during polymerisation, a second methyl substituent was introduced onto the α-carbon of RAFT agent 3, in an attempt to attain a more stable secondary radical. But-3-yn-2-ol was thus reacted with tosyl chloride to yield its activated alcohol, which was immediately reacted further with potassium ethyl xanthate, through nucleophilic substitution, to afford product 4. Compounds 2 and 4 were then coupled through CuAAC to attain RAFT agent 5 (Scheme 3.4).

Scheme 3.4 Synthesis of RAFT agent 5; (a) Tosyl chloride, KOH, diethyl ether, 0 oC to r.t.; (b) THF, r.t.; (c) CuSO4.5H2O, sodium ascorbate, DMF, r.t.

RAFT agent 5 was employed to control the polymerisation of NVP at 60 oC, using AIBN as a thermal initiator. Various ratios of RAFT agent to AIBN were investigated to determine the optimal ratio. In all three experiments, the target DP was 200 and Table 3.2 summarises these results.

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26

Table 3.2 Polymerisation conditions and results for PVP polymerised using RAFT agent 5.

DP α a (%) RAFT/ AIBN Reaction Time (hr) Reaction Temp (oC) Mn, theo, (g/mol) Mn, SECb (g/mol) Mn,NMRc (g/mol) Ð 200 26 5:1 15 60 6100 4080 6000 1.20 200 24 4:1 15 60 5710 3440 5550 1.17 200 25 3:1 15 60 6040 3910 5850 1.19 a_conversion b _M

n, SEC based on PMMA standards

c _M

n, NMR determined by integrating the xanthate signal versus the polymer backbone signal

The difference in molar mass between SEC and 1H NMR spectroscopy can be attributed to the difference in hydrodynamic volume of poly(methyl methacrylate) (PMMA) compared to that of PVP. In all three cases, 1H NMR spectroscopy showed that the end-groups were retained and the low dispersities correlated with a controlled polymerisation. The RAFT/AIBN ratio of 4:1 showed the lowest dispersity and through repeated experiments, shone as the optimal ratio.

In order to optimise the deprotection conditions of the acetal, model compound 6 was synthesised (Scheme 3.5). For this, phenyl acetylene was coupled with 2 through CuAAC to attain model cyclic acetal 6.

Scheme 3.5 Synthesis of model cyclic acetal 6; (a) CuSO4.5H2O, sodium ascorbate, DMF, r.t.

It is well known that cyclic acetals are acid labile and can be converted into aldehydes under fairly mild conditions. Model 6 was thus treated with 1 M HCl in acetone to form the aldehyde. The acetone acts as a donor to accept the released ethylene glycol, from which the acetal was originally fashioned. The reaction was tracked using thin layer chromatography (TLC) and after 24 hours, no sign of reaction was observed. 1H NMR spectroscopy confirmed that no reaction had taken place. This reaction was then repeated under refluxing conditions,

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27 but 1H NMR spectroscopy revealed structure degradation. Alternative deprotection methodologies were subsequently investigated.

In this regard, Sun et al. reported an extremely fast and efficient method for the deprotection of acetals and ketals, catalysed by 10 mol % iodine in acetone.10 Compound 6 was thus treated under these conditions and again, TLC only showed starting material. In an alternative procedure, Dodd and co-worker Oehlschlager described refluxing the cyclic acetal in an acetone/water (85/15) mixture, containing a large excess of tosyl chloride.11 Again, 6 was subjected to these conditions and after the reaction was worked up, 1H NMR spectroscopy revealed only the starting compound.

After a consultation with an organic chemist, Dr. Gareth Arnott (Stellenbosch University), it was suggested that the acetal, in close proximity to the triazole-ring, could be the source of the problem (Scheme 3.6). Two scenarios are possible and both are acid-catalysed. In order for the acetal to be deprotected, one of the oxygen molecules within the acetal-ring, needs to donate an electron pair to the acid. This protonates the oxygen and initiates the ring-opening deprotection sequence (A). Alternatively, the sp2 basic nitrogen, within the triazole-ring, donates an electron pair to the acid. This is the more likely scenario, as it is highly unlikely that both protonations will occur, as there is a high thermodynamic penalty for molecules that are doubly charged. The choice of employing a compound with a cyclic acetal in close proximity to an sp2 nitrogen, therefore happened to be an unfortunate selection.

Scheme 3.6 Acetal deprotection.

3.2.2 Linear-acetal RAFT systems

Initially, the cyclic acetal was chosen because it was inexpensive to order and was shown to be easily deprotected according to literature. Two strategies were thus envisioned to make the

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28 acetal deprotection more labile: a) extend the distance between the acetal and the nitrogen source (triazole-ring) or b) make use of a linear acetal. After a thorough search, a compound meeting both these requirements was found and in addition, was inexpensive and available, namely 1-amino-2,2-diethoxypropane. The first challenge was to convert a primary amine to an azide. In this regard, Goddard-Borger and Stick reported the synthesis of a stable diazotransfer reagent to convert primary amines to azides, namely imidazole-1-sulfonyl azide hydrochloride.12

Initially, a model of the linear acetal was synthesised to make sure that it had a higher degree of lability to the previous model compound 6 (Scheme 3.7). Diazotransfer reagent 7 was thus synthesised according to literature.12 1-Amino-2,2,-diethoxypropane was converted to its azide 8, using reagent 7. Finally, phenyl acetylene was coupled to 8 through CuAAC to achieve linear acetal 9.

Scheme 3.7 Synthesis of model acetal 9; (a) Tosyl chloride (TsCl), KOH, 0 oC, imidazole, HCl; (b) CuSO4.5H2O, sodium ascorbate, DMF, r.t.

Linear acetal 9 was treated with 1M HCl in acetone and the reaction was tracked via TLC. After 1 hour, a new spot had appeared and after 4 hours the reaction was worked up by way of solvent extraction and multiple washing steps. 1H NMR spectroscopy confirmed the deprotection has been successful (Figure 3.1). A new peak (j) appeared at 9.80 ppm, which is a result of the aldehyde formation. Protons g, h and i and were also lost, confirming the deprotection of the linear acetal.