Cell-penetrating poly(disulfide)s: focus on sidechain engineering

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Thesis

Reference

Cell-penetrating poly(disulfide)s: focus on sidechain engineering

MORELLI, Paola

Abstract

The cell-penetrating poly(disulfide)s (CPDs) developed by the Matile group in 2013 have shown excellent cellular uptake properties. Indeed, their mechanism of cell entry relies on the interaction with exofacial anions and thiols, due to the presence in their sidechain of guanidinium groups and in their backbone of disulfides, respectively. Therefore, their uptake is counterion- and thiol-mediated and the combination of the two has enabled the delivery of model fluorophores as well as proteins, antibodies and quantum dots. In order to study the effect of modifications in the CPD structure, new monomers need to be sythesized and then polymerized, through disulfide-exchange, with conditions that need to be optimized each time for even the smaller changes in the monomer structure. This, therefore, called for the development of a strategy that would allow to install different functionalities starting form the same batch of polymer. In the first approach, hydrazone exchange was envisioned as a strategy to perform CPD sidechain functionalization. Due to the low yield of functionalization that was obtained, we decided to adopt a [...]

MORELLI, Paola. Cell-penetrating poly(disulfide)s: focus on sidechain engineering . Thèse de doctorat : Univ. Genève, 2018, no. Sc. 5195

DOI : 10.13097/archive-ouverte/unige:104164 URN : urn:nbn:ch:unige-1041646

Available at:

http://archive-ouverte.unige.ch/unige:104164

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Section de chimie et biochimie

Département de chimie organique Professeur Stefan Matile

Cell-Penetrating Poly(disulfide)s:

Focus on Sidechain Engineering

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention chimie

par Paola MORELLI

de Firenze (IT) Thèse N° 5195

GENEVE

Atelier Repromail – Uni Mail 2018

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Acknowledgements

I would first like to thank Prof. Stefan Matile for allowing me to carry out the PhD work in his group. These four years have enabled me to grow scientifically and as a person as well. A special thanks also goes to Dr.

Naomi Sakai for all her precious advise.

I am also very thankful to Prof. Carsten Schmuck and Dr. Fabien Cougnon for having accepted to judge this work. My gratitude also goes to Dr. Santiago Lascano, Dr. Eline Bartolami, Dr. Javier Lopez and Alessandro Morelli for the corrections they provided.

I am very grateful to all the members of the Matile group, in particular the loosests, for the wonderful atmosphere I have always encountered in the lab. I was very lucky to be part of the “Kindergarten Lab” and I especially thank Queen, Jaga, Yoyo and Fwanny for all the happy memories. A big thanks also to all the colleagues from the whole department that have come to Bout to share necessary spritzs, riojas, beers and vieux pirates.

I would also like to thank my friends for their support during these years abroad, in particular my “Gorditas” Costanza, Elisa, Laura, Susi, Chia and Bene, as well as Giuseppe, Lisa, Giovanni, Pau and Gianluca. A special thanks goes to Antonella, Michela, Javi, Eline, Quentin and Yoann for always being there, in the most difficult moments.

A warm thank you also goes to Adriana, Josefito, Coqui, Clemente and Julien for accepting me into their family making Geneva feel more like home.

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decision I made. To my grandparents, Claudio, Adriana, Alfiero and Rina, because the thought of trying to make them proud was always with me.

Finally, I would like to thank Santi for believing in me, being by my side and pushing me to do better. He has always supported me, giving me so much strength and love. I could not be more grateful to have had the occasion to meet him and get to know him during this PhD.

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List of Publications

1. Chuard, N.; Fujisawa, K.; Morelli, P.; Saarbach, J.; Winssinger, N.;

Metrangolo, P.; Resnati, G.; Sakai, N.; Matile, S. “Activation of Cell- Penetrating Peptides with Ionpair-π Interactions and Fluorophiles”, J. Am.

Chem. Soc. 2016, 138, 11264-11271.

2. Morelli, P.; Martin-Benlloch, X.; Tessier, R.; Waser, J.; Sakai, N.; Matile, S.

“Ethynyl Benziodoxolones: Functional Terminators for Cell-Penetrating Poly(disulfide)s”, Polym. Chem. 2016, 7, 3465-3470.

3. Morelli, P.; Matile, S. “Sidechain Engineering in Cell-Penetrating Poly(disulfide)s”, Helv. Chim. Acta 2017, 100, e1600370.

4. Morelli, P.; Bartolami, E.; Sakai, N.; Matile, S. “Glycosylated Cell- Penetrating Poly(disulfide)s: Multifunctional Cellular Uptake at High Solubility”, Helv. Chim. Acta 2018, 101, e1700266.

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Summary

The cell-penetrating poly(disulfide)s (CPDs) developed by the Matile group in 2013 have shown excellent cellular uptake properties. Indeed, their mechanism of cell entry relies on the interaction with exofacial anions and thiols, due to the presence in their sidechain of guanidinium groups and in their backbone of disulfides, respectively. Therefore, their uptake is counterion- and thiol-mediated and the combination of the two has enabled the delivery of model fluorophores as well as proteins, antibodies and quantum dots.

In order to study the effect of modifications in the CPD structure, new monomers need to be sythesized and then polymerized, through disulfide- exchange, with conditions that need to be optimized each time for even the smaller changes in the monomer structure. This, therefore, called for the development of a strategy that would allow to install different functionalities starting form the same batch of polymer.

In the first approach, hydrazone exchange was envisioned as a strategy to perform CPD sidechain functionalization. The hydrazone polymer S1 was synthesized and exchange with the fluorescent aldehyde S2 was performed to give the modified CPD S3, as shown in Figure S1. Due to the low yield of functionalization that was obtained, we decided to adopt a more robust strategy, that is copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Indeed, by preparing the azide polymer S4, CuAAC could be performed with alkynes S5 to obtain modified CPDs S6 with excellent yields, reproducibility and versatility.

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Figure S1. Sidechain engineering in CPDs using a) hydrazone exchange and b) CuAAC.

Sidechain functionalization through CuAAC allowed to introduce a wide variety of alkynes, as shown in Figure S2. In particular, by introducing ring tension alkynes S7-S8 and cumulative charges S9-S11, the effects on the CPD cellular uptake of thiol- and counterion-mediated mechanisms, respectively, could be studied. Moreover, saccharides S12-S14 were also introduced on the sidechain to achieve multifunctional uptake through carbohydrate-mediated cell entry.

S2

S S

S n S S

m

S1 S3

O

+ NH

N

S S

S n S S

m

+ NH

N

O +

S S

S n

+ N3

S S

S n

+ NN N

S4

S5

S6 b)

a)

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Figure S2. Sidechain functionalization of the CPDs to study a) thiol-, b) counterion- and c) carbohydrate-mediated uptake.

Glycosylation was successful to improve the cellular uptake by integrating a new mechanism to the mode of action of the CPDs. It also enabled the efficient delivery of streptavidin by performing glycosylation at the sidechain of biotinylated polymers.

The compatibility of CuAAC with the poly(disulfide) backbone of the polymers was also exploited for terminator engineering. At first, new reagents were screened to improve the yield of termination in the polymerization of the CPDs. Then, two strategies were developed for the conjugation of the polymers

S S

S n

+ N3

S4

O S S

NH

S7

S8 S S

O NH

O HO

O O

O O

HO OH

HO OH

HO OH

S14 S13

O HO HO

HO O OH O

HO HO

OH

O HO

S12

NH2 NH O NH

H2N NH2

S9 : m = 1 S10 : m = 2 S11 : m = 3

m

a) Thiol-mediated Uptake b) Counterion-mediated Uptake

c) Carbohydrate-mediated Uptake

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As shown in Figure S3, disulfide exchange and thio-alkylation were evaluated by designing CPD S16-S17, respectively. The reaction with S15 was followed by UV-Vis and FRET measurements revealing the quantitative formation of the doubly-labelled polymers S18-S19.

Figure S3. CPD functionalization at the terminator level by disulfide exchange and thio-alkylation.

SS N S16 :

I O O

SH

S15 S S

S n

+

=

S17 : =

S S

S n

+

SS S18 : =

S19 : = S

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Résumé

Les poly(disulfure)s à pénétration cellulaire (ou CPDs) développés par le groupe Matile en 2013 ont montré d’excellentes propriétés de captation cellulaire. En effet, leur mécanisme d’entrée cellulaire dépend d’intéractions avec des anions et des thiols exofaciaux, dûs à la présence de groupes guanidinium et de disulfures, respectivement sur leurs chaînes latérales et sur la chaîne polymérique principale respectivement. La combinaison de ces deux types d’intéractions a permis l’administration cellulaire de fluorophores modèles ainsi que de protéines, anticorps et points quantiques.

Afin d’étudier l’effet de modifications dans la structure des CPDs, de nouveaux monomères doivent être synthétisés puis polymérisés par échange de disulfures, dans des conditions devant être optimisées à chaque fois et ceci même pour de petits changements dans la structure du monomère. Cet état de fait démontrait le besoin de développer une stratégie que permettrait d’installer différentes fonctionnalités à partir d’un même lot de polymère.

Dans la première approche, l’échange d’hydrazones fut envisagé en tant que stratégie pour obtenir une fonctionnalisation des chaînes latérales des CPDs. Le polymère S1, contenant des hydrazones, fut synthétisé et soumis à l’échange d’hydrazone avec l’aldéhyde fluorescent S2 pour obtenir le polymère modifié S3, comme montré dans la Figure S1. Le bas rendement d’échange obtenu nous poussa cependant à adopter une stratégie plus robuste, c’est-à-dire la cycloaddition azoture-alcyne catalysée au cuivre (I) (CuAAC). En effet, après préparation du polymère S4 contenant des azotures, sa réaction avec l’alcyne S5 conduit à l’obtention du polymère modifié S6 avec d’excellents rendements, en restant reproductible et versatile.

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Figure S1. Optimisation exploratoire des chaînes latérales de CPDs via a) échange d’hydrazones et b) CuAAC.

La fonctionnnalisation des chaînes latérales via CuAAC a permit l’introduction d’une large gammes d’alcynes, tel que montré dans la Figure S2.

En particulier, l’introduction d’alcynes à tension de cycle S7-S8 et à charges supplémentaires S9-S11, facilita l’étude de l’effet des méchanismes d’administration thiol- et contre-ion-dépendant sur la pénétration cellulaire des CPDs. Qui plus est, les saccharides S12-S14 furent aussi introduits dans les chaînes latérales afin d’obtenir une captation cellulaire multifunctionelle en ajoutant aux méchanismes précédents celui carbohydrate-dépendent.

S2

S S

S n S S

m

S1 S3

O

+ NH

N

S S

S n S S

m

+ NH

N

O +

S S

S n

+ N3

S S

S n

+ NN N

S4

S5

S6 b)

a)

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Figure S2. Fonctionnalisation des chaînes latérales de CPDs pour l’étude de la captation cellulaire a) thiol-, b) contre-ion et c) carbohydrate-dépendante.

La glycosylation eut comme effet d’améliorer la captation cellulaire en intégrant un nouveau méchanisme au mode d’action des CPDs. Elle a permit aussi l’administration efficace de streptavidine lorsqu’elle fut effectuée sur les chaînes latérales de polyméres biotinylés.

La compatibilité de la CuAAC avec la chaîne polymérique centrale à base de poly(disulfure)s fut aussi exploitée pour l’optimisation exploratoire du terminateur. Dans un premier temps, de nouveaux réactifs furent évalués afin d’améliorer le rendement de termination lors de la polymérisation des CPDs.

S S

S n

+ N3

S4

O S S

NH

S7

S8 S S

O NH

O HO

O O

O O

HO OH

HO OH

HO OH

S14 S13

O HO HO

HO O OH O

HO HO

OH

O HO

S12

NH2 NH O NH

H2N NH2

S9 : m = 1 S10 : m = 2 S11 : m = 3

m

a) Thiol-mediated Uptake b) Counterion-mediated Uptake

c) Carbohydrate-mediated Uptake

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niveau du terminateur avec les thiols de fluorophores modéles S15 contenant des cystéines. Comme montré dans la Figure S3, l’échange de disulfures et la thio- alkylation furent évaluées en concevant les CPDs S16-S17, respectivement. La réaction avec S15 fut suivie par spectroscopie UV-Vis et par des mesures de FRET montrant ainsi la formation quantitative des polymères doublement marqués S18-S19.

Figure S3. Fonctionnalisation des CPDs au niveau de terminateur par échange de disulfures et par thio-alkylation.

SS N S16 :

I O O

SH

S15 S S

S n

+

=

S17 : =

S S

S n

+

SS S18 : =

S19 : = S

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Table of Contents

CHAPTER 1 1

INTRODUCTION 1

1.1. Cell-Penetrating Poly(disulfide)s 1

1.1.1. Counterion-Mediated Uptake 3

1.1.2. Thiol-Mediated Uptake 8

1.2. Polymer Sidechain Modification 21

1.2.1. Non-Covalent and Dynamic Covalent Strategies 21

1.2.2. Covalent Strategies 26

1.3. Cellular Uptake Involving Carbohydrates 34 1.3.1. General Considerations on Carbohydrates 34

1.3.2. Glycodelivery Systems 39

1.4. Protein-Polymer Conjugation 46

1.4.1. Bioorthogonal Ligation 47

1.4.2. Focus on Thiol Reactivity 54

CHAPTER 2 59

OBJECTIVES 59

CHAPTER 3 61

RESULTS AND DISCUSSION 61

3.1. Sidechain Engineering in Cell-Penetrating Poly(disulfide)s 61 3.1.1. Sidechain Engineering with Hydrazone Exchange 62 3.1.2. Sidechain Engineering with Azide/Alkyne

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3.1.3. Sidechain Screening 92

3.1.3.1. Introducing Ring Tension 93

3.1.3.2. Introducing Extra Charges 105

3.1.3.3. Introducing Solubilizing Groups 124 3.2. Glycosylated Cell-Penetrating Poly(disulfide)s 133

3.2.1. Sidechain Modification 133

3.2.2. Mechanistic Investigations 139

3.2.3. Protein Delivery with Glyco-CPDs 153 3.3. Terminator Engineering in Cell-Penetrating Poly(disulfide)s 164 3.3.1. Azide/Alkyne Reactive Terminators 164

3.3.2. Thiol Reactive Terminators 177

CHAPTER 4 190

PERSPECTIVES 190

CHAPTER 5 195

EXPERIMENTAL SECTION 195

5.1. General 195

5.1.1. Reagents, Solvents and Equipment 195

5.1.2. Equipment for Characterization 195

5.1.3. Equipment Used for Experiments 197 5.2. Sidechain Engineering with Hydrazone Exchange 196

5.2.1. Synthesis 198

5.2.2. Polymerization 203

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5.2.3. Hydrazone Exchange 205 5.3. Sidechain Engineering with Azide/Alkyne

Cycloaddition 206

5.3.1. Synthesis 206

5.3.2. Calibration Standards 222

5.3.3. Polymerization 225

5.3.4. Sidechain Engineering 226

5.3.5. Cellular Uptake 229

5.3.5.1. Confocal Laser Scanning Microscopy 229 5.3.5.2. Cellular Uptake in the Presence of

Ellman’s Reagent 231

5.3.5.3. Colocalization Experiments 232 5.4. Glycosylated Cell-Penetrating Poly(disulfide)s 234

5.4.1. Synthesis 234

5.4.2. Calibration Standards 239

5.4.3. Polymerization 239

5.4.4. Sidechain Engineering 240

5.4.5. Conjugation with Streptavidin 241

5.4.6. Cellular Uptake 242

5.4.6.1. Confocal Laser Scanning Microscopy 242 5.4.6.2. Cellular Uptake in the Presence of

Inhibitors 243

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5.4.6.4. Flow Cytometry 244

5.4.6.5. Mechanistic Investigations 245

5.4.6.6. Kinetics 245

5.4.6.7. Toxicity Assay 246

5.5. Terminator Engineering in Cell-Penetrating

Poly(disulfide)s 247

5.5.1. Azide/Alkyne Reactive Terminators 247

5.5.1.1. Synthesis 247

5.5.1.2. Polymerization 248

5.5.1.3. CuAAC Reaction 249

5.5.1.4. FRET Measurement and

Depolymerization 249

5.5.1.5. Cellular Uptake 250

5.5.2. Thiol Reactive Terminators 250

5.5.2.1. Synthesis 250

5.5.2.2. Polymerization 251

5.5.2.3. CuAAC Reaction 251

5.5.2.4. Disulfide Exchange 252

5.5.2.5. Thiol Alkylation 252

5.6. Abbreviations 254

CHAPTER 6 257

REFERENCES 257

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Chapter 1

INTRODUCTION

1.1. Cell-Penetrating Poly(disulfide)s

Efficient delivery of biologically relevant cargos, whether drugs or probes, large or small molecules, represents one of the key challenges in life sciences. In order to address this issue, efforts are continuously made towards the development of new delivery systems. The goal of a delivery system is to enable the cargo it is transporting to overcome the barriers that inhibit its translocation to then release it to the site of action. Among the characteristics that are under current investigation for the more sophisticated delivery technologies,[1] we can find the controlled release of the cargo over time using polymeric matrixes or hydrogels,[2] as well as specificity towards one cell or organ type over another (typically cancerous versus healthy)[3] and finally the self-degradation once the system has achieved its purpose in order to minimize toxicity.[4] Liposomes,[5]

nanoparticles,[6] micelles,[7] carbon nanotubes[8] or polymers[9] are among the delivery systems that can be found in the literature with a mechanism of uptake that mainly relies on endocytosis.[10]

Endocytosis is a form of transport that usually concerns large molecules (from nm to µm scale) which involves an active participation of the cell membrane itself to achieve internalization.[11] The term ‘endocytosis’ is very general and actually encompasses a wide variety of mechanisms in which different proteins are involved in the transport process, e.g. clathrin or caveolin, and according to the different cargo size, pino or phagocytosis (Figure 1).[12]

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late endosomes characterized by an acidic pH of approximately 5.5. The last compartments of the endocytic pathway are lysosomes which are responsible for the digestion of cellular waste products and therefore need to be avoided or escaped when the target of the cargo is located in the cytosol or the nucleus.[10]

While endocytosis is an active form of transport, and therefore requires energy, its counterpart is energy-independent and is called passive diffusion.

Physiologically, it concerns small molecules with the right hydrophobicity which enables them to pass through the cell membrane driven by a concentration gradient. Among the few delivery systems which make use of this type of direct transport such as small dendrimers or metal clusters,[11-12] cell-penetrating peptides have over the years received the most attention.[13]

Figure 1. Mechanisms of entry of common delivery systems. Image from reference.[12]

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1.1.1. Counterion-Mediated Uptake

Cell-penetrating peptides (CPPs) are a class of polycationic transporters which have the ability to bypass the cell membrane predominantly through direct translocation (Figure 2). They were first introduced in 2000 by Rothbard and coworkers[14] taking inspiration from the fact that certain protein subunits, which were highly basic, could enter cells in an energy-independent manner.

Indeed, in 1988 it was shown that the Tat protein of the HIV-1 virus, unlike other proteins, was able to readily pass through the membrane of different cell lines.[15-16] Ten years later,[17] it was shown that the α-helical region of the protein, bearing both hydrophobic and hydrophilic residues, was not necessary for the cell entry, but that, counter-intuitively, the cation-rich sequence (Tat49-57: RKKRRQRRR) was responsible for the direct translocation and it was proven that endocytosis was not the main mechanism involved. At this point, Tat started to be used to achieve cellular entry of various cargos, as depicted in Figure 2, such as the delivery of a 120 kDa β-galactosidase protein across the blood brain barrier.[18] It was, finally, in 2000 that the guanidinium groups present in the arginine-rich sequence of the protein were determined to be responsible for its diffusion.[14] Since then, the optimal structural requirements were studied in detail such as the presence of spacers, the stereochemistry and the number of the arginine residues required for cell entry or the introduction of other cationic amino acids (histidines and lysines).[14]

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Figure 2. The evolution of cell-penetrating peptides. Figure adapted from reference.[12]

As for the mechanism of entry of these arginine-rich transporters, it is hypothesized that it involves ion-pairing with anionic species. In particular, Wender and coworkers proposed a model which relies on the ability of the guanidinium cations to form strong bidentate hydrogen bonds with the anionic phosphates, carboxylates and sulfates that are located on the cell surface.[19] This bifurcated motif would explain the difference with less active ammonium cations coming from lysine groups which on the other hand can only react with anions through random electrostatic interactions. On the other hand, Matile and coworkers have also proposed that the strong ion-pairing with arginine residues stems from the very low acidity of the guanidinium group.[20] Indeed, the strong binding of anions to arginine residues compared to lysines originates from a proximity effect, as shown in Figure 3.[21] In nature, the proximity of ammonium groups of lysine residues (pKa ≈ 10.5) causes charge repulsion that can be eliminated by the deprotonation of the vicinal cation (with pKa ≈ 7). In the case of proximal arginines, due to the weak acidity of the guanidinium group (pKa ≈ 12.5) the charge repulsion cannot be eliminated by deprotonation and therefore ion-pairing with anions always occurs. In the case of the arginine-rich transporters, the ion-pairing between the guanidinium groups to the exofacial anions binds them to the cell surface increasing their hydrophobicity and enabling direct translocation. It is noteworthy to mention that depending on the size of the transporter, endocytosis is in competition or dominates with direct

Tat49-57 (RKKRRQRRR)

: Cargo

Tat Rn

NH NH2 H2N

NH NH2 H2N NH

H2N H2N

NH NH2 NH2

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translocation and many CPP-like systems can be found to enter either using both pathways or only one of the two.

Figure 3. Charge repulsion elimination through ion-pairing for arginine residues and deprotonation for lysine residues. Image from reference.[21]

Once the ion-pairing occurs, direct translocation through the membrane enables the cell entry. However, the mechanism of this type of transport is still under debate. Many models have been proposed to explain how the entry occurs,[22] as shown in Figure 4. Among the various theories we can find the formation of transient micellar pores (carpet model),[23] the loosening of the cell membrane packing,[24] pore formation (barrel-stave model)[25] or an influence of the different protonation and deprotonation state of fatty acids.[26] Effort is also oriented towards activating these cationic transporters rendering them more efficient through the use of more hydrophobic counter-anions such as pyrenebutyrate[27] or ion-pair-π interactions and fluorophiles[28] to gain

N N NH

H H H

H

N N NH

H H

H H

pKa ≈ 12.5 +2

O OH

N N NH

H H H

H

N N NH

H H

H H

+1

O O

pKa ≈ 10.5 +2

NH3

NH3

- H+

pKa < 7.0 +1

NH3

NH2

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Figure 4. Models postulating how CPPs may perturb membranes and enter into the cell. Image from reference.[22]

As mentioned earlier, oligoarginines 1 were first used as CPPs, however further investigations led also to the replacement of the peptidic backbone with a great variety of scaffolds, depicted in Figure 5, such as PNAs,[29] glycosides introduced by Tor et al.,[30] polymers such as oligophosphoesters 2[31] and oligocarbonates 3[32] developed by Wender et al. or polyprolines 4.[33] Variations of the positive charge have also been studied, replacing the guanidinium group with ammoniums 5,[34] phosphoniums 6,[35] or sulfoniums 7 introduced by Deming et al.[36] leading to less toxicity in most cases, but also less proficient ion-pairing with the exofacial anions. On the other hand, Schmuck and coworkers have shown that guanidinocarbonylpyrroles 8 can lead to better transfection efficiencies compared to the guanidinium analogues thanks to the better anion binding motif.[37]

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Figure 5. Original CPP oligoarginine 1 and CPP mimics 2-8.

Even though many CPPs have been developed throughout the years leading to the efficient delivery of a wide range of cargos not only in cultured cells but also in animal models,[38] the two major drawbacks that can be encountered are endosomal entrapment and cytotoxicity. The introduction of pH-responsive residues like histidines with a weakly basic imidazole ring, such as in 9 (Figure 6),[39] can help to escape the endosomes due to the slightly acidic pH found in these vesicles (so-called proton-sponge effect). Indeed, the presence of the guanidinium groups enables 9 to enter the cell through a counterion-mediated

HN O

NH O HN H2N NH2

n

NH NH2 H2N

1

P O OO

O

NH NH2 H2N O H

5 n

2

NH O

O HN

H2N NH2

NH2

n

3

N O O N

O

n

4 O

NH NH2 H2N

HN O

H3N

n

5

O O

O

n

6 O

P

O H N

SR

7

n

n

NH O

NH NH O NH2

H2N O

8

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escape. On the other hand, a common strategy to address CPP cytotoxicity is to enable their degradation as in the self-immolative oligo-α-aminoesters 10.[40]

Indeed, Waymouth et al. recently reported the dynamic CPP 10 which can undergo a rearrangement once it enters the cell, leading to the formation of the neutral piperazine 11. Moreover, it was shown that it allows the progressive reduction of the charge aiding to escape possible endosomes, releasing the cargo it carries and thus eliminating toxicity. Another common strategy involves the introduction of cleavable linkers such as disulfides like the one contained in transporter 9, which can be reduced in the cytosol of the cell by the high concentration of glutathione (GSH).

Figure 6. pH-responsive CPP 9 and biodegradable CPP mimic 10.

1.1.2. Thiol-Mediated Uptake

While counterion-mediated uptake relies on the formation of ion-pairs with exofacial anions, thiol-mediated uptake exploits the interaction with exofacial thiols. As previously mentioned, disulfides can be introduced in transporters to achieve the release of the cargo through glutathione-mediated reduction.

However, it was also found that the introduction of disulfides could improve the cellular uptake. Most of the examples in literature coupling the presence of

10 O

O H2

N O n O

O H2

N O n-2+ N N HO

O

O

OH 11

N N

H O

SS HN

O N

HN NH2

NH2 N

N

NH O

SS HN

O

9

n m

10

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disulfides or thiols to efficient cellular uptake were overlooked,[41] until Sagan and coworkers proposed in 2009 that cell-surface thiols mediated the entry of a disulfide-engineered cell-impermeable peptide, a protein kinase C inhibitor (PKCi). Figure 7 shows how cell entry can be achieved only when a disulfide bridge is used to conjugate two molecules of PKCi. Moreover, a decrease in uptake was observed when the exofacial thiols were oxidized using DTNB (5,5- dithio-bis-(2-nitrobenzoic acid)), the so-called Ellman’s reagent.[42]

Figure 7. Cellular uptake of PKCi achieved without (left) and with (right) the presence of a disulfide bond. Image from reference.[42]

Moreover, in 2012, Gait and coworkers showed that the activity of a PNA containing a cysteine residue was enhanced compared to an analogue in which the thiol of the cysteine was alkylated (Figure 8).[43] These findings strongly suggested that the thiol was responsible for the increase in activity and led Gait to hypothesize that disulfide exchange between thiolated transporters and exofacial thiols could occur, leading to the concept of thiol-mediated uptake.[41]

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Figure 8. Cellular uptake of a PNA sequence modified without (red) and with a free (blue) or an alkylated cysteine (green). Image from reference.[43]

Disulfide bridges have also been introduced in polymeric materials such as the capsules developed by Caruso and coworkers in 2011.[44] As shown in Figure 9, the capsules could be delivered into HeLa cells only when cross-linked with disulfides and not when thioethers or amides were used.

Figure 9. Structure and cellular uptake of polymeric capsules cross-linked with a) disulfides, b) thioethers and c) amides. Figure from reference.[44]

a) b) c)

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Polymers containing disulfides, that is poly(disulfide)s, have been most commonly used as delivery systems to introduce bioreducible bonds to enable the release of the cargo they are carrying.[45] In particular, in the case of nucleic acid delivery, cationic poly(disulfide)s are used to form polyplexes that protect the nucleic acids, mediate their transport through mainly endocytosis and release them into the cytoplasm or the nucleus due to the high concentration of GSH that reduces the disulfide bonds, as shown in Figure 10.[46]

Figure 10. Nucleic acid delivery using cationic poly(disulfide)s. Image from reference.[46]

Poly(disulfide)s can be synthesized using Michael polyaddition of disulfide containing monomers, like commercially available bisacrylamide 12, to primary or secondary aliphatic amines to give poly(amido amine) 13 (Figure 11).[47]

Another strategy involves the use of disulfide containing crosslinkers, such as imido ester 14, on polyamines to give permanently charged amidinate salts 15.[48]

Controlled radical polymerizations, such as RAFT (reversible addition- fragmentation chain transfer) are also employed to obtain polymers with well- defined end groups containing thiols, such as 16, which can then undergo a post-

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synthesis of cationic polypeptides, like 17, and then the thiols oxidized using mild oxidizing agents, such as dimethylsulfoxide.[50]

Figure 11. Common methods for the synthesis of cationic poly(disulfide)s: a) Michael polyaddition, b) disulfide crosslinking, c) RAFT polymerization and d) polypeptide synthesis. Figure adapted from reference.[46]

Matile and coworkers introduced in 2013 a different type of strategy to obtain cationic poly(disulfide)s that uses disulfide exchange. The procedure takes inspiration from the so-called self-organizing surface-initiated polymerization (SOSIP) developed in the group in 2011. As shown in Figure 12, the disulfide-containing initiator 18, which is bound to an indium tin oxide (ITO) electrode, is activated to the corresponding thiolate 19 under basic conditions.

Polymerization occurs by using derivatives of asparagusic acid 20, a strained cyclic disulfide, through disulfide exchange, generating new thiolates that propagate the reaction to build a polymeric architecture.[51]

12 NH

O

SS HN

O

H2N R

13 NH O

SS HN

O n

a)

b)

14

O SS O

NH2

NH2 H2N R

15 HN SS N

NH2 H

NH2 Cl

Cl

Cl

Cl

c)

O O

N

O O

N SH HS

n m

16

d)

17

HS N

H O NH2

HN SH O COOH

NH R

R R

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Figure 12. Self-organizing surface-initiated polymerization. Figure adapted from reference.[51]

The concept of substrate-initiated disulfide exchange polymerization was then applied to obtain cationic poly(disulfide)s as novel CPP mimics, named cell- penetrating poly(disulfide)s (CPDs). As shown in Figure 13, a thiolated initiator is used to attack the disulfide-containing monomer. The monomer contains a strained cyclic disulfide and, when the new disulfide bond with the initiator is formed, the ring tension is released and a thiolate generated which will in turn attack the disulfide of a second monomer unit and, therefore, propagate the polymerization. To terminate the exchange, a thiol-alkylation reaction is performed using a iodoacetamide derivative.[52]

N N

O O O

O

NHN N

NNH N N

N

NH O

NH O

O O

O O

O

O O

O

HN

NH N

H HN

O O

O O

P P

SR O

O SR

P P O OO O

OO P P O OO

O O O

O O O

OO O

P P O

O O O OO N

N

O O O

O N

H O

S S O NHN

HN O N HN O SS

18: R = StBu 19: R =

N N

O O O

O N

H O

S S O NHN

HN O N HN O SS

20

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Figure 13. Substrate-initiated disulfide exchange polymerization to obtain cell- penetrating poly(disulfide)s.

The CPP-mimic characteristic of the CPDs originates from the presence of a guanidinium group in the monomer leading, after polymerization, to a cation- rich poly(disulfide). The most commonly used initiators are thiolated fluorophores, such as 21-22, green-emitting and red-emitting respectively, in order to track the cellular uptake of the resulting CPDs. While commercially available iodoacetamide 23 is used as the terminator of the polymerization.

Among the different monomers that were tested over the years, the ones that gave CPDs with the highest activity are lipoic acid derivatives 24-25, as shown in Figure 14.

Initiator S S S S S

NH NH2 H2N

NH NH2 H2N

S S NH NH2 H2N

I N

H

O Terminator

polymerization

S S

S n

O NH NH

NH2 H2N

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Figure 14. Building blocks used to synthesize cell-penetrating poly(disulfide)s:

initiators 21-22, terminator 23 and monomers 24-25.

Interestingly, it was found that small structural modifications of the lipoic acid sidechain, such as the introduction of aromatic groups, led to a loss of cellular uptake efficiency.[53] Moreover, in order to study the effect of a wide variety of functionalities, such as branching, π-acidic surfaces or binding to glycosaminoglycans using boronic acids, co-polymerization was developed, as shown in Figure 15.[54]

O

HO O

COO

NH O O

O SH I O NH2

22 O

N N

COO

NH O O

O SH

21 23

S S NH

O OO HN

H2N

NH2 S S

NH H O N NH2 H2N

24 25

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Figure 15. Substrate-initiated co-polymerization.

As mentioned previously, fluorescent CPDs grown using monomers 24-25 were able to reach the cytosol, nucleus and nucleoli of HeLa cells after only 15 minutes of incubation at 37 °C using 500 nM concentration (Figure 16).

Moreover, compared to CPP reference poly-arginine 1, no toxicity could be observed up to 10 µM concentration suggesting that, similar to other poly(disulfide) scaffolds, the high concentration of GSH enables the depolymerization of the polymer and results in lack of cytotoxicity.[53]

Mechanistic investigations revealed insensitivity towards endocytosis inhibitors, such as chlorpromazine (which is a marker for clathrin-mediated), wortmannin (macropinocytosis) and methyl-β-cyclodextrin (caveolae- mediated). Interestingly, sensitivity towards exofacial thiols was observed when thiol-mediated uptake was inhibited using DTNB. Together these findings suggested that, in addition to counterion-mediated uptake due to the presence of the guanidinium groups, thiol-mediated uptake also strongly contributed to the efficient cell entry of the CPDs.[53]

S S S S S I NH2 O

polymerization

S S S S

O NH2 S

n m

n m

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Figure 16. Cellular uptake of best-performing CPDs into HeLa cells: a) confocal laser microscopy (CLSM) images, b) cytotoxicity (poly-arginine ¡, CPD ¨) and c) DTNB inhibition. Figure from reference where CPDs 1, 4 bear propagators 24 and 25, respectively.[53]

Further studies on the cellular uptake of the CPDs revealed dependency of the intracellular localization on the molecular weight: longer CPDs were able to reach the nucleus and nucleoli, while shorter ones either stayed trapped in endosomes or localized preferentially in the cytosol.[55] Indeed, CPDs bind to the cell surface by counterion exchange with exofacial anions like common CPPs, but the shortest ones cannot outcompete endocytosis and therefore remain trapped in endosomes. With increasing length, however, thiol-mediated uptake becomes more important since CPDs, bearing a poly(disulfide) backbone, can also covalently bind exofacial thiols inducing a faster formation of transient micellar pores which enables them to reach the cytosol (Figure 17). Moreover, if the polymers are long enough, they can escape complete depolymerization by GSH, reaching nucleus and nucleoli. [21]

a) b) c)

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Figure 17. Dual mechanism of entry of cell-penetrating poly(disulfide)s:

counterion-mediated (left) and thiol-mediated (right). Image adapted from reference.[21]

An important application of the CPDs is the delivery of proteins through the use of biotin-streptavidin technology. As shown in Figure 18, the biotinylated green-emitting polymer 26 was interfaced with streptavidin 27.[56] Streptavidin is a 53 kDa tetramer of β-barrels which is known for its high affinity with biotin (KD = 10-15 M). In this study, the four sites of the protein were labelled with both red-emitting biotinylated fluorophore 28 and CPD 26 to give complex 29. The complex reached the nucleus and nucleoli of HeLa cells and this general strategy was used for the delivery of larger cargos, such as quantum dots and nanobodies by the Matile group,[57] but also proteins, monobodies and nanoparticles by the Yao group.[58-59]

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Figure 18. General strategy for protein delivery with CPDs with complex 29 obtained using biotinylated CPD 26 and streptavidin 27 labelled with biotinylated fluorophore 28. Figure adapted from reference.[56]

The contribution of thiol-mediated uptake to the efficient delivery of cargos was investigated further by Matile and coworkers also using small molecules. In 2015, they were able to demonstrate that by increasing the ring tension, in this case the C-S-S-C dihedral angle of cyclic disulfides, uptake efficiency in HeLa cells increased accordingly. In a first study, the more strained asparagusic acid 30, depicted in Figure 19, with a dihedral angle of 27° was found to have superior uptake compared to less strained lipoic acid 31 (35°).Inhibition was observed when the exofacial thiols were oxidized or alkylated while uptake was promoted when exofacial disulfides were reduced, suggesting the occurence of disulfide

[60]

HN O

NH O H

N

S S S

O NH2

HN OO O

NH NH2 NH2 O

S NH HN

O

n

HN

O HO

O COO

26

27 28

29

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entry. Moreover, the conjugation of asparagusic acid to a cell-impermeable peptide enables it to be delivered and to exercise its apoptotic function.[61]

Recently, the highest degree of ring tension was achieved with epidithiodiketopiperazine (ETP) 32 (dihedral angle of near 0°) with a 20-fold increase in the uptake efficiency of the model fluorophore to the cytosol and nucleus compared to 30 (Figure 19).[62]

Figure 19. Thiol-mediated uptake with increasing ring tension from lipoic acid 30 to asparagusic acid 31 and ETP 32.

In conclusion, this first chapter shows that there are two mechanisms that can be exploited to enable cellular uptake: counterion-mediated, exploiting ion- pairing with exofacial anions by using positive charges like guanidiniums, and

SS

S S HN

O O NH

S 35°

S 27°

S S S S

S S

S S

SS S S

S

S S N N

HN O

O O

30 31 32

Increased Uptake with Increased Strain

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thiol-mediated, exploiting disulfide exchange with exofacial thiols by using disulfides. When dealing with polymers, such as the CPDs, motifs that can enable cellular uptake through these described mechanisms could be installed through, among others, modification of their sidechain. The next chapter will deal with strategies that can be adopted for polymer sidechain engineering.

1.2. Polymer Sidechain Modification

Polymer sidechain modification is developed to screen the effect of different functionalities on a comparable backbone. Instead of synthesizing the monomers and then optimizing polymerization conditions each time, different functionalization techniques can be adopted to reduce the number of steps needed to prepare a wide variety of structures with even small differences.

Over the years, many strategies have been developed to functionalize polymers at the sidechain level, mainly using non-covalent, dynamic covalent and covalent approaches which will be discussed in the following chapter.

1.2.1. Non-Covalent and Dynamic Covalent Strategies

Taking inspiration from the complex assemblies that can be found in Nature, the use of non-covalent bonds, such as the hydrogen bond, has now been implemented in a wide range of disciplines, such as polymer functionalization.[64]

The use of supramolecular chemistry, that is the use of reversible and directional interactions, to build materials with growing degrees of complexity, can enable to overcome the synthetic difficulties which occur using traditional covalent bonds.[63]

Supramolecular polymers are abundantly found in Nature, with DNA as a striking example, but also in synthetic polymers like nylons which are held together by cooperative hydrogen bonding.[64] This field is growing

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interactions, such as anion-π or chalcogen bonds.[65] However, supramolecular stategies are not only used to synthesize new polymers, but also to modify polymers held together by covalent interactions. Indeed, the polymer backbone can be covalently linked together and contain molecular recognition motifs to achieve functionalization using a non-covalent strategy.[66]

Sidechain functionalization can occur before or after polymerization, be single or multiple and involve the use of one or more non-covalent strategies.

The most common non-covalent force that is used for sidechain engineering is hydrogen bonding which can be weakened or strengthened according to the function of the resulting material. The first example was shown in 1989 by Kato and Fréchet who functionalized liquid crystalline polymer 33 by introducing benzoic acids in the sidechain that formed hydrogen bonds with pyridine 34 (Figure 20).[67] More Nature-inspired recognition motifs have been developed by Rotello and coworkers by introducing triazines in the sidechain of poly(styrene) 35 which could then be post-functionalized through hydrogen bonding with gold nanoparticle 36 possessing thymine residues.[68]

Figure 20. Examples of polymer sidechain functionalization using hydrogen bonding. Adapted from reference.[66]

N O

O O

H O O O

O O

n

33 34

n

m O

N N NHN

H

HNH O O N N

H S

10

SS S

SS

35 36

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Another common non-covalent interaction used for sidechain modification is metal coordination. The use of metal-ligand interactions can enable to screen a wide variety of polymers with catalytic or magnetic properties and the combination with hydrogen-bonding can solve solubility issues, as for the ruthenium aqueous micelles of Schubert and coworkers.[69] However, there are certain disadvantages to using metals for sidechain engineering, such as incompatibility with most polymerization methods or lack of reversibility if not through ligand displacement.[66]

Even though non-covalent interactions, and especially hydrogen bonding, are useful for self-assembly and the building of complex architectures with well- known recognition motifs, an excellent compromise between the reversibility of non-covalent interactions and the robustness of covalent ones is given by dynamic covalent bonds (DCB). Indeed, DCBs are strong, stable and permanent under certain conditions, while rapidly forming, breaking and exchanging in others. The disulfides described in the previous chapter, for example, are labile and can exchange in basic conditions, while being robust in acidic ones. These characteristics have also led to the use of dynamic covalent chemistry in polymer sidechain functionalization.

Montenegro and coworkers reported, indeed, in 2016 the use of DCBs for polymer functionalization.[70] By introducing a hydrazide group in the scaffold, they screened different amphiphilic polymers for siRNA delivery through hydrazone formation. Starting from poly(acryloyl hydrazide) 37 (Figure 21), sidechain engineering was performed using different molar ratios of guanidinium-containing aldehyde 38 and more hydrophobic aldehydes, such as 39. At neutral pH, hydrazides are weakly protonated and can react with aldehydes to form hydrazones, like 40, that are stable at physiological conditions.

Thanks to the presence of the positive charge, 40 was used to form polyplexes with negatively charged siRNA for gene transfection.[70]

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Figure 21. Polymer sidechain functionalization using dynamic covalent bonds:

polymer 40 is obtained by hydrazone formation between hydrazide 37 and aldehydes 38-39. Figure adapted from reference.[70]

Sidechain functionalization with hydrazones is also very attractive since it tolerates the presence of a second DCB, such as the disulfide bond. The orthogonality between the two different bonds enables the construction of fully dynamic covalent systems. In the SOSIP methodology developed by Matile and coworkers for example, hydrazones are introduced on the poly(disulfide)- containing backbone for post-modification in a strategy called templated stack exchange (TSE), shown in Figure 22. Indeed, they exploit the stability of hydrazones in basic conditions where the disulfide polymer is formed, and viceversa, can form new hydrazone bonds to introduce different functionalities in acidic conditions while keeping the poly(disulfide) scaffold intact.

In this example, a benzaldehyde hydrazone is introduced in the monomer which, after disulfide-exchange polymerization to give structure 41, is cleaved with excess hydroxylamine to give the free hydrazide 42. The hydrazides can then react with the desired aldehydes to form hydrazones 43. The SOSIP-TSE methodology was extensively used to introduce different properties to the photosystems starting from the same scaffold each time.[71]

n

37

38

39 O

O

SH NH O NH2

H2N N H NH2

NH O

H O

H O

n

40 HO

O

NH O

SH O NH

(44)

Figure 22. SOSIP-TSE methodology for post-functionalization using hydrazone formation in the presence of a poly(disulfide) backbone. Figure adapted from reference.[71]

There is extensive use of disulfides and hydrazones in the literature to achieve orthogonal systems by simple adjustment of pH. The dynamic nature of these bonds is not only used to construct systems, but also to impart certain

S SS SONN

S H S S

SS

SONN S H SS

ONN H S

SS S

S SONN H

S S

N

N O

O

O O O

HN N

NH O

n-2

NH O

S

S S

S

n

41

N

ITO

S SS SONNH2

S H S SS

S SONNH2 S H SS

ONNH2 H S

SS S

S SONN H

S S

n-2

42

S SS SONN

S H S SS

S SONN S H SS

ONN H S

SS S

S SONN H

S S

n-2

43 O

Figure

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