Nanoparticle As Supramolecular Platform for Delivery and Bioorthogonal Catalysis

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Doctoral Dissertations Dissertations and Theses

November 2017

NANOPARTICLE AS SUPRAMOLECULAR PLATFORM FOR DELIVERY AND BIOORTHOGONAL CATALYSIS

Gulen Yesilbag Tonga University of Massachusetts Amherst

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Recommended Citation Yesilbag Tonga, Gulen, "NANOPARTICLE AS SUPRAMOLECULAR PLATFORM FOR DELIVERY AND BIOORTHOGONAL CATALYSIS" (2017). Doctoral Dissertations. 1141. https://doi.org/10.7275/10521708.0 https://scholarworks.umass.edu/dissertations_2/1141

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NANOPARTICLE AS SUPRAMOLECULAR PLATFORM FOR DELIVERY AND BIOORTHOGONAL CATALYSIS

A Dissertation Presented

GULEN YESILBAG TONGA

Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

September 2017

Department of Chemistry

NANOPARTICLE AS SUPRAMOLECULAR PLATFORM FOR DELIVERY AND BIOORTHOGONAL CATALYSIS

A Dissertation Presented

GULEN YESILBAG TONGA

Approved as to style and content by:

______Vincent M. Rotello, Chair

______Richard W. Vachet, Member

______James J. Chambers, Member

______Barbara A. Osborne, Member

______Richard W. Vachet, Department Head Department of Chemistry

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DEDICATION

To my husband Murat and my son Ali Eray, and to my family.

ACKNOWLEDGEMENTS

First, I would like to thank to my research advisor, Professor Vincent M. Rotello for his support and guidance during my PhD studies. As a mentor and advisor, he contributed a lot to my scientific skills and understanding. I have learned how to approach and solve scientific problems, perform multiple projects, and be multitasking and independent researcher. Under his guidance, I did not only improve my scientific skills but also my social skills such as managing my time as well as managing relationships with my colleagues and collaborators. I always admired him for how well he manages such a big group and endless number of projects. I appreciate the opportunity he gave me to work in his group. When I looked behind, I think I achieved a lot during my PhD and I truly believe this is because I was in Rotello group. I am so proud of being a member of Rotello group and I believe wherever I go I will carry the excellent impacts of Rotello group for my future career and I will never forget Professor Vincent`s precious advices.

I would like to thank my committee, Professor Richard W. Vachet, Professor Barbara A.

Osborne and Dr. James J. Chambers for their valuable comments on my research. I appreciate their time and effort advising and helping me.

I also would like to thank all the past and present research group members including all the visiting students and scholars for sharing their thoughts, cultures, and scientific advice. They were all my friends and colleagues. Especially, I would like to mention some of them here,

Chaekyu, Youngdo, Riddha, Roberto, Mine, Ying, Ngoc Le, Moumita, Rubul, and Ryan.

Lastly, I would like to express my gratitude to my family and friends. I always felt their support and love during my PhD. Also, I would like to thank my husband Murat and our son Ali

Eray for their unconditional support and love. They are the best thing that ever happened to me.

ABSTRACT

NANOPARTICLE AS SUPRAMOLECULAR PLATFORM FOR DELIVERY AND

BIOORTHOGONAL CATALYSIS

SEPTEMBER 2017

GULEN YESILBAG TONGA, B.S., BOGAZICI UNIVERSITY

M.S., BOGAZICI UNIVERSITY

Ph.D., UNIVERSITY OF MASSACHUSETTS AMHERST

Directed by: Professor Vincent M. Rotello

Nanoparticles (NPs) are being investigated widely for many applications including imaging, drug delivery, therapeutics, materials, and catalysis due to their unique and tunable physical and chemical properties. Among NPs, gold nanoparticles (AuNPs) have attracted great attention due to ease of synthesis and surface functionalization, inertness of the core, biocompatibility, and functional versatility. Introducing supramolecular chemistry into the nanoparticle-based platforms brings out controllable properties, dynamic self assembly processes, and adjustable performance. My research has focused on the synthesis of AuNPs bearing different surface functionalities and their host-guest interactions with synthetic small molecules or commercially available hydrophobic catalysts for delivery and therapeutic applications. My research is consisted of two main sections. First part is the regulation of the exocytosis of AuNPs using host-guest interactions and characterization of these interactions in solution using isothermal titration calorimetry and inside cells using laser desorption/ionization/matrix assisted laser desorption/ionization mass spectrometry. Second part is about encapsulating various hydrophobic transition metal catalysts into the monolayer of AuNPs. These catalyst-embedded

AuNPs were used to catalyze industrially important reactions in aqueous environment. For biological applications, this system was called as ‘nanozyme’ because it was used as an enzyme mimic to perform bioorthogonal activation of profluorophores and prodrugs for imaging and therapeutic applications, respectively. Using the host-guest chemistry, intracellular catalysis was supramolecularly regulated. Lastly, different monolayer designs were engineered to increase the catalyst loading and improve catalytic efficiency.

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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... v

ABSTRACT ...... vi

LIST OF TABLES ...... xii

LIST OF FIGURES ...... xiii

LIST OF SCHEMES...... xxiii

CHAPTER

1. GOLD NANOPARTICLES IN DELIVERY AND THERAPEUTICS ...... 1

1.1 Introduction ...... 1 1.2 Application of Gold Nanoparticles in Drug Delivery and Therapeutics ...... 3 1.3 Supramolecular Chemistry ...... 5 1.3.1 Supramolecular Chemistry in Drug Delivery ...... 5 1.3.2 Supramolecular Platforms Using Gold Nanoparticles ...... 7 1.3.3. Characterization and Monitoring of Host-Guest Interactions in Solution and Living Cells ...... 9 1.4 Bioorthogonal Catalysis ...... 11 1.4.1 Bioorthogonal Catalysis Using Transition Metal Catalysts ...... 13 1.4.2 Non-Covalent Incorporation of Hydrophobic Transition Metal Catalysts into AuNP Monolayers ...... 16 1.5. Dissertation Overview...... 17 1.6. References ...... 21 2. REGULATING EXOCYTOSIS OF NANOPARTICLES VIA HOST-GUEST CHEMISTRY ...... 32

2.1 Introduction ...... 32 2.2 Results and Discussion...... 34 2.2.1 Complexation between AuNP-TBen and CB[7] ...... 34 2.2.2 Inducing Assemblies of AuNP-TBen-CB[7] ...... 35 2.2.3 Controlling Exocytosis of AuNP-TBen-CB[7] Inside Cells ...... 38 2.2.4 Tracking Exocytosis of CB[7]-complexed Nanoparticles Inside Cells Using Inductively Coupled Plasma Mass Spectrometry ...... 39 2.2.5 Cellular Viability of the Induced Assemblies ...... 41 2.3 Summary and Future Outlook ...... 41 2.4 Synthesis of Materials and Experimental Methods ...... 42 2.4.1 General ...... 42 2.4.2 Synthesis of Ligands and Their Characterization ...... 42 2.4.3 Synthesis of Benzyl-Ligand Protected Gold Nanoparticle (AuNP-TBenz) ...... 46

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2.4.4 Monitoring CB[7]-Nanoparticle Complexation Using Proton and NOESY 2D NMR ...... 48 2.4.5 ICP-MS Sample Preparation and Measurements ...... 51 2.4.6 Preparation of Cellular TEM Samples and Their Measurements ...... 51 2.4.7 Cell Culture and Cytotoxicity Measurements ...... 52 2.5. References ...... 53 3. BINDING STUDIES OF CUCURBIT[7]URIL WITH GOLD NANOPARTICLES BEARING DIFFERENT SURFACE FUNCTIONALITIES ...... 56

3.1 Introduction ...... 56 3.2 Result and Discussion ...... 57 3.2.1 Isothermal Titration Calorimetry (ITC) Measurements of NP1-NP8 and Thermodynamic Values ...... 58 3.2.2 ITC Measurements of NP9-NP13 and Effects of Surface Functionality on Binding Affinity ...... 61 3.2.3 Monitoring Binding of Free Ligand to the Host Molecule ...... 61 3.3 Summary and Future Outlook ...... 62 3.4 Synthesis of Materials and Experimental Methods ...... 63 3.4.1 Synthesis of Ligand ...... 62 3.5 References ...... 79 4. MASS SPECTROMETRIC DETECTION OF NANOPARTICLE HOST-GUEST INTERACTIONS IN CELLS ...... 84

4.1 Introduction ...... 84 4.2 Results and Discussion...... 86 4.2.1 Detection of Host-Guest Complexes in Aqueous Solution ...... 86 4.2.2 Detection of Host-Guest Complexes inside Cells ...... 88 4.2.3 Dissociation of Host-Guest Complexes Using a Competitive Guest Molecule ...... 90 4.2.4 Multiplexed Detection of Association and Dissociation of Host-Guest Complexes ...... 90 4.3 Summary and Future Outlook ...... 92 4.4 Experimental Section ...... 92 4.4.1 Cell Culture Experiments ...... 92 4.4.2 Treatment of Competitive Guest Molecule, ADA ...... 92 4.4.3 Cell Sample Preparation for MALDI-MS ...... 93 4.4.4 Sample Preparation for ICP-MS and Analysis of the Cellular Uptake ...... 96 4.4.5 Synthesis of Ligands and AuNPs ...... 97 4.4.6 Complexation of AuNP with CB[7] ...... 105 4.5 References ...... 105 5. SOLUBILIZATION OF HYDROPHOBIC CATALYSTS USING NANOPARTICLE HOSTS ...... 111

5.1 Introduction ...... 110 5.2 Results and Discussion...... 112

5.2.1 Encapsulation of Hoveyda-Grubbs 2nd Generation (HG2) Catalyst into AuNP Monolayer ...... 112 5.2.2 Ring Opening Metathesis Polymerization in Aqueous Solution Using AuNP-HG2 ...... 116 5.2.3 Cleavage of Allylcarbamate Bonds Using Ruthenium or Palladium Catalyst Embedded AuNP ...... 118 5.2.4 Hydrogenation of Alkenes Using Wilkinson’s Catalyst Encapsulated AuNP ...... 120 5.3 Summary and Outlook ...... 123 5.4 Experimental Section ...... 123 5.4.1 Materials and Instruments ...... 123 5.4.2 Synthesis of TMA and TTMA Coated AuNPs ...... 123 5.4.3 Encapsulation of the Catalysts ...... 124 5.4.4 Quantification of the Catalysts...... 124 5.4.5 Laser Desorption/Ionization Mass Spectrometry (LDI-MS) Instrumentation: ...... 125 5.4.6 ROMP Reaction in Water by Using AuNP-HG2 ...... 125 5.4.7 Allyl Carbamate Bond Cleavage Reaction by Using AuNP-Cp*Ru(cod)Cl or AuNP-(Pd(dppf)Cl2) ...... 129 5.4.8 Hydrogenation of Sodium 4-vinylbenzenesulfonate via AuNP-Wilkinson Catalyst ...... 129 5.5 References ...... 130 6. ENHANCING CATALYTIC ACTIVITY OF NANOZYME BY MONOLAYER ENGINEERING ...... 136

6.1 Introduction ...... 136 6.2 Results and Discussion...... 138 6.2.1 Design of AuNP Monolayer ...... 138 6.2.2 Encapsulation of Hydrophobic Cp*Ru(cod)Cl Catalyst into AuNP ...... 138 6.2.3 Catalytic Activity of Nanozymes in Solution ...... 140 6.2.4 Catalytic Activity inside Living Cells ...... 141 6.3 Summary and Future Outlook ...... 143 6.4 Experimental Section ...... 143 6.4.1 General ...... 143 6.4.2 Synthesis of Ligands ...... 144 6.4.3 Synthesis of Gold Nanoparticles and Characterization ...... 152 6.5 References ...... 154 7. SUPRAMOLECULAR REGULATION OF BIOORTHOGONAL CATALYSIS IN CELLS USING NANOPARTICLE-EMBEDDED TRANSITION METAL CATALYSTS ...... 156

7.1 Introduction ...... 156 7.2 Results and Discussion...... 158 7.2.1 Design and Synthesis of Nanozymes ...... 158 7.2.2 Catalytic Efficacy of NP_Ru Nanozymes in Solution ...... 160 7.2.3 Supramolecular Control of Catalysis ...... 162 7.2.4 Kinetic Analysis of the NP_Ru Nanozymes Using Lineweaver–Burk Analysis ...... 164 7.2.5 Catalytic Efficacy of NP_Pd Nanozymes in Solution ...... 166

7.2.6 Catalytic Activity inside Living Cells ...... 168 7.2.7 NP_Pd Nanozymes for Prodrug Activation ...... 172 7.3 Summary and Future Outlook ...... 176 7.4. Synthesis of Materials and Experimental Sections ...... 177 7.4.1 Synthesis of the Benzyl Ligand ...... 177 7.4.2 Synthesis of Benzyl-Ligand-Protected Gold Nanoparticle (AuNP) ...... 182 7.4.3 Mass Spectrometric Characterization of Ligand Composition ...... 183 7.4.4 Catalyst Encapsulation into the Monolayer of AuNPs ...... 184 7.4.5 Transmission Electron Microscopy (TEM) Measurement of the Nanoparticle Before and After the Encapsulation of the Catalysts ...... 185 7.4.6 Size and Zeta Potential of the NP ...... 185 7.4.7 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Instrumentation for Ruthenium Catalyst ...... 186 7.5 References ...... 187 BIBLIOGRAPHY ...... 192

LIST OF TABLES

Table Page

3.1: Thermodynamic data of NP1-8 for binding to CB[7]...... 60

3.2: Thermodynamic data of NP9-13 for binding to CB[7]..………………………...…….……...61

4.1: Detail p values for the comparison between different NP-CB[7] complexes...... 94

4.2: The ion intensity ratios of MALDI-MS analysis of AuNP-CB[7] complexes in solutions...... 95

5.1: Quantification of encapsulated catalysts in the 2 nm TTMA-AuNP...... 125

6.1: Size and zeta potential of nanozymes used in the study ...... 153

7.1: Ruthenium amount in the nanozyme using ICP-MS measurement...... 186

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LIST OF FIGURES

Figure Page

1.1: Schematic illustration of monolayer-protected gold nanoparticles. The rigid inorganic core is protected by a soft organic monolayer that can be chemically tailored for various applications...... 2

1.2: Top scheme-synthesis of Pt-DNA-AuNP. Bottom a) Live cell imaging of HeLa cells after incubation with platinum-tethered Cy5-DNA-Au NPs for 6 h and (b) 12 h, and (c) colocalization of the particles with the cytoplasmic microtubules...... 4

1.3: Schematic representation of hierarchical self-assembly of the supramolecularly engineered polymers to yield micelles and drug release after being exposed to three different triggers...... 6

1.4: (A) Six channel-output in a single well. The fluorescence of FPs is quenched when the NP-FP complexes are formed. Upon addition of cell lysates, three emission channels are obtained from the released FPs. In the same well, CB[7] is added to obtain three additional channels from the three FPs as a result of changed interactions between the analyte and newly formed complex, NP-CB[7]. (B) Linear Discriminant Analysis (LDA) with the fluorescence responses from all six channels showed well separation of all cell types...... 8

1.5: (A) Schematic showing the activation of AuNP-NH2-CB[7] cytotoxicity by dethreading of CB[7] from the nanoparticle surface by ADA. (B) Cytotoxicity of AuNP-NH2 and AuNP-NH2-CB[7] measured by Alamar blue assay after 24 h incubation in MCF-7. (C) Triggering cytotoxicity using ADA. After 3h incubation of AuNP-NH2-CB[7] (2 µM) in MCF-7 cell, different concentrations (0, 0.2 and 0.4 mM) of ADA in medium added and further incubated at 37 ºC for 24 h. The cell viability was then determined by using an Alamar blue assay...... 9

1.6: (A) The copper-catalyzed azide–alkyne cycloaddition. (B) The Cu-free click reaction of azides and DIFOs...... 12

1.7: (A) Comparison of Cu-free click chemistry with Cu-catalyzed click chemistry. (B) Schematic showing cell surface labeling using Cu-free click chemistry. (C) Comparison of Cu-free click chemistry with other bioorthogonal ligations using alkyne containing Alexa Fluor 488. (D) Labeling using alkyne containing biotin...... 12

1.8: Bioorthogonal nanocatalysis designed and demonstrated by Meggers (2006, 2012), Bradley and Unciti-Broceta (2014, 2016) and Rotello (2015)...... 13

1.9: Fluorescence microscopy images of uncaging process of allylcarbamate-protected rhodamine 110 (pro-fluorophore) inside HeLa cells using ruthenium nanocatalyst. a) Right after catalyst addition and b) after 15 min. c)–f): Fluorescence generation after indicated times. Cells were incubated with pro-fluorophore and the membrane

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carbocyanine dye DiIC18(5) for 30 min and then treated with [Cp*Ru(cod)Cl] and thiophenol...... 14

1.10: Scheme of the bioorthogonal reactions taking place inside the cell internalized PdNP@PStyr composite. (A) Allylcarbamate deprotection (cleavable unit highlighted in red). (B) Confocal microscope image of cell stained through the generation of highly fluorescent Rhodamine 110 after the allylcarbamate cleavage process. (C) Scheme of the Susuki-Miyaura transformation on the fluorescein triflate ester taking place on the surface of the cell internalized PdNP@PStyr composite. (D) Confocal image of the generated fluorescein derivative staining mitochondria...... 15

1.11: (A) Delivery of payload to cell due to change in hydrophobicity through monolayer- membrane interactions (B) Structure of particles and guest molecules: Bodipy, TAF, and LAP, the number of encapsulated guests per particle, and log P of the guests...... 17

2.1: Controlling exocytosis of AuNPs by using intracellular host-guest complexation. (a) Inhibition of AuNP-TBen exocytosis by the threading of CB[7] onto the nanoparticle surface. (b) Formation of the AuNP-TBen-CB[7] assemblies...... 34

2.2: Inducing nanoparticle assemblies upon binding with CB[7] and the effect of surface functional group on the assembly formation. (a) Size changes of nanoparticle assemblies at different NP:CB[7] ratios. (b) AuNP-ADA and AuNP-TMC6 induce the larger assemblies upon binding with CB[7], but no assembly formation was observed for AuNP-TMOH and AuNP-TMNH2 with polar end groups...... 36

2.3: NOESY 2D NMR shows the interaction of benzyl moiety with TEG and C11 units, indicating head group is bending in towards the monolayer (red circle)...... 37

2.4: CB[7] treatment induced the formation of large assemblies. AuNP-TBen-CB[7] were treated with excess of 1-adamantyl amine (ADA), ADA triggered the particle assemblies to disassemble, making the particles soluble back in PBS...... 38

2.5: Cellular uptake and intracellular behavior of the AuNPs. (a) TEM images of MCF-7 cells incubated with AuNP-TBen. The cationic AuNP-TBen is trapped in organelles (e.g. endosome, red circle). TEM images MCF-7 cells incubated with AuNP-TBen and then washed and further incubated with (b) only cell culture media or (c) culture media with CB[7] for 24 h. (d) Quantification of the amount of gold retained in cells at different time after incubation with free media or media containing CB[7]. Cellular uptake experiments with each gold nanoparticle were repeated 3 times, and each replicate was measured 5 times by ICP-MS. Error bars represent the standard deviations of these measurements...... 39

2.6: Effect of surface functional groups on exocytotic behavior of AuNPs. ICP-MS measurements of (a) AuNP-TTMA, (b) AuNP-TMOH, (c) AuNP-TMC6, and (d) AuNP-ADA. Quantification of exocytosis of the AuNPs was determined by analyzing ICP-MS on MCF-7 cell with same experimental condition carried out for the AuNP-TBen. Error bars represent the standard deviations of these measurements...... 40

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2.7: Cytotoxicity of AuNPs. After 3h incubation of AuNPs (200 nM), MCF-7 cells were washed off and further incubated with media and media of CB[7] (0.2 mM) at 37 ºC for 24 h. As a control, cell viability of CB[7] (0.2 mM) was measured after 24 h incubation...... 41

2.8: Synthesis of benzyl-ligand (compound 6) for functionalizing AuNP-TBen...... 42

2.9: MALDI-MS spectrum of AuNP-TBenz. The molecular ion (MH+) was detected at m/z =498...... 46

2.10: MALDI-MS spectrum of AuNP-TBenz. The molecular ion (MH+, m/z =498) was detected, and the disulfide ion formed by the benzyl ligand and the original pentanethiol was also detected at m/z 600...... 47

2.11: Transmission electron micrograph of AuNP-TBen...... 47

2.12: 1H-NMR of AuNP-TBen showing the ligand attachment on AuNP surface...... 48

1 2.13: H-NMR of AuNP-TMNH2 in D2O...... 49

1 2.14: H-NMR of AuNP-TMNH2-CB[7] in D2O...... 49

2.15: NOESY 2D NMR of AuNP-TMNH2...... 50

2.16: NOESY 2D NMR of AuNP-TMNH2-CB[7] showing the host-guest interactions between CB[7] and AuNP-TMNH2 (black circles)...... 50

3.1: Structures of DMBA and MMBA derivatives used in the study. The schematic represents the binding event between AuNP and CB[7]...... 57

3.2: ITC measurements of NP1, NP3, and NP8 bearing DMBA head group with H, CH2NH2 and NO2 functionalities at the para position of the benzene ring showing similar binding constants...... 58

3.3: a) Schematic representation of binding between DMBA ligand and CB[7]. b) ITC measurement indicated a 1:1 binding stoichiometry between free ligand and CB[7]...... 62

3.4: Synthesis of compound 2 from compound 1...... 63

3.5: Synthesis of compound 3 from compound 2...... 63

3.6: Synthesis of compound 4 from compound 3...... 64

3.7: Synthesis of compound 2 from compound 1...... 64

3.8: Synthesis of compound 3 from compound 4...... 65

3.9: Synthesis of compound 4 from compound 3...... 66

3.10: Synthesis of compound 2 from compound 1...... 66

3.11: Synthesis of compound 4 from compound 3...... 67

3.12: Synthesis of compound 5 from compound 4...... 68

3.13: Synthesis of compound 2 from compound 1...... 68

3.14: Synthesis of compound 3 from compound 2...... 69

3.15: Synthesis of compound 4 from compound 3...... 70

3.16. Synthesis of compound 2 from compound 1...... 70

3.17: Synthesis of compound 3 from compound 2...... 71

3.18: Synthesis of compound 4 from compound 3...... 71

3.19: Synthesis of compound 2 from compound 1...... 72

3.20: Synthesis of compound 3 from compound 2...... 73

3.21: Synthesis of compound 2 from compound 1...... 73

3.22: Synthesis of compound 2 from compound 1...... 74

3.23: Synthesis of compound 2 from compound 1...... 75

3.24: Synthesis of compound 2 from compound 1...... 75

3.25: Synthesis of compound 3 from compound 2...... 76

3.26: Synthesis of compound 2 from compound 1...... 77

3.27: Synthesis of compound 3 from compound 2...... 77

3.28: Synthesis of compound 2 from compound 1...... 78

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4.1: (a) Structures of the surface functionalities on the AuNPs used in this work. (b) The mass-to-charge (m/z) ratios of ligands and their corresponding supramolecular complexes monitored by MALDI-MS. Letter code key: molecular ions of the surface ligands (L ions), disulfide ions (D ions) formed by surface ligands and pentanethiol ligands, supramolecular complex ions (C ions) formed by surface ligand and CB[7], and disulfide ions formed by CB[7] and D ions (DC ions)...... 86

4.2: Monitoring AuNP-CB[7] interaction in solution using MALDI-MS. (a) AuNP 1-CB[7]. (b) AuNP 2-CB[7]. (c) AuNP 3-CB[7]. [AuNP]=2 µM, [CB[7]]=400 µM. See the Figure 2 caption for the identities of the L, D, C, and DC ions...... 88

4.3: Monitoring AuNP-CB[7] interactions using MALDI-MS. (a) Detection of AuNP 1 in cells after incubation with 250 nM AuNP. (b) Detection of AuNP 1-CB[7] ([AuNP]=250 nM, [CB[7]] = 50 µM) taken up by the cells. (c) The dissociation of the host-guest complex by adding ADA (4 µM) to the cells containing AuNP 1-CB[7]...... 89

4.4: Monitoring the dissociation of three AuNP-CB[7] complexes in cells. (a) Typical mass spectrum of cell samples incubated with a mixture of three AuNP-CB[7] complexes. (b) Normalized ion intensity ratios indicating the relative amount of the remaining supramolecular complexes after ADA treatments. n.s., no significant difference. **, 0.001

4.5: Various AuNPs which bind CB[7] that have been successfully characterized by MALDI- MS. The ones in the red box have been utilized for the selective dissociation study...... 93

4.6: Mass spectra of the AuNP-CB[7] complexes. (a) AuNP 4-CB[7], (b) AuNP 5-CB[7], (c) AuNP 6-CB[7], (d) AuNP 7-CB[7]...... 94

4.7: Cellular uptake amount per well of AuNP-CB[7] complexes measured by ICP-MS...... 95

4.8: 400 MHz 1H NMR spectra of compound 1 in chloroform-D (D, 99.8%)...... 97

4.9: 400 MHz 1H NMR spectra of compound 2 in chloroform-D (D, 99.8%)...... 98

4.10: 400 MHz 1H NMR spectra of compound 3 in chloroform-D (D, 99.8%)...... 99

4.11: 400 MHz 1H NMR spectra of compound 4 in chloroform-D (D, 99.8%)...... 100

4.12: 400 MHz 1H NMR spectra of benzyl ligand in chloroform-D (D, 99.8%)...... 101

4.13: 400 MHz 1H NMR spectra of dimethyldiaminohexane ligand in chloroform-D (D, 99.8%)...... 102

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4.14: 400 MHz 1H NMR spectra of benzyl ligand in chloroform-D (D, 99.8%)...... 102

4.15: 400 MHz 1H NMR spectra of diaminohexane ligand in chloroform-D (D, 99.8%)...... 103

4.16: TEM image of pentanethiol (C5) coated AuNPs...... 104

5.1: a) The structure of NP hosts (core 2 and 7 nm) and the structure of TTMA and TMA ligands. b) The encapsulation efficacy of various NP hosts that have different core sizes and monolayer structures for HG2...... 113

5.2: a) Structure of the TTMA functionalized AuNP, b) NMR spectrum of HG2 catalyst encapsulated AuNP, c) Enlarged section of NMR spectrum in the range from 6 to 8 ppm...... 114

5.3: TEM images of HG2 catalyst encapsulated AuNPs...... 115

5.4: Ion intensity ratio of TMA and TTMA coated 2 and 7 nm AuNPs...... 116

5.5: a) The structure of 2nd generation Hoveyda-Grubbs catalyst (HG2). b) The reaction scheme of ROMP using NP host and water soluble monomer. c) The molecular weight and PDI values of polymers in terms of reaction time. d) While the free HG2 in acetone/ water mixture failed to produce the polymers, the polymers formation was detected in the presence of the AuNP-HG2, showing increased molecular weight in terms of reaction time. e) GPC result of NP host-HG2-monomer mixture after 24 h. f) GPC result of free catalyst-monomer mixture after 24 h, no polymerization was observed...... 117

5.6: The structure of a) Cp*Ru(cod)Cl and b) Pd(dppf)Cl2 catalyst. c) The reaction scheme of ruthenium-induced (or palladium-induced) allylcarbarmate cleavage using NP hosts and non-fluorescent Rhodamine 110 derivative. d) Catalytic activity of NP host- Cp*Ru(cod)Cl and only NP host. e) Photographs of the NP host reaction with and without ruthenium catalyst under UV-irradiation. f) Catalytic activity of NP host- Pd(dppf)Cl2 versus substrate and NP host. g) Photograph of the palladium catalysis under UV-irradiation with the control reactions...... 118

5.7: Catalytic activity of NP host-Pd(dppf)Cl2 versus substrate and NP host after 24 h and 7 days...... 119

5.8: Fluorescence increased further for AuNP-Pd(dppf)Cl2 while controls (only substrate and AuNP host + substrate without catalyst) showed no fluorescence after one week...... 119

5.9: a) The structure of Wilkinson catalyst. b) The reaction scheme of hydrogenation using NP host and sodium 4-vinylbenzenesulfonate. c) Hydrogenation conversion profile in terms of reaction time for AuNP-Wilkinson. d) Reusability test of AuNP-Wilkinson system for five consecutive cycles...... 120

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5.10: 1H-NMR spectra of the hydrogenation reactions in water at the different times (1, 3, 6, 24, and 48 h)...... 121

5.11: 1H-NMR spectra of hydrogenation reactions for recyclability test, yielding 4- ethylbenzenesulfonate as the final product. All of samples were measured after 24 h reaction...... 122

5.12: Photos of the reaction mixtures including a) NP Host or b) NP Host-HG2 after 24 h reactions...... 126

5.13: GPC spectra of the polymers that were synthesized by ROMP in water at different time intervals (1, 4, 6, and 24 h)...... 127

5.14: GPC spectra of reactions that were performed using free catalysts in acetone/water mixture at different time intervals (1, 4, 6, and 24 h)...... 128

6.1: Monolayer structures of nanozymes used in the study...... 138

6.2: Catalyst encapsulation into the monolayer of nanozyme...... 139

6.3: Amount of catalyst in nanozymes. All experiments were carried out in triplicate, and error bars represent standard deviation...... 139

6.4: Activation of bisallyloxycarbonyl rhodamine 110 catalyzed by AuNP_Ru nanozyme...... 140

6.5: Catalytic activity of nanozymes under ambient temperature. All experiments were carried out in triplicate, and error bars represent standard deviation...... 141

6.6: Activation of pro-Dox using ruthenium catalyst embedded nanozyme ...... 141

6.7: Cell viability of Dox versus pro-Dox (Alloc-Dox)...... 142

6.8: Viability of cells incubated with nanozymes with different monolayer structures and pro-Dox...... 143

6.9: Synthesis of compound S2 from compound S1...... 144

6.10: Synthesis of pyrene ligand...... 145

6.11: Synthesis of C16 ligand...... 149

7.1: Bioorthogonal nanozyme design and supramolecular regulation of intracellular catalysis. a) AuNPs, catalyst embedded AuNPs, and CB[7] capped catalyst embedded AuNPs used in study. b) Endosomal uptake of nanozymes. c) Intracellular catalysis

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with NP_Ru converting substrate into product. d) CB[7] complexation with the ligand headgroup to provide NP_Ru_CB[7] inhibits catalyst activity. e) Nanozyme activity is restored through addition of the competitive guest 1-adamantylamine (ADA). f) Structures of the NP platform with the surface ligand bearing a dimethylbenzylammonium group to bind the CB[7] gatekeeper, CB[7] gatekeeper, and ADA, a competitive guest molecule for CB[7] binding. g) Structures of the non- fluorescent substrate (rhodamine 110 derivative), fluorescent product (rhodamine 110) obtained after catalysis, and embedded catalyst for allylcarbamate cleavage...... 158

7.2: TEM images of AuNP with (a) without (b) encapsulation of the ruthenium catalysts. No size change or aggregation of nanoparticles was observed from TEM images, indicating no morphological change occurred during encapsulation process...... 159

7.3: Bar graph of intensities of NP_Ru and NP_Ru_CB[7] at 5 different time points for 2 h. ... 160

7.4: Photo of the reaction mixtures in water with NP_Ru and NP_Ru_CB[7] under UV light at 0 h...... 161

7.5: Reaction mixtures including a, the nanozyme and caged rhodamine 110 and b, AuNP and caged rhodamine 110 after 24 h...... 161

7.6: Photo of the reaction mixtures in water with NP_Ru and NP_Ru_CB[7] under UV light after 5 days...... 161

7.7: ITC titration of CB[7]s into the NP solution. The circles represent the integrated heat changes during complex formation and the lines represent the curve fit to the binding isotherm...... 162

7.8: Catalytic activity of nanozymes in solution. a) Fluorescence was generated by NP_Ru after the cleavage of profluorophore bis-Alloc-rhodamine 110, while NP_Ru_CB[7] showed no significant change. b) After adding ADA, catalytic activity of NP_Ru_CB[7] was restored and no significant effect was observed for the activity of NP_Ru. c) The reaction rates of NP_Ru and NP_Ru_CB[7] before and after adding ADA showing catalytic activity for NP_Ru_CB[7] was fully recovered after the addition of ADA. The reaction rate experiments were performed in triplicate. Error bars represent standard deviations of these measurements. d) Photo of the reaction mixtures in water with NP_Ru and NP_Ru_CB[7] under UV light...... 163

7.9: Activity assay of allylcarbamate cleavage of the [Cp*Ru(cod)Cl] in acetone/water (1:1 v/v). No catalytic activity change was observed in presence of CB[7] or CB[7] + ADA, indicating CB[7] or ADA cannot affect the catalytic activity of the catalysts directly...... 164

7.10: Lineweaver-Burk plot showing competitive binding of CB[7] to nanozyme. Kinetic studies of NP_Ru and NP_Ru_CB[7] in sodium phosphate buffer (5 mM, pH 7.4) indicate that CB[7] inhibits catalyst activity through a competitive inhibition mechanism, with CB[7] affinity and stoichiometry consistent with ITC binding studies. Numbers

calculated on a per particle basis. Kinetic experiments with each nanozyme were repeated in triplicate. Error bars represent standard deviations of these measurements...... 165

7.11: The dependence of reaction rates on the concentration of the CB[7] (a)0 μM; b) 4 μM; c) 16 μM; and d) 80 μM). e) The fitting curve of the reaction rate vs. concentration of the substrates...... 165

7.12: The dependence of reaction rate on the concentration of CB[7]...... 166

7.13: The reaction curve for relative activity versus [CB[7]]/[NP] ...... 166

7.14: Photos of the reaction mixtures in water with NP_Pd and NP_Pd_CB[7] under UV light at a) 0 h and b) 6 h...... 167

7.15: Kinetics of palladium catalyst embedded nanozymes. Fluorescence generation by NP_Pd and NP_Pd_CB[7] a) before ADA addition and b) after ADA addition...... 168

7.16: Nanozyme uptake assay by tracking gold amount through ICP-MS...... 168

7.17: Cytotoxicity of the NP_Ru and NP at various concentrations...... 169

7.18: Triggered allylcarbamate cleavage in living cells using gated nanozymes. a) Flow cytometry of NP_Ru, NP_Ru_CB[7], and controls (only cell and NP) revealing NP_ Ru showed significant increase in fluorescence while NP_Ru_CB[7] was completely inhibited. b) Addition of ADA to NP_Ru_CB[7] treated cells recovered the catalysis and resulted in increase in fluorescence. c-f) Confocal microscopy images of HeLa cells showing the supramolecularly regulated intracellular chemical reactions, a punctate fluorescence was observed for NP_Ru and NP_Ru_CB[7] + ADA treated cells as the indication of catalysis while no fluorescence was obtained for only substrate and NP_Ru_CB[7] (scale bars = 10 μm)...... 170

7.19: The confocal images of the cell treated with a) the nanozyme, substrate, and lysotracker; b) the nanozyme-bound-CB[7], substrate, ADA, and lysotracker...... 171

7.20: Confocal microscopy images of HeLa cells incubated with only substrate (a), NP_ Ru + substrate (b), and NP_Ru nanozyme + free CB[7] + substrate (c) (scale bars = 20 μm). Fluorescence intensities were obtained from confocal images using ImageJ program (d)...... 171

7.21: Intracellular gated-catalysis using NP_Pd/NP_Pd_CB[7] (scale bars = 10 μm)...... 172

7.22: Prodrug activation in living cells using gated nanozymes. a) Structures of pro-5FU, 5FU and the palladium catalyst used for prodrug activation. b) Viability of cells treated with 5FU and pro-5FU at various concentrations, showing a nice therapeutic window was obtained between 5FU and pro-5FU. c) NP_Pd and ADA treated NP_Pd_CB[7] showed increasing intracellular toxicity as a result of more conversion of prodrug into

xxi

5FU drug at higher pro-5FU concentrations, while NP_Pd_CB[7] did not show any toxicity at any prodrug concentration used due to the blocking catalysis. Also, only prodrug, NP_Pd, NP_Pd_CB[7], and NP_Pd_CB[7] + ADA did not cause any toxicity into the system at zero prodrug concentration. Cell viability experiments were performed in triplicate. Error bars represent standard deviations of these measurements...... 174

7.23: Cleavage of propargyl functionality of prodrug was monitored using MALDI-MS at (a) 15, (b) 48, and (c) 72 h...... 175

1 7.24: H NMR spectrum (400 MHz) of compound 2 in CDCl3 (D, 99.8%)...... 178

1 7.25: H NMR spectrum (400 MHz) of compound 3 in CDCl3 (D, 99.8%)...... 179

7.26: 1H NMR spectrum (400 MHz) of compound 4 in CDCl3 (D, 99.8%)...... 180

1 7.27: H NMR spectrum (400 MHz) of compound 4 in CDCl3 (D, 99.8%)...... 181

1 7.28: H NMR spectrum (400 MHz) of benzyl ligand in CDCl3 (D, 99.8%)...... 182

1 7.29: H NMR spectrum (400 MHz) of benzyl-AuNPs in D2O (D, 99.8%)...... 183

7.30: MALDI-MS spectrum of benzyl-AuNP...... 184

7.31: Characterization of the functionalized AuNP. a) Size (diameter) of AuNP was measured by DLS before and after catalyst encapsulation. DLS measurement shows that size of the NP after catalyst encapsulation stays same. b) Zeta potential of AuNP was measured by DLS. The overall charge of AuNP is measured as 25.5 ± 1 mV from three independent replicates...... 185

xxii

LIST OF SCHEMES

Scheme Page

4.1: (a) Schematic illustration of the MALDI-MS detection process of supramolecular complexes in cells. AuNP-CB[7] complexes are measured as complex ions between CB[7] and AuNP surface ligands, and these ions appear at m/z values above 1600. (b) Monitoring the selective dissociation of the supramolecular complexes after adding the competitive binding molecule ADA. The addition of ADA dissociates some AuNP- CB[7] complexes and also leads to a new ADA-CB[7] complex ion at m/z 1314...... 85

4.2: Synthesis scheme of dimethyldiaminohexyl ligand (DMAH, L2)...... 96

5.1: a) The encapsulation process of hydrophobic catalysts in water soluble NP hosts. b) Schematic illustrating catalytic reaction within NP monolayer...... 112

6.1: Schematic showing the increase in the catalyst loading in three nanozymes used in the study...... 137

7.1: Synthetic scheme of the benzyl ligand for the functionalization of the nanoparticle...... 177

xxiii

CHAPTER 1

GOLD NANOPARTICLES IN DELIVERY AND THERAPEUTICS

1.1 Introduction

Nanoparticles with tunable properties are promising platforms for myriad applications in delivery,1 therapeutic, sensing,2 materials,3 and catalysis.4 Especially monolayer protected inorganic nanoparticles have been widely employed in biotechnology and nanotechnology due to their tunable and controllable core and surface properties. Monolayer protected nanoparticles are hybrid materials where metal core is protected with self-assembled monolayers (SAMs) of organic ligands. The SAMs are chemisorbed onto metal/semiconductor core surfaces to make the nanoparticle stable, soluble in water, biocompatible and chemically diverse.5,6 The applications of these nanoparticles take advantages of properties of both rigid core material and soft monolayer ligands.

Nanoparticles exhibit a number of special properties relative to bulk material.

Nanoparticles possess unique chemical and physical properties that can be tailored for the intended application. Among the physicochemical properties of inorganic nanoparticles, size, shape and surface functionality are the key attributes that dictate the behaviour of nanoparticles and their fate within biosystems.7 Tuning these three features during chemical synthesis of nanoparticles bring out distinct optical, electronic, catalytic, or magnetic properties.8 At the same time, uptake, distribution, cytotoxicity, and interaction with biomolecules are highly affected by these three features.9 Especially, decorating the surface of nanoparticles with a wide range of appropriate functionalities reveals structurally and dynamically well-defined architectures that provide efficient drug delivery platforms.10 Addition to size, shape, and surface functionality, surface area to volume ratio in nanoparticles has a significant effect on the nanoparticles properties. Nanoparticles have a very high surface area to volume ratio compared to larger particles and this allows multiple copies of a ligand to be attached per nanoparticle resulting in

1 multivalency, a crucial feature that provides enhanced interaction with the surrounding environment. These unique properties of nanoparticles have made them attractive platforms for many applications from studying the nanoparticle-biomolecule interactions to their use in delivery and therapeutics.

Among the inorganic nanoparticles, gold nanoparticles (AuNPs) have attracted great attention due to ease of synthesis and surface functionalization, inertness of the core, biocompatibility and functional versatility (Figure 1.1). After the pioneering work of Brust et al., thiol-protected AuNPs has been the mostly investigated monolayer protected nanoparticle system.11 AuNPs have been widely studied for many purposes such as catalysis,12 sensing,13 delivery,14 and therapeutics.15

Figure 1.1: Schematic illustration of monolayer-protected gold nanoparticles. The rigid inorganic core is protected by a soft organic monolayer that can be chemically tailored for various applications.

The most common way of functionalization of AuNPs is performed using Brust-

Schriffrin reduction followed by Murray place-exchange reaction16 with appropriate ligands that are synthesized for the intended application. After the place exchange reaction and purification steps that involve washing/centrifugation cycles and dialysis, nanoparticles that are of commensurate size to many biological components such as DNA, protein, and cell membrane are

2 obtained. This biomimetic size provides multiple applications at the bio-nano interface such as gene transfection, regulating DNA transcription, protein-protein interactions, etc. This thesis uses

AuNPs as a scaffold for catalysis, delivery and therapeutic applications using supramolecular chemistry. Therefore, as a background to following chapters, AuNPs in delivery and therapeutics, supramolecular interactions between small molecules and the monolayer of AuNPs, bioorthogonal catalysis, and some applications of AuNPs related to this thesis will be discussed in this chapter.

1.2 Application of Gold Nanoparticles in Drug Delivery and Therapeutics

AuNP-based drug delivery systems are highly attractive platforms for the efficient delivery and release of drugs to targeted tissues and cells. There are two major strategies for conjugating drugs to AuNPs: covalent or non-covalent association.17 Mirkin et al. have employed a covalent attachment strategy to conjugate paclitaxel (a potent chemotherapeutic drug) to AuNPs via DNA linkers, thereby enhancing the solubility and overall effectiveness of the drug.18 Also,

Zubarev et al. have attached a flexible hexaethylene glycol linker to the C-7 position of paclitaxel to conjugate it to phenol-terminated AuNPs.19 Using this strategy, 70 molecules of paclitaxel were able to be loaded onto an AuNP in a controlled manner. Mirkin, Lippard and coworkers have combined the properties of DNA, AuNPs and Pt(IV) prodrugs into a single agent for drug delivery using the covalent association approach. The particles were incubated with HeLa cells for different time periods. After 6 h, they had localized in vesicles (Figure 1.2a), and after 12 h, they were released into the cytosol (Figure 1.2b). Oregon Green 488 taxol bisacetate, which stains microtubules, showed colocalization of these nanoparticles with the microtubules in HeLa cells

(Figure 1.2c). Overall, they have demonstrated that the acidic environment in cancer cells facilitate reduction of the Pt(IV) and yield the cytotoxic Pt(II) species.

Figure 1.2: Top scheme-synthesis of Pt-DNA-AuNP. Bottom a) Live cell imaging of HeLa cells after incubation with platinum-tethered Cy5-DNA-Au NPs for 6 h and (b) 12 h, and (c) colocalization of the particles with the cytoplasmic microtubules.

Rotello et al. have used the hydrophobic interior of AuNP monolayers to non-covalently encapsulate and deliver a hydrophobic drug molecule.20 Depending on the strategy used, the release of a drug from its carrier can be triggered by a wide range of stimuli, such as hydrophobicity, pH, temperature, magnetic fields, enzymes, UV-light, and NIR photoluminescence.21 For instance, Burda et al. have shown that a photodynamic therapy (PDT) drug such as silicon phthalocyanine 4 could be successfully adsorbed on PEGylated AuNPs and used as an effective PDT drug delivery vector for both in vitro and in vivo applications.22

Furthermore, inorganic nanomaterials can be designed to improve drug efficacy through enhanced targeting delivery.23 For instance, El-Sayed et al. have quantified the degree of tumor cell uptake of Au nanorods covalently conjugated to tumor-targeting peptides.24

1.3 Supramolecular Chemistry

Supramolecular chemistry studies the non-covalent interactions in and between molecules, as well as the interactions between formed multimolecular complexes.25 Natural molecules, such as proteins, oligonucleotides and lipids have been the source of inspiration for supramolecular chemistry and supramolecular chemists aim to design and synthesize novel supramolecular architectures using various building blocks that can reach the complexity and functionality of these natural molecules. Supramolecular chemistry mainly focuses on how molecules recognize each other, assemble and function on a molecular scale.26 It brings a bottom up approach where nanoscale systems are generated from self assembling molecules using hydrogen bonding, host–guest recognition, metal coordination or electrostatic interactions..27

Supramolecular chemistry has been involved in many applications ranging from biology to materials science.28

1.3.1 Supramolecular Chemistry in Drug Delivery

Non-covalent interactions provide a flexible method of engineering various building blocks with tailored properties. Due to the reversible nature of the non-covalent interactions, the supramolecularly engineered systems have exhibited unique features such as facile preparation and functionalization, controllable morphologies and structures, dynamic self assembly processes, and adjustable performance.29 Furthermore, the self-assembled supramolecular structures have been applied in various biomedical fields including bioimaging,30 gene transfection,31 protein delivery,32 regenerative medicine,33 tissue engineering, sensing, and drug delivery.34 Especially, when drug delivery is considered, excellent biocompatibility and biodegradability, responsive nature, and possibility of incorporating multiple arrays of different functional units through intermixing of different building blocks render supramolecular platforms very promising delivery systems.35 All of these features are of great importance for designing and producing drug carriers.

Compared to those of the covalent assemblies, the self-assembly and disassembly processes of

5 supramolecularly engineered assemblies are easier to control due to the existence of reversible non-covalent interactions, which makes the supramolecular assemblies an ideal vehicle for drug delivery. For example, Scherman et al. reported triple stimuli-responsive supramolecular double hydrophilic block copolymer micelles for controlling drug release (Figure 1.3).36 In their design, a stimuli responsive micelle was fabricated by connecting a thermo-responsive polymer (poly(N- isopropylacrylamide), PNIPAM) and a pH-responsive polymer

(poly(dimethylaminoethylmethacrylate), PDMAEMA) together using a host molecule, cucurbituril[8] (CB[8]). These micelles were loaded with Doxorubicin (Dox) and its release was triggered by the disassembly of the carrier by decreasing the temperature, lowering the pH or adding competitive guests. Compared to the covalently connected micelles, these supramolecular micelles exhibited a faster release rate and a tunable release profile that was controlled using these three stimuli.

Figure 1.3: Schematic representation of hierarchical self-assembly of the supramolecularly engineered polymers to yield micelles and drug release after being exposed to three different triggers.

1.3.2 Supramolecular Platforms Using Gold Nanoparticles

AuNPs with a large number of surface ligands are promising scaffolds as the building blocks of self assembly process. Proper design of the interactions between AuNPs and complementary units such as proteins, polymers, and small molecules results in unique materials with tunable properties and desirable functions. Various supramolecular interactions including electrostatic attractions,37 hydrogen bonding,38 π-π stacking and van der Waals interactions between nanoparticle and complementary units have been designed to control nanoparticle self- assemblies. Rotello et al. have developed a ‘chemical nose’ sensing system using supramolecular interactions between functionalized AuNPs, transducers (fluorescent proteins), and analytes. The sensor was generated by non-covalent conjugation of AuNPs with fluorescent proteins (EBFP2,

EGFP and tdTomato). The fluorescent proteins serve the dual roles of exhibiting differential supramolecular affinities with the particle and transducing the binding events. In these AuNP– fluorescent protein supramolecular complexes, the cationic AuNP binds strongly with the anionic fluorescent proteins, resulting in quenching of the fluorescent protein fluorescence by the particle core. The binding equilibria between AuNP and the fluorescent proteins (FBs) are altered in the presence of analytes due to the competitive binding to analytes, resulting in rapid

(seconds/minutes) displacement of fluorescent proteins from the particle surface, with consequent restoration of the fluorescence. The fluorescence ‘turn-on’ of the emission channels differs considerably depending on the signatures of the analyte surfaces. Specific signal patterns of this system can be used for identifying proteins, cell surface glycomic signatures,39 bacteria,40 and cancer drug mechanisms.41 Introducing host-guest chemistry into the sensor system doubled the information content of the array-based sensor for cancer diagnostics. In this approach, the interaction of the particle with both the fluorescent protein transduction elements and the cell lysate analytes were modulated by non-covalent modification of nanoparticle surface with a complementary cucurbit[7]uril (CB[7]) moiety.42 As a result, the change in competitive binding effectively resulted in doubling the number of output channels from three to six while

7 maintaining the one-well configuration with 100 % accuracy and minimal sample quantity (200 ng, ~1000 cells) (Figure 1.4).

Figure 1.4: (A) Six channel-output in a single well. The fluorescence of FPs is quenched when the NP-FP complexes are formed. Upon addition of cell lysates, three emission channels are obtained from the released FPs. In the same well, CB[7] is added to obtain three additional channels from the three FPs as a result of changed interactions between the analyte and newly formed complex, NP-CB[7]. (B) Linear Discriminant Analysis (LDA) with the fluorescence responses from all six channels showed well separation of all cell types.

Rotello and coworkers used supramolecular chemistry to regulate the therapeutic efficiency of AuNPs. This supramolecular system includes cucurbit[7]uril (CB[7]), a synthetic host molecule and AuNPs with head group functionality which is complementary to CB[7]

(Figure 1.5). After forming complexes of AuNPs and CB[7], a non-toxic assembly that is readily taken up by cells was obtained. This host-guest complex can be disassembled intracellularly by using an orthogonal competitive molecule, 1-adamantylamine (ADA), which has a very high affinity for CB[7]. Intracellular removal of CB[7] from the nanoparticle surface results in endosomal escape of toxic AuNPs and concomitant cell death. This result shows the utility of supramolecular host-guest interactions in drug delivery and therapeutics.

Figure 1.5: (A) Schematic showing the activation of AuNP-NH2-CB[7] cytotoxicity by dethreading of CB[7] from the nanoparticle surface by ADA. (B) Cytotoxicity of AuNP-NH2 and AuNP-NH2-CB[7] measured by Alamar blue assay after 24 h incubation in MCF-7. (C) Triggering cytotoxicity using ADA. After 3h incubation of AuNP-NH2-CB[7] (2 µM) in MCF-7 cell, different concentrations (0, 0.2 and 0.4 mM) of ADA in medium added and further incubated at 37 ºC for 24 h. The cell viability was then determined by using an Alamar blue assay.

1.3.3. Characterization and Monitoring of Host-Guest Interactions in Solution and Living Cells

Characterization of intermolecular interactions and their binding equilibria in solution are performed using techniques such as NMR,43 fluorescence, UV/Vis spectroscopy,44 and surface plasmon resonance (SPR).45 However, all these techniques have certain drawbacks such as the requirement of large quantities of the samples, the necessity to introduce labels, or long measurement times. ITC (isothermal titration calorimetry), a useful analytical tool, is widely used by biochemists and supramolecular chemists to characterize interacting systems and study host−guest interactions.46 In contrast, sample preparation is very easy for ITC and also it offers a fast calorimetric response and thermal equilibration. The most important features of ITC include the determination of the entire thermodynamic profile (ΔH, ΔS, ΔG, KB, and the stoichiometry n)

9 of an interaction in only one experiment.47 This method is based on the repetitive addition of a solution of one of the interacting molecules (ligand or host molecule), via automated injection at constant temperature, into a cell containing a solution of the second molecule (guest molecule).

The heat exchange is measured and reveals quantitative information about the interaction between molecules. The binding stoichiometry and thermodynamic parameters of binding (host-guest complexation) including association and dissociation constants as well as changes in enthalpy, entropy, and free energy can be derived from a single ITC titration. Supramolecular interactions of protein-protein,48 small molecule/drug-protein,49 protein-polymer50, biomolecule- nanoparticle,51 and enzyme kinetics have already been successfully examined using ITC.52 Self- assembling monocomponent systems can also be studied by ITC to monitor the dissociation event. A concentrated solution of the self-assembled compound is placed in the syringe and injected into the cell containing pure solvent. If the final concentration is low enough, the supramolecular assemblies are dissociated and the heat exchange is characteristic of the dissociation event. This procedure has been used to measure the critical micelle concentration of low-molecular-weight surfactants,53 to study amphiphilic polyelectrolytes,54 and to derive the partition coefficient of solutes in lipid vesicles.55

Due to the complex environments in cells, the aforementioned methods including ITC cannot be used to analyze supramolecular interactions in biological systems. Mass spectrometry

(MS) is an effective tool for characterizing host-guest interactions in solution56 and in biological environment.57 For example, electrospray ionization (ESI) MS and matrix assisted laser desoption/ionization (MALDI) MS have been utilized for the detection of host-guest complexes.58

Nau and coworkers investigated host-guest inclusion complexes between a macrocyclic host, cucurbit[n]urils (CBn, n=6–8) and the bicyclic azoalkanes by mass spectrometry, and studied thermally activated, selective retro-Diels–Alder reactions in the gas phase.59

Among MS techniques, laser desorption/ionization (LDI)-MS has been shown to selectively detect ligands on intact NPs in complex biological samples.60 This method relies on the selective desorption/ionization process of the surface ligands on NPs under the laser irradiation. It is a highly sensitive and selective method that enables to detect NP monolayer in cell lysates.61 Supramolecular complexes formed by the surface ligands of NPs and host molecules serve as “mass barcodes” to indicate complex formation inside cells. By this method, host-guest interactions can be monitored without using an additional labeling process.

1.4 Bioorthogonal Catalysis

Bioorthogonal chemistry is a powerful strategy that allows to do artificial chemistries in the same physical space where biological processes take place continuously, yet do not interfere with each other.62,63 Bertozzi`s benchmark works demonstrated the potential of bioorthogonal chemical reactions in studying biomolecules in their native settings.64,65 Visualization of biomolecules and/or dynamic cellular processes in real time and in real life has been achieved by bioorthogonal platforms that utilize reactions such as Staudinger ligation and Cu-free click chemistry. Staudinger ligation which forms an amide bond between the azide and an ester- derivatized phosphine is highly biocompatible in cells and living animals. However, slow reaction kinetics and oxidation issues of the phosphine reagents66 limit the use of this bioorthogonal ligation method to detect low-abundance species or to visualize rapid biological processes. On contrary, click chemistry which is the azide-alkyne cycloaddition reaction, is highly reactive and sensitive bioorthogonal functionalization method. Click chemistry has been developed by

Sharpless and coworkers67 and Meldal and colleagues68 using copper catalyst (Figure 1.6). This copper mediated cycloaddition has been used for probing enzyme activities in cell lysates69 or visualizing biomolecules in fixed cells.70 However, for dynamic processes in living systems, this system was not suitable due to the cytotoxic nature of copper catalyst.

Figure 1.6: (A) The copper-catalyzed azide–alkyne cycloaddition. (B) The Cu-free click reaction of azides and DIFOs.

Bertozzi et al. designed a bioorthogonal reaction for dynamic cellular imaging that

combines the biocompatibility of the Staudinger ligation with the fast reaction kinetics of click

chemistry (Figure 1.7). They explored the use of cyclooctynes as an alternative to activating

alkynes for [3+2] cycloaddition with azides. Due to the ring strain, they achieved bioorthogonal

labeling of azides without using copper catalyst.71 To increase the sensitivity of the cyclooctynes

for azide detection, they incorporated an electron withdrawing group, difluoromethylene (DIFO)

moiety.72

Figure 1.7: (A) Comparison of Cu-free click chemistry with Cu-catalyzed click chemistry. (B) Schematic showing cell surface labeling using Cu-free click chemistry. (C) Comparison of Cu-free click chemistry with other bioorthogonal ligations using alkyne containing Alexa Fluor 488. (D) Labeling using alkyne containing biotin.

Beside these applications, bioorthogonal chemistry has been widely used for the intracellular73 and extracellular74 generation of molecules for therapeutic,75 imaging,76-77 and sensing applications.78-79 Bioorthogonal catalysis enables scientists to selectively activate molecules for the aforementioned applications under complex environments with high efficiency.

However, these bioorthogonal systems must possess fast rates under physiological conditions and be inert to myriad of functionalities found in vitro and in vivo.

1.4.1 Bioorthogonal Catalysis Using Transition Metal Catalysts

Transition metal catalysts (TMCs) are excellent candidates for use in bioorthogonal chemistry, as they provide platforms that rapidly catalyze a wide range of transformations where enzymes are not able to perform.80 In some studies, TMCs are used to build up artificial metalloenzymes where the naturally occurring metalloenzyme was redesigned by replacement of the native metal with a suitable transition metal to develop highly new selective hybrid catalysts for several asymmetric catalytic reactions.81 However, in some studies, TMCs are incorporated in totally synthetic platforms or used directly to perform bioorthogonal selective transformations

(Figure 1.8).82,83

Figure 1.8: Bioorthogonal nanocatalysis designed and demonstrated by Meggers (2006, 2012), Bradley and Unciti-Broceta (2014, 2016) and Rotello (2015).

Meggers and co-workers were pioneers to report the first example of bioorthogonal catalysis by cleaving protecting groups using nanocatalysts based on organometallic catalysts in cellular environment.84,85 In one of their work, they found that ruthenium(II) half-sandwich complex [Cp*Ru(COD)Cl] (Ru1), with Cp* = pentamethylcyclopentadienyl and COD = 1,5- cyclooctadiene can catalyze the uncaging reaction of allylcarbamates under bio-relevant conditions (Figure 1.9). To evaluate the catalytic efficiency in living mammalian cells, they chose a non-fluorescent profluorophore N,N-bis-allyloxycarbonyl protected rhodamine 110 as a cellular probe. HeLa cells were incubated with the substrate for 30 minutes followed by washing with phosphate buffered saline (PBS). Upon addition of Ru-catalyst and thiophenol, rapid increase in fluorescence was observed due to cleavage of the both allylcarbamate-protecting groups (Figure

1.9 c-f).

Figure 1.9: Fluorescence microscopy images of uncaging process of allylcarbamate-protected rhodamine 110 (pro-fluorophore) inside HeLa cells using ruthenium nanocatalyst. a) Right after catalyst addition and b) after 15 min. c)–f): Fluorescence generation after indicated times. Cells were incubated with pro-fluorophore and the membrane carbocyanine dye DiIC18(5) for 30 min and then treated with [Cp*Ru(cod)Cl] and thiophenol.

The direct application of TMC-mediated reactions in living cells is challenging due to issues of biocompatibility, water solubility/stability, and rapid efflux from living cells.86 In order to overcome such limitations, Bradley et al. pioneered on the uses of nanoparticles for carrying

14 out bioorthogonal catalysis.87 They investigated several bioorthogonally chemical processes using

3 nm palladium nanoparticles entrapped in a 500 nm polystyrene microsphere.88 After a day of interaction, 75% of HeLa cells engulfed one or more of microspheres and 91% of them were viable after 48 h. The cells internalized in microparticles were used to carry out several catalytic processes such as allylcarbamate cleavage and C-C bond formation through Susuki-Miyaura cross coupling reaction (Figure 1.10). More recently, Bradley, Unciti-Broceta and coworkers used palladium catalyst loaded polystyrene composite with particle size of 150 μm for extracellular activation of prodrugs such as propargyl caged 5-Flurouracil (5-FU).74 Other cytotoxic drugs like

Gemcitabine89 and Floxuridine90 were also transformed into prodrugs that are activated by Pd chemistry.

Figure 1.10: Scheme of the bioorthogonal reactions taking place inside the cell internalized PdNP@PStyr composite. (A) Allylcarbamate deprotection (cleavable unit highlighted in red). (B) Confocal microscope image of cell stained through the generation of highly fluorescent Rhodamine 110 after the allylcarbamate cleavage process. (C) Scheme of the Susuki-Miyaura transformation on the fluorescein triflate ester taking place on the surface of the cell internalized PdNP@PStyr composite. (D) Confocal image of the generated fluorescein derivative staining mitochondria.

1.4.2 Non-Covalent Incorporation of Hydrophobic Transition Metal Catalysts into AuNP Monolayers

Non-covalent drug delivery utilizing NPs involves either an encapsulation mechanism or a stabilizing pocket.91 Efficient release of the cargo in the hydrophobic pocket is dictated by external changes in hydrophobicity. If the NP carrier encounters more hydrophobic environment such as a cell membrane, drug molecules can simply diffuse into the membrane. AuNPs posses some hydrophobic interior (pockets) in the monolayer that can be used to encapsulate various hydrophobic cargos such as transition metal catalysts, cancer drugs, dye molecules, or antibiotics.92 These hydrophobic pockets are formed due to the hydrophobic section of the ligands that are used to functionalize AuNPs. Structurally, the radial nature of the monolayer results in a decrease in ligand density as one goes farther from the core of small AuNP cores (< ~6 nm).93

Consequently “hydrophobic pockets” are created inside the monolayer of AuNP into which hydrophobic materials can be partitioned. Pasquato et al. demonstrated the encapsulation of radical probes in AuNP monolayers, using EPR spectroscopy to monitor the partition of the lipophilic probe between a monolayer of AuNP and bulk water.94 As expected, smaller particles featuring more strongly radial monolayers favor guest encapsulation.

Rotello et al. have utilized hydrophobic pockets of AuNP monolayers to encapsulate highly hydrophobic dyes/therapeutics.95 They have demonstrated that hydrophobic dyes/drugs can be stably entrapped in a hydrophobic pocket of small sized AuNPs (hydrodynamic diameter

∼10 nm) coated with non-interacting monolayers (zwitter ionic layers). They have chosen three different hydrophobic guest compounds: 4,4-diﬂuoro-4-bora-3a,4a-diaza-s-indacene (Bodipy) as a ﬂuorescent probe and the highly hydrophobic therapeutics tamoxifen (TAF) and-lapachone

(LAP) as the drugs (Figure 1.11). The nanoparticle-payload conjugates (AuNPZwit-Bodipy, TAF, and LAP) were prepared by a solvent displacement method.96 Payloads have released into the cell by membrane-mediated diffusion without uptake of the carrier nanoparticle. Importantly, the small size of these nanocarriers coupled with their biocompatible surface functionality should

16 provide long circulation lifetimes and preferential accumulation in tumor tissues by the enhanced permeability and retention (EPR) effect.97

Figure 1.11: (A) Delivery of payload to cell due to change in hydrophobicity through monolayer- membrane interactions (B) Structure of particles and guest molecules: Bodipy, TAF, and LAP, the number of encapsulated guests per particle, and log P of the guests.

Burda et al. have also utilized hydrophobic pocket concept to deliver a hydrophobic photodynamic therapy drug Pc 4 using PEG functionalized AuNPs, and reported an increase in drug tumor accumulation. 98 The drug was found to reside deep within the PEG layer, and only after the delivery and release, the fluorescence of the drug was observed. However, when a covalent attachment of Pc 4 to Au surface is used, almost no drug was found to be delivered.

1.5. Dissertation Overview

Incorporating supramolecular chemistry into AuNPs combines the unique features of both systems into a single platform. While dynamic nature of supramolecular interactions brings reversibility, tunable physical and chemical properties of AuNPs render them as controllable platforms with potential applications in delivery and bioorthogonal catalysis. My research has

17 been oriented toward the fabrication of functional AuNPs scaffolds using host-guest interactions.

In some of my studies, ligand head groups on AuNPs serve as the guest molecules that are ready to interact with synthetic host molecules. In other studies, I used AuNP as a host for hydrophobic transition metal catalyst (guest molecules) encapsulation and solubilization. To achieve my goals,

I have combined organic synthesis, surface science, analytical chemistry, and biology to develop supramolecularly engineered AuNP platforms. In the following chapters, details of designing functionalized AuNPs and their use in delivery as well as catalysis are described.

In chapter 2, regulation of exocytosis of AuNPs using a simple host-guest interaction will be discussed. AuNPs carrying quaternary ammonium head groups were internalized into the cells through endocytosis. Subsequent in situ treatment of a complementary cucurbit[7]uril (CB[7]) to the head groups induced the particle-CB[7] complexation inside cells, rendering the particles assembled each other. This complexation followed by the concomitant formation of larger assemblies resulted in the inhibition of exocytosis of particles without creating toxicity, due to the sequestered particles in endosomes. This approach would provide a potential strategy for prolonged retention of drug carriers within endosome, enabling sustained release of the loaded therapeutics in the carriers.

In chapter 3, quantification of host–guest interactions between CB[7] molecule and

AuNPs using ITC titrations will be described. In this study, AuNPs featuring ligands with a monomethyl-benzylammonium (MMBA) or dimethyl-benzylammonium (DMBA) head groups were served as guest scaffold and CB[7] as the host molecule. We have investigated the changes in the binding event considering two features: (1) the effect of different functionalities at the para position of the benzene ring and (2) the effect of carrying a permanent positive charge on the head group.

Chapter 4 will discuss a new mass spectrometric approach for the characterization and detection of supramolecular host-guest interaction in complex environment such as cell lysates.

Using this simple method, both association and dissociation of host-guest complexes between

AuNPs and synthetic macrocyclic molecules were monitored inside cells. Also, a selective triggering process for the dissociation of host-guest interactions of three AuNPs with different surface functionalities was successfully demonstrated.

Chapter 5 will discuss the solubilization of hydrophobic transition metal catalysts through encapsulation into the water soluble nanoparticle hosts. We report the solubilization of various hydrophobic catalysts achieved by using hydrophobic pockets of water soluble gold nanoparticles. Besides preserving original catalyst activity, this nanoparticle platform provides a protective environment for the hydrophobic catalyst. Grubbs, Cp*Ru(cod)Cl, Pd(dppf)Cl2, and

Wilkinson catalysts encapsulated in the water soluble AuNPs are used for ring opening metathesis polymerization, allyl carbamate cleavage and hydrogenation of alkenes, respectively.

Recyclability of a nanoparticle-catalyst system is examined for the Wilkinson catalyst. This work demonstrates a versatile platform for the encapsulation of different hydrophobic catalysts, allowing the utilization of a wide range of catalysis in water.

Chapter 6 will describe the engineering of AuNP monolayer to increase the catalyst loading. Hydrophobic catalyst was encapsulated into the hydrophobic pocket in the monolayer.

The extension of hydrophobic layer of surface monolayer and introduction of aromatic functional group resulted in the increase of the loading amount of transition metal catalysts per particle as well as the catalytic activity of nanozyme in aqueous solution and inside cells.

In chapter 7, supramolecular regulation of bioorthogonal catalysis in cells will be discussed. This AuNP-catalyst complex is named as “nanozyme” as it mimics the allosteric regulation of enzymes and possesses biomimetic size and surface functionalities. Hydrophobic

19 transition metal catalysts were encapsulated into the hydrophobic pockets in the monolayer to create active sites for bioorthogonal catalysis. Effectively delivering and regulating the activity of protein-sized, catalytic systems is challenging due to both the complex intracellular environment and catalyst instability. The activity of these catalysts could be reversibly controlled by binding a supramolecular cucurbit[7]uril ‘gate-keeper’ onto the monolayer surface, providing a biomimetic allosteric control mechanism. The efficacy of this gated nanozyme was demonstrated by the triggered cleavage of protecting units on the substrates (profluorophores and prodrugs) inside living cells, providing a new type of nanozyme for use in imaging and therapeutic applications.

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CHAPTER 2

REGULATING EXOCYTOSIS OF NANOPARTICLES VIA HOST-GUEST CHEMISTRY

2.1 Introduction

Drug delivery systems (DDSs) improve the efficacy of conventional pharmaceutics through enhanced pharmacokinetics and biodistribution.1 Finely tuned and engineered nanoparticle platforms2 are used to design DDSs to achieve the release of drugs at a controlled rate.3 Trigger sensitive release mechanisms of covalently4 or non-covalently5 attached drugs are widely employed for on-target site activation strategies.6 Furthermore, much effort has focused on increasing the uptake of the carrier into targeted tissues passively through the enhanced permeability and retention (EPR) effect7 and/or actively by using targeting modalities.8 However, for effective DDSs, the sustained therapeutic effect inside cells relies not only on the cellular uptake of nanocarriers but also on their subsequent long-term retention in the cells.5c, 9

One of the main obstacles of DDSs is the rapid removal of the internalized drug carriers through the exocytosis before the drug release.10 Exocytosis is the process of expelling wastes and other large molecules out of the cells,11 which is also commonly observed with a wide variety of drug carriers.12 As an example, internalized poly (D,L-lactide-co-glycolide) nanoparticles undergo exocytosis to the extent of 65 % at 30 min and 85 % at 6 h.13 Reducing exocytosis of the nanoparticulate drug carriers thus prolong their retention time and concurrently release the loaded drugs gradually inside cells, enhancing the therapeutic efficiency of the DDSs. Developing strategies for orthogonal control of exocytosis in the cellular environment is a major challenge because of the intracellular chemical complexity and a potential toxicity of relevant reagents.

Supramolecular chemistry generates controlled assemblies from molecular building blocks through non-covalent interactions including hydrogen bonding, hydrophobic, and van der

Waals interactions. Due to their reversible modularity, supramolecular complexes are useful for

32 creating responsive host-guest systems for many therapeutic applications. Cucurbit[n]rils (CB[n]) are water-soluble macrocyclic hosts with a hydrophobic cavity that form strong inclusion complexes with many types of guests, including positively charged ligands on the gold nanoparticle (AuNP) surface. Various CB[n]-guest systems have been developed to create delivery vectors for therapeutic materials including drugs and an actuator system to control catalytic activity of an enzyme. Moreover, engineering host-guest systems provide the capability of actuation for the regulation of therapeutics in living cells. Among the cucurbituril family, cucurbit[7]ril (CB[7]) is attractive as a building block for the construction of supramolecular architectures due to its remarkable guest binding behavior in aqueous media and non-toxic behavior in vitro and in vivo.

In this work, we describe a new approach to regulate the exocytosis of AuNPs by using host-guest interactions between AuNPs and CB[7] molecules. Quaternary ammonium functionalized cationic gold nanoparticles (AuNP-TBen) were readily taken up by the cells via endocytosis (Figure 2.1). Afterward, in situ treatment of complementary CB[7] molecules resulted in threading of CB[7]s on the terminal functionalities of AuNP-TBen inside the cells, resulting in AuNP-TBen-CB[7] complexes. This complexation rendered the surface of the particles less hydrophilic, inducing the self-assembly of AuNP-TBen-CB[7] sequestered in the endosomes. Exocytosis of the AuNP-TBen-CB[7] was then blocked by the increased size of the induced assemblies. This approach provides a new strategy for improving efficacy of drug delivery systems through prolonged retention of drug carriers within the cells.

Figure 2.1: Controlling exocytosis of AuNPs by using intracellular host-guest complexation. (a) Inhibition of AuNP-TBen exocytosis by the threading of CB[7] onto the nanoparticle surface. (b) Formation of the AuNP-TBen-CB[7] assemblies.

2.2 Results and Discussion

2.2.1 Complexation between AuNP-TBen and CB[7]

Exocytosis of nanoparticles is dependent on their size9a and surface functionality.14

Compared to their smaller counterparts, larger nanoparticles (more than 100 nm in diameter) tend to undergo exocytosis at slower rate and lower amount. Therefore, an efficient way of regulating exocytosis could be inducing in situ growth of assemblies from the internalized individual nanoparticles entrapped in endocytic vesicles by using a host-guest supramolecular system. The exocytosis of the particles then would be blocked by the increased size of the induced assemblies15 and the host-guest system provides an orthogonal stimulus, allowing temporal control of the exocytosis (Figure 2.1 (b)).

We have synthesized a water-soluble AuNP-TBen featuring a tetra(ethylene glycol) and quaternary benzyl amine head group prepared via a Murray place-exchange reaction.16 The detailed syntheses and characterization of the AuNPs are available at the end of this chapter. The gold core had an average size of 2.1 ± 0.5 nm with hydrodynamic diameter of the AuNP-TBen being 9.7 ± 0.1 nm determined by transmission electron microscope (TEM) and dynamic light scattering (DLS), respectively. The AuNP-TBen had a zeta potential of + 14.2 mV. The terminal quaternary benzyl amine moiety serves as a recognition unit for the formation of a host-guest inclusion complex with CB[7] with the association constant of ~ 108 M-1. This higher binding constant is strong enough for the complexes to remain stable under biological conditions.

2.2.2 Inducing Assemblies of AuNP-TBen-CB[7]

The complexation between AuNP-TBen and CB[7] was investigated by performing DLS experiments whose results are shown in Figure 2.2 (a). At the molar ratio of 1:1 and 1:2 (AuNP-

TBen:CB[7]), the hydrodynamic diameter of the AuNP-TBen slightly increased from 9.7 ± 0.1 to

11.6 ± 0.5 nm. At a ratio of 1:4 (AuNP-TBen:CB[7]), the particles began to assemble together and completely aggregated at the ratio of 1:10 (AuNP-TBen:CB[7]) to give a clear solution with a precipitation on the bottom of vial as shown in Figure 2.1 (b). The hydrodynamic sizes of control nanoparticles including AuNP-TTMA and AuNP-TCOOH showed no observable change upon addition of CB[7] because both particles have no significant binding affinities to CB[7].

Figure 2.2: Inducing nanoparticle assemblies upon binding with CB[7] and the effect of surface functional group on the assembly formation. (a) Size changes of nanoparticle assemblies at different NP:CB[7] ratios. (b) AuNP-ADA and AuNP-TMC6 induce the larger assemblies upon binding with CB[7], but no assembly formation was observed for AuNP-TMOH and AuNP- TMNH2 with polar end groups.

The formation of the assemblies upon binding with CB[7] was carried out with AuNPs having different surface functional groups (Figure 2.2 (b)). AuNP-ADA and AuNP-TMC6 behaved similar to AuNP-TBen and induced the large assemblies upon binding with CB[7] at the ratio of 1:10 (AuNPs:CB[7]). In contrast, AuNP-TMOH and AuNP-TMNH2 did not show any aggregate formation although the NP-CB[7] complexes were formed. This indicates that the surface end group of the AuNPs plays an important role in inducing the assembly formation.

The amphiphilic nanoparticle, AuNP-TBen (also AuNP-ADA and AuNP-TMC6) is soluble in both aqueous and organic solvents (e.g. dichloromethane). In the aqueous solution, the hydrophobic benzyl units on the particles are hidden from the external environment, making the surface of the particles more hydrophilic and soluble in the aqueous media. NOESY NMR showed the interaction of benzyl peaks with tetraethylene glycol and 11-undecane peaks as an indication of bending over of benzyl head group (Figure 2.3). Addition of CB[7] resulted in a binding competition of pulling AuNP-TBen from its hydrophobic shell to be encapsulated by

CB[7] host molecules. Upon binding with CB[7], the benzyl units on the particle became

36 stretched out along the particle, rendering the surface of the particle less hydrophilic. This process decreases particle solubility and induces the formation of large assemblies/aggregates (Figure 2.1

(b)). AuNP-TMOH and AuNP-TMNH2 can bind with CB[7] but the hydroxyl and amine end groups presumably keep the surface of particles hydrophilic enough even after binding with

CB[7] and therefore no assembled structures were observed. It indicates that inducing assemblies is dependent on not only CB[7] binding but also end group surface functionality of AuNPs.

Figure 2.3: NOESY 2D NMR shows the interaction of benzyl moiety with TEG and C11 units, indicating head group is bending in towards the monolayer (red circle).

When the particle assemblies of AuNP-TBen-CB[7] were treated with excess of 1- adamantyl amine (ADA), a competitive guest molecule for CB[7] binding, CB[7] dethreaded from the NP surface through creation of more favorable 1:1 ADA-CB[7] complexes (Ka = 1.7 x

1012).21c Addition of ADA triggered disassembly of the particles, indicating that host-guest interaction can be used to control the solubility (hydrophobicity) of AuNPs by using the precise

‘lock and key’ modulation over their molecular-level interactions (Figure 2.4)

Figure 2.4: CB[7] treatment induced the formation of large assemblies. AuNP-TBen-CB[7] were treated with excess of 1-adamantyl amine (ADA), ADA triggered the particle assemblies to disassemble, making the particles soluble back in PBS.

2.2.3 Controlling Exocytosis of AuNP-TBen-CB[7] Inside Cells

Cellular uptake behaviour of the AuNPs was investigated by TEM analysis of the cell.

After 3 h incubation of the cationic AuNP-TBen (200 nM), TEM images showed that the AuNPs were trapped in the endosomal vesicles in cytoplasm as shown in Figure 2.5(a). This observation is consistent with an endocytotic behaviour of other cationic nanoparticles previously reported in literature.17, 18 To study the exocytotic behaviours of the nanoparticle, the cells were washed off after 3 h incubation with the AuNP-TBen and incubated with fresh media or CB[7] (0.2 mM) containing media for an additional 24 h. When the cells were treated by fresh media, the number of the particle-entrapped in vesicles was significantly decreased (Figure 2.5 (b)). Only a few particles remained within the vesicles or dispersed in various organelles, indicating a major portion of the AuNP-TBen are removed from the cells through exocytosis. In contrast, when the cells were treated by the CB[7] containing media, the AuNPs remained trapped in the endosomes

(Figure 2.5 (c)). These results indicate that treatment of CB[7] caused the intracellular assembly formation of the AuNP-TBen-CB[7] as the CB[7] can cross the cell membrane19 and the large bulky assemblies remained sequestered in endocytic vesicles without exocytosis.

Figure 2.5: Cellular uptake and intracellular behavior of the AuNPs. (a) TEM images of MCF-7 cells incubated with AuNP-TBen. The cationic AuNP-TBen is trapped in organelles (e.g. endosome, red circle). TEM images MCF-7 cells incubated with AuNP-TBen and then washed and further incubated with (b) only cell culture media or (c) culture media with CB[7] for 24 h. (d) Quantification of the amount of gold retained in cells at different time after incubation with free media or media containing CB[7]. Cellular uptake experiments with each gold nanoparticle were repeated 3 times, and each replicate was measured 5 times by ICP-MS. Error bars represent the standard deviations of these measurements.

2.2.4 Tracking Exocytosis of CB[7]-complexed Nanoparticles Inside Cells Using Inductively Coupled Plasma Mass Spectrometry

The exocytosis of the nanoparticles was further quantified by using inductively coupled plasma mass spectrometry (ICP-MS). After 3 h incubation of AuNP-TBen (200 nM), the cells were completely washed off and replaced by fresh cell culture media or CB[7] (0.2 mM) containing media. The cells were then further incubated for different time intervals (0 h, 3 h, 12 h, and 24 h). The amount of the AuNPs retained by the cells was determined by using ICP-MS.

Retention of the AuNP-TBen inside cells treated by free cell culture media decreased to 34 % at

24 h while no significant change was observed for cells treated with CB[7] containing media

(Figure 2.5 (d)). Along with the observed TEM results, ICP-MS data shows that the treatment of the CB[7] on the cells effectively inhibited the exocytosis of the AuNP-TBen.

The effect of the AuNP end group on the exocytosis was further investigated and the retention of other AuNPs with different functional head groups in the cells was measured by using ICP-MS. AuNP-TTMA and AuNP-TMOH (Figure 2.6 (a) and (b)) exhibited no significant difference of exocytotic behavior for both free media and media containing CB[7], showing no effect of CB[7] on exocytosis of these AuNPs.

Figure 2.6: Effect of surface functional groups on exocytotic behavior of AuNPs. ICP-MS measurements of (a) AuNP-TTMA, (b) AuNP-TMOH, (c) AuNP-TMC6, and (d) AuNP-ADA. Quantification of exocytosis of the AuNPs was determined by analyzing ICP-MS on MCF-7 cell with same experimental condition carried out for the AuNP-TBen. Error bars represent the standard deviations of these measurements.

Regardless of CB[7] treatment, retention of the AuNP-TTMA and AuNP-TMOH decreased to ~ 62 % and ~ 70 % at 24 h, respectively. On the other hand, exocytosis of AuNPs including AuNP-ADA and AuNP-TMC6 was blocked when the cells were treated by the media containing CB[7] similar to the AuNP-TBen (Figure 2.6 (c) and (d)). Amount of the retained

AuNP-ADA and AuNP-TMC6 in the cells reduced to ~ 75 % and ~ 70 % at 24 h for the media treatment. Both AuNP-ADA and AuNP-TMC6 induced assemblies of the particle upon binding

40 with CB[7]. This result indicates that CB[7] itself does not affect cellular uptake of the particles and exocytosis of the particles was regulated due to the increased size of the assemblies.

2.2.5 Cellular Viability of the Induced Assemblies

Cellular proliferative activity was measured by the Alamar blue assay to evaluate possible toxicity that can arise from retained nanoparticles in the cells. As shown in Figure 2.7, all the nanoparticles exhibited no decrease in cell viability for the treatment of both free media and media containing CB[7]. This result indicates that the induced assemblies of the particles sequestered in endocytic vesicles do not affect cell viability.

Figure 2.7: Cytotoxicity of AuNPs. After 3h incubation of AuNPs (200 nM), MCF-7 cells were washed off and further incubated with media and media of CB[7] (0.2 mM) at 37 ºC for 24 h. As a control, cell viability of CB[7] (0.2 mM) was measured after 24 h incubation.

2.3 Summary and Future Outlook

In conclusion, we have demonstrated a strategy for regulating exocytosis of the internalized nanoparticles. Using a supramolecular host-guest system on the AuNPs induced the assemblies of the particles in the living cells, preventing their exocytosis without any observed cytotoxicity. This approach provides a potential strategy for prolonged retention of drug carriers within endosomes, enabling sustained therapeutic effect of the carriers. We are currently

41 exploring this strategy with AuNPs featuring prodrugs tethered with labile linkages that can be degraded by external stimuli. Additionally, this approach also will be applied to other nanomaterials with potential utility of their prolonged transplantation in a wide variety of cells for in vivo cellular tracking20 and tumor-targeted delivery of therapeutic systems.21

2.4 Synthesis of Materials and Experimental Methods

2.4.1 General

All the chemicals were purchased from Sigma-Aldrich or Fisher Scientific unless otherwise specified. The chemicals were used as received. AuNPs used in this work have been reported previously.22 1H NMR spectra were recorded at 400 MHz on a Bruker AVANCE 400 machine. A Hewlett-Packard 8452A UV-Vis spectrophotometer was used to record UV-Vis spectra. Dynamic light scattering (DLS) was measured by Zetasizer Nano ZS. The fluorescence from the Alamar blue assay was measured in a SpectraMax M5 microplate spectrophotometer.

2.4.2 Synthesis of Ligands and Their Characterization

Figure 2.8: Synthesis of benzyl-ligand (compound 6) for functionalizing AuNP-TBen.

Compound 2: 11-bromo-1-undecanol (8.22 g, 32.74 mmol) was dissolved in 80 mL 1:1 ethanol/toluene mixture. Triphenylmethanethiol (10.86 g, 39.29 mmol) dissolved in 80 mL 1:1 ethanol/toluene was added to 11-bromo-1-undecanol in solution. Then, sodium hydroxide (1.96 g,

49.11 mmol) was dissolved in 2 mL water and added to the mixture. The reaction mixture was stirred for 24 hours at 50oC. Upon completion, the reaction mixture was extracted twice with a satrated solution of sodium bicarbonate (NaHCO3) The organic layer was extracted, dried over

42 sodium sulfate (Na2SO4), and concentrated using a rotavapor. The crude product was purified by column chromatography over silica gel using hexane/ethyl acetate (1:1, v/v) as the eluent. The solvent was removed in vacuum to obtain compound 2 as colorless oil (Yield: 13.88 g, 95%). 1H

NMR (400 MHz, CDCl3, TMS) of Compound 2 : δ 7.48-7.40 (m, 6H, HAr-), 7.37-7.27 (m, 6H,

HAr-), 7.26-7.18 (m, 3H, HAr-), 3.65 (t, J = 6.7Hz, 2H,CH2OH), 2.16 (t, J = 7.2Hz, 2H,-CH2-),

1.66-1.52 (m, 2H, -SCH2CH2) , 1.44-1.12 (m, 16H, -CH2CH2OH + -(CH2)8 CH2OH).

Compound 3: Compound 2 (13.88 g, 31.1 mmol) in 150 mL dry dichloromethane

(DCM) was mixed with triethylamine (TEA) (4.72g, 6.48 mL, 46.65 mmol), followed by dropwise addition of methanesulfonyl chloride (3.92 g, 2.65mL, 34.21 mmol) in ice bath. After

30 minutes the reaction mixture was warmed to room temperature and stirred for 12 hr. After the reaction was completed (by TLC), solvent was evaporated. The compound was diluted again with

100 mL DCM and extracted with 100 mL 0.1 M HCl twice. The organic layer was collected, neutralized with a saturated NaHCO3 solution, and washed with water three times. Following extraction, the organic layer was dried over Na2SO4 and concentrated at reduced pressure. The crude product was purified by column chromatography over silica gel using hexane/ethyl acetate

(1:1, v/v) as the eluent. Solvent was removed in vacuum to obtain the mesylated compound as

1 light yellow oil (yield: 15 g, 92%). H NMR (400 MHz, CDCl3, TMS) of intermediate mesylation product: δ 7.48-7.40 (m, 6H, HAr-), 7.34-7.27 (m, 6H, HAr-), 7.26-7.19 (m, 3H, HAr-), 4.24 (t, J

= 6.8Hz, 2H, -CH2SO3CH3), 3.01 (s, 3H, -SO3CH3), 2.16 (t, J = 7.6Hz, -SCH2-), 1.76 (p, J =

6.8Hz, 2H, -CH2CH2SO3CH3), 1.41 (p, J = 7.2Hz, 4H, -SCH2CH2- + -SCH2CH2CH2-), 1.35-1.1

(m, 12H, -(CH2)6 CH2CH2SO3CH3).

To synthesize compound 3, NaOH (1.37 g, 34.3 mmol) solution (1 mL) was added to

99.24 mL of tetraethyleneglycol (TEG) (111.15 g, 57.22 mmol) and stirred for 2 hr at 80 °C. To this reaction mixture, 15 g of 11-(tritylthio)undecyl methanesulfonate was added and stirred for

48 hr at 100 °C. The product was extracted in hexane/ethyl acetate (4:1, v/v) six times. Then, the

43 organic layer was concentrated at reduced pressure and the crude product was purified by column chromatography over silica gel using ethyl acetate as the eluent. The solvent was removed in vacuum to obtain compound 3 as light yellow oil (yield: 15.28 g, 68%). 1H NMR (400 MHz,

CDCl3, TMS) of Compound 3: δ 7.47-7.40 (m, 6H, HAr-), 7.34-7.26 (m, 6H, HAr-), 7.25-7.19

(m, 3H, HAr-), 3.77-3.57 (m,16H, -CH2-(OCH2CH2)4-OH), 3.46 (t, J = 6.8 Hz, 2H, -CH2-

(OCH2CH2)4-OH), 2.95 (br, s, 1H, -TEG-OH), 2.15 (t, J = 7.2Hz, -SCH2-), 1.59 (p, J = 7.2Hz,

2H, -CH2CH2TEG-OH), 1.4 (p, J = 7.6Hz, 2H, -SCH2CH2-), 1.35-1.13(m, 14H, -(CH2)7

CH2CH2TEG-OH).

Compound 4: Triethylamine (3.26g, 4.49 mL, 32.2 mmol) was added to compound 3 (10 g, 16.1 mmol) in 100 mL dry DCM in an ice bath. Methanesulfonyl chloride (2.77 g, 1.87 mL,

24.1 mmol) was added dropwise to the reaction mixture in ice-bath. After 30 minutes the reaction mixture was warmed up to room temperature and stirred overnight. The reaction mixture was worked up and the organic layer was extracted. The extracted DCM layer was dried over Na2SO4 and concentrated at reduced pressure. The crude product was purified by column chromatography over silica gel using ethyl acetate as the eluent. Solvent was removed in vacuum to obtain

1 compound 4 as light yellow oil (yield 10.7 g, 95 %). H NMR (400 MHz, CDCl3, TMS) of

Compound 4: δ 7.44-7.37 (m, 6H, HAr-), 7.31-7.23 (m, 6H, HAr-), 7.22-7.16 (m, 3H, HAr-),

4.40-4.34 (m, 2H, -CH2OSO3CH3), 3.78-3.54 (m, 14H, CH2-(OCH2CH2)3-CH2CH2OSO3CH3),

3.44 (t, J = 6.8Hz, 2H, CH2-CH2-(OCH2CH2)3-), 3.07 (s, 3H, -OSO3CH3), 2.12 (t, J = 7.2Hz, 2H,

-SCH2-), 1.56 (p, J = 7.2Hz, 2H, -CH2CH2TEG-N(CH3)2), 1.38 (p, J=7.6Hz, 2H, -SCH2CH2-),

1.32-1.11 (m, 14H, -(CH2)7CH2CH2TEGOSO3 CH3).

Compound 5: Compound 4 (1.075 g, 1.53 mmol) was added to dimethylbenzylamine

(0.62 g, 0.7 ml, 4.6 mmol) in 10 mL ethanol. The reaction mixture was stirred at 40 oC for 72 hr.

After evaporating ethanol at reduced pressure, the light yellow residue was purified by successive washings with hexane (10 mL, 4 times) and hexane/diethylether (1:1 v/v, 10 mL, 6 times) and

44 then dried in high vacuum. The product formation was quantitative and was confirmed by NMR.

1 H NMR (400MHz, CDCl3, TMS) of Compound 5: δ 7.64-7.58 (m, 2H, HAr-), 7.38-7.32 (m, 9H,

HAr-), 7.24-7.17 (m, 6H, HAr-), 7.16-7.09 (m, 3H, HAr-), 4.9 (s, 2H, -CH2-C6H5), 3.94 (s, br, 2H,

-OCH2CH2N(CH3)2-), 3.8 (s, br, 2H, -OCH2CH2N(CH3)2-), 3.77-3.22 (m, 12H, -(OCH2CH2)3-

CH2CH2N(CH3)2-), 3.33 (t, J = 6.8Hz, 2H, -CH2CH2O-), 3.23 (s, 6H, -N(CH3)2-), 2.06 (t, J =

7.2Hz, 2H, -SCH2-), 1.51-1.42 (p, J = 6.8Hz, 2H, -CH2CH2O-), 1.36-1.28 (p, J = 7.6Hz, 2H, -

SCH2CH2-), 1.24-1.08 (m, 14H, -(CH2)7 CH2CH2O-).

Compound 6: An excess of trifluoroacetic acid (TFA, 20 equivalents, 3.69 g, 2.5 mL,

32.4 mmol) was added to compound 5 (1.2 g, 1.62 mmol) in 10 mL dry DCM. The color of the solution turned yellow upon addition of TFA. Then, triisopropylsilane (TIPS, 3 equivalents,

0.77g, 1 mL, 4.86 mmol) was added to the reaction mixture. The reaction mixture was stirred for

12 hr under N2 at room temperature. The solvent, most of TFA, and TIPS were evaporated under reduced pressure. The yellow residue was purified by repeated washing with hexane (10 mL, 4 times) and dried in high vacuum. The final product formation was quantitative and was confirmed

1 by NMR spectroscopy. H NMR (400 MHz, CDCl3, TMS) of Compound 6: δ 7.57-7.47 (m, 5H),

4.61 (s, 2H, -CH2-C6H5), 4.01 (s, br, 2H, -OCH2CH2N(CH3)2-), 3.74-3.48 (m, 14H, -(OCH2CH2)3-

CH2CH2N(CH3)2-), 3.41 (t, J = 6.8Hz, 2H, -CH2CH2O-), 3.14 (s, 6H, -N(CH3)2-), 2.52 (q, J =

7.2Hz, HSCH2-), 1.65-1.48 (m, 4H, -CH2CH2O-,+ HSCH2CH2-), 1.43-1.20 (m, 15H, -(CH2)7

13 CH2CH2O- + HS-). C NMR(400 MHz, CDCl3, TMS) of Compound 6: δ 132.92, 131.11,

129.39, 126.65, 116.69, 114.10, 71.51, 70.31, 70.21, 70.03, 69.92, 69.78, 64.59, 63.34, 50.69,

34.01, 29.49, 29.45, 29.37, 29.34, 29.18, 29.02, 28.31, 25.91, 24.60.

+ Figure 2.9: MALDI-MS spectrum of AuNP-TBenz. The molecular ion (MH ) was detected at m/z =498.

2.4.3 Synthesis of Benzyl-Ligand Protected Gold Nanoparticle (AuNP-TBenz)

The gold salt was purchased from Strem Chemicals Inc. We followed two-step method for synthesizing AuNP-TBen, where a gold nanoparticle core was synthesized followed by place- exchange with the ligand of interest. First, pentanethiol-coated AuNPs with core diameter ~2 nm were synthesized using the Brust-Schiffrin two-phase synthesis protocol.23,24 Subsequently,

Murray place-exchange method25 was followed to obtain the benzyl-ligand protected AuNPs.

Pentanethiol conjugated AuNPs (10 mg) and compound 6 (27 mg) was dissolved in a mixture of

5 mL dry DCM, and 1 mL methanol and stirred under N2 atmosphere for 72 hr at room temperature. Then, solvents were removed under reduced pressure and the resulting precipitate was washed with hexane (10 mL) three times and with DCM (10 mL) twice. Then the precipitate was dissolved in distilled water and dialyzed for 72 hr (membrane molecular weight cut-off

=10,000) to remove excess ligands, pentanethiol, acetic acid, and other salts present in the nanoparticle solution. After dialysis, the particle was lyophilized to yield a solid brownish product. The particles were then redispersed in deionized water. The presence of ligands on

1 AuNP was also confirmed by mass spectrometry (Figure 2.10). H NMR-spectra in D2O showed substantial broadening of the proton peaks with no sign of free ligands (Figure 2.12).

Figure 2.10: MALDI-MS spectrum of AuNP-TBenz. The molecular ion (MH+, m/z =498) was detected, and the disulfide ion formed by the benzyl ligand and the original pentanethiol was also detected at m/z 600.

Figure 2.11: Transmission electron micrograph of AuNP-TBen.

1 Figure 2.12: H-NMR of AuNP-TBen showing the ligand attachment on AuNP surface.

2.4.4 Monitoring CB[7]-Nanoparticle Complexation Using Proton and NOESY 2D NMR

AuNP-TMNH2 was chosen to run NMR experiments before and after CB[7]

1 complexation. 5 µM nanoparticle solution was prepared in D2O and H-NMR and NOESY 2D

NMR of AuNP-TMNH2 was obtained (Figure 2.13 and Figure 2.15). Mixing AuNP-TMNH2 with CB[7] yielded AuNP-TMNH2-CB[7], CB[7] peaks around 4.3, 5.5, and 5.7 ppm were appeared in the 1H-NMR and NOESY 2D NMR of AuNP-TMNH2-CB[7] (Figure 2.14 and

Figure 2.16).

1 Figure 2.13: H-NMR of AuNP-TMNH2 in D2O.

1 Figure 2.14: H-NMR of AuNP-TMNH2-CB[7] in D2O.

Figure 2.15: NOESY 2D NMR of AuNP-TMNH2.

Figure 2.16: NOESY 2D NMR of AuNP-TMNH2-CB[7] showing the host-guest interactions between CB[7] and AuNP-TMNH2 (black circles).

2.4.5 ICP-MS Sample Preparation and Measurements

ICP-MS measurements were performed on a Perkin Elmer Elan 6100. Operating conditions of the ICP-MS are listed below: RF power: 1200 W; plasma Ar flow rate: 15 L/min; nebulizer Ar flow rate: 0.96 L/min; isotopes monitored: 197Au; dwell time: 50 ms; nebulizer: cross flow; spray chamber: Scott. AuNPs (200 nM, 0.5 ml) were incubated with pre-seeded MCF-7 cell line in 24 well plates (20,000 cells/well). After 3 h incubation, cells were washed three times with

PBS buffer and then 0.5 mL of media or media of CB[7] (0.2 mM) was added to the cells. The wells of the plates then connected to a peristaltic pump which provides a continuous flow of the media or media of CB[7] to remove exocytosed nanoparticles. The cells were then incubated for different additional times (0 h, 3 h, 12 h, and 24 h). Cells were washed three times with PBS buffer and then a lysis buffer (300 l) was added to the cells. The resulting cell lysate was digested overnight using 3 mL of HNO3 and 1 mL of H2O2. On the next day, 3 mL of aqua regia was added and then the sample was allowed to react for another 2-3 h. The sample solution was then diluted to 100 mL with de-ionized water and aqua regia. The final AuNP sample solution contained 5% aqua regia. The AuNP sample solution was measured by ICP-MS under the operating conditions described above. Cellular uptake experiments with each gold nanoparticle were repeated 3 times, and each replicate was measured 5 times by ICP-MS. A series of gold standard solutions (20, 10, 5, 2, 1, 0.5, 0.2, 0 ppb) were prepared before each experiment. Each gold standard solution contained 5% aqua regia. The resulting calibration line was used to determine the gold amount taken up in the cells in each sample.

2.4.6 Preparation of Cellular TEM Samples and Their Measurements

For a preparation of cellular TEM samples, MCF-7 cells (100,000 cells per well in a 24 well plate) were seeded and incubated on 15 mm diameter Theramanox® coverslips (Nalge Nunc

International, NY) in 1 mL of serum containing media for 24 h prior to the experiment. The media was replaced by 0.5 mL of 200 nM AuNP-TBen in serum containing media and incubated

51 for 3 h. The cells were completely washed with PBS buffer three times and then 0.5 mL of media or media of CB[7] (0.2 mM) was added to the cells. After 24 h incubation, the cells were then fixed in 2 % glutaraldehyde with 3.75 % sucrose in 0.1 M sodium phosphate buffer (pH 7.0) for

30 min and then washed with 0.1 M PBS containing 3.75% sucrose three times over 30 min. The cells were postfixed in 1 % osmium tetroxide with 5 % sucrose in 0.05 M sodium phosphate buffer solution (pH 7.0) for 1 hr and then rinsed with distilled water three times. They were dehydrated in a graded series of acetone (10 % per step) and embedded in epoxy resin. The resin was polymerized at 70 °C for 12 h. Ultrathin sections (50 nm) obtained with a Reichert Ultracut E

Ultramicrotome and imaged under a JEOL 100S electron microscopy.

2.4.7 Cell Culture and Cytotoxicity Measurements

MCF-7 cells were grown in a cell culture flask using low glucose Dulbecco's Modified

Eagle Medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2. For cytotoxicity testing, MCF-7 cells were seeded at 20,000 cells in 0.2 mL per well in a 96-well plate 24 h prior to the experiment. During the experiment, old media was replaced by 0.2 mL of AuNPs (200 nM) in serum containing media and the cells were incubated for 3 h at 37°C in a humidified atmosphere of 5 % CO2. The cells were then completely washed with PBS buffer three times and media or media of CB[7] (0.2 mM) was added to the cell. After 24 h of incubation, the cells were then completely washed off and 10% Alamar Blue in serum containing media was added to each well and further incubated at 37 °C for 4 h. The cell viability was then determined by measuring the fluorescence intensity at 570 nm using a

SpectraMax M5 microplate spectrophotometer.

2.5. References

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CHAPTER 3

BINDING STUDIES OF CUCURBIT[7]URIL WITH GOLD NANOPARTICLES BEARING DIFFERENT SURFACE FUNCTIONALITIES

3.1 Introduction

The chemist`s motive for the synthesis of nanoscale structures through the non-covalent interactions has led the development of supramolecular chemistry.1-5 Molecular recognition,6,7 self-assembly,8 lock-key modality9 and reversibility10 are among the most important features that enabled supramolecular chemistry to be employed in many applications including but not limited to catalysis,11,12 delivery,13,14 sensing,15-17 and imaging.18 Supramolecular chemistry has been described as a lego-chemistry in which each lego piece represents a molecular building block that are held together by intermolecular interactions including electrostatic interactions,19 hydrogen bonding,20 π–π interactions,21 ion-dipole interactions,22,23 hydrophobic or solvophobic effects.24

The choice of building blocks in host-guest chemistry is critical as they determine the selectivity, reversibility and tunability of the supramolecular architectures. Gold nanoparticles

(AuNPs) are useful platforms to play either host or guest role due to their unique features such as tunable core size,25 monodispersity,26 large surface to volume ratio,27 and easy surface functionalization.28,29 Rotello et al. have used AuNP as a host scaffold to encapsulate hydrophobic guest molecules into the engineered monolayer.30 On the other hand, proper surface functionalization of AuNPs renders them to be recognized by macrocyclic compound such as crown ethers,31,32 calixarenes,33,34 cyclodextrins,35,36 pillararenes37 and cucurbiturils.38-40

Cucurbit[7]uril (CB[7])41,42 is a member of cucurbituril family with a heptameric macrocyclic structure self-assembled from an acid catalyzed condensation reaction of glycoluril and formaldehyde. CB[7] is a water soluble molecule with well-established host properties.43,44 It has a pumpkin-shaped structure45 consisting of a hydrophobic cavity of 7.3 Å diameter and two identical carbonyl-laced portals. While the hydrophobic interior provides an encapsulation site for

56 hydrophobic guest molecules,46 the polar carbonyl groups at the portals allow CB[7] to bind ions47 and charged molecules48,49 by forming charge-dipole and/or hydrogen bonding interactions.

In this work, we demonstrated the host-guest interactions between CB[7] molecule and

AuNPs bearing ligands with a monomethyl-benzylammonium (MMBA) or dimethylbenzylammonium (DMBA) head group. We used AuNPs with core diameters of ~2.5 nm as the guest scaffold while CB[7] served as a host molecule. We kept the monolayer design same with the exception of the head group functionality. We have investigated the changes in the binding event considering two features: 1) the effect of different functionalities at the para position of benzene ring and 2) the effect of carrying a permanent positive charge on the head group.

3.2 Result and Discussion

We have designed a series of nanoparticles with benzylammonium terminal group possessing different functionalities at the para position of the benzene ring, which is the least sterically hindered position for binding process based on the reported crystal structures of cucurbiturils.50,51 Eight DMBA and five MMBA derivatives (Figure 3.1) were synthesized to represent a wide range of functionality that brings at least one of the effects into the system: hydrophobicity, hydrophilicity, electron-withdrawing, electron-donating, and bulkiness.

Figure 3.1: Structures of DMBA and MMBA derivatives used in the study. The schematic represents the binding event between AuNP and CB[7].

3.2.1 Isothermal Titration Calorimetry (ITC) Measurements of NP1-NP8 and Thermodynamic Values

Isothermal titration calorimetry (ITC) is an extremely powerful and sensitive technique that measures the heat taken up or evolved depending on the nature of the reaction when one solution is titrated against the other solution.52 ITC experiments were performed at 30 oC in 5 mM phosphate buffer at pH 7.4. During the experiment, the reference cell was filled with only 5 mM phosphate buffer and the sample cell was containing the AuNP solution (1 µM). Then, CB[7] solution (2 mM) was titrated into the sample cell. All AuNPs showed multiple host/guest bindings while free ligand (DMBA) showed a binding stoichiometry of 1:1.

Binding affinities of DMBA head group bearing AuNPs (NP1-NP8) were in the order of

104-6 M-1 (Table 1). NP1 having H atom at the para position showed a binding constant of

1.60.32 x 105 M-1 (Figure 3.2).

Figure 3.2: ITC measurements of NP1, NP3, and NP8 bearing DMBA head group with H, CH2NH2 and NO2 functionalities at the para position of the benzene ring showing similar binding constants.

For the NPs with electron donating groups including CH3, CH2NH2, and OMe, binding affinities were similar to NP1 except the NP2 with a binding constant of 6.160.15x 104 M-1. The

NP2 has tBu group located at the para position and although tBu functionality has weakly electron donating property, it brings steric hindrance into the system and thus resulting in a lower

58 binding affinity. On the other hand, the NP3 with CH2NH2 functionality demonstrated slightly higher association constant compared to NP1 (Figure 3.2 and Table 3.1). This could be originated from the hydrogen bonding interactions between the carbonyl portals of CB[7] and amine part of

CH2NH2 functionality. The NP4 with weakly electron donating methyl group exhibited a binding constant of 1.260.18 x 105 M-1 which is slightly lower than that of NP1 as expected.

Hydrophobic interactions within the inner cavity of cucurbituril play an important role in binding event. Electron donating groups make the benzene ring less electron deficient and therefore weaken the interaction of benzene ring with the inner cavity of CB[7]. However, the same effect was not observed for NP5 carrying a stronger electron donating ‘methoxy group’.

Thermodynamic values for NP1-8 were listed in Table 1.Titration of CB[7] into NP solution represented an exothermic process with negative enthalpy change (H) resulting from the robust van der Waals forces inside the hydrophobic cavity of CB[7] as well as additional forces including hydrogen bonding and ion-dipole interactions between the carbonyl portals of

CB[7] and guest molecules. As the sites available on the surface of the AuNPs become progressively occupied by CB[7] during the titration, the exothermicity of the peaks decreases and eventually saturates (Figure 2). H values for NP1-NP5 were found similar to each other and not very large; they were changing between -6.6 and -11.29 kcal/mol. From the entropic standpoint, complexation of CB[7] with NP1, NP2, and NP3 is less favorable as -TS values were positive. However, overall entropy of complexation for NP4 and NP5 is slightly favorable as -TS values were negative but very close to zero.

Table 3.1: Thermodynamic data of NP1-8 for binding to CB[7]

AuNPs with electron withdrawing groups at the para position rendered the benzene ring more electron deficient, resulting in more stable inclusion complexes. Fluorine, nitro, and nitrile functionalities (NP6, NP7, and NP8) were used to bring electron withdrawing property into the system. All the three NPs showed somewhat higher binding towards CB[7] molecule compared to

NP1 whereas the binding for NP6 and NP7 was not greatly so. However, NP8 bearing CN group exhibited an association constant of 1.320.69 x 106 M-1 which is about 10 times higher than that of NP1. The negative H values for these NPs indicated an exothermic binding process.

Numerically they were all quite similar to each other but smaller than that of NPs carrying electron donating groups. S values were positive indicating favorable complexation between

CB[7] and NPs bearing electron withdrawing groups. The complexation process for NP1-NP8 was found spontaneous which is confirmed by negative Gibbs free energy change (G).

3.2.2 ITC Measurements of NP9-NP13 and Effects of Surface Functionality on Binding Affinity

Binding experiments of MMBA derivatives revealed a drop in the binding affinities; Ka values were mostly in the order of 104 M-1. This result indicated that a permanent positive charge played an essential role in the binding event as ion-dipole interactions between CB[7] and AuNP were dominating over the hydrogen bonding interactions. NP9 showed no complexation to CB[7] while both the absence of positive charge and bulkiness of the tBu group prevented the

60 recognition event (Table 3.2). On the other hand, NP11 with the methoxy group showed an

5 -1 unexpected Ka of 1.200.15 x 10 M . H values for NP9-13 were very similar to each other, and negative showing exothermic binding events. Complexation between CB[7] and NP9-13 was found spontaneous as indicted by negative G.

Table 3.2: Thermodynamic data of NP9-13 for binding to CB[7]

3.2.3 Monitoring Binding of Free Ligand to the Host Molecule

We have synthesized a ligand containing a DMBA head group same as in NP1 to investigate the behavior of free ligand. ITC measurement revealed a binding constant of

3.690.25 x 105 M-1 when 100 µM of ligand solution was titrated with 2 mM CB[7] in 5 mM phosphate buffer Figure 3.3). This value was in the same order with the binding constant of NP1

5 -1 (Ka= 1.60.32 x 10 M ) but still 2.3 times higher in magnitude. This result showed that attaching the ligand on AuNP showed no dramatic decrease in the binding process between CB[7] and head group of the ligand.

Figure 3.3: a) Schematic representation of binding between DMBA ligand and CB[7]. b) ITC measurement indicated a 1:1 binding stoichiometry between free ligand and CB[7].

3.3 Summary and Future Outlook

We have used isothermal titration calorimetry to monitor the binding interactions of

CB[7] with thirteen different AuNPs. Binding studies revealed that NP1-NP8 possessing DMBA head groups showed a higher binding affinity towards CB[7] compared to NP9-NP13 having

MMBA head groups. Insertion of tBu group at the para position of the benzene ring led to a lower binding due to the steric effect. Although not very strong difference in Ka constants was observed between electron donating and withdrawing groups, a slight decrease in the binding was monitored for electron donating groups compared to withdrawing derivatives. Methoxy functionality behaved differently than expected, computational studies may help to understand the process better.

3.4 Synthesis of Materials and Experimental Methods

3.4.1 Synthesis of Ligand

Dimethyl OMe and Dimethyl NO2 ligands and NPs were synthesized according to the reported literature.53 We have followed the reported literature for the synthesis of AuNP core and place exchange reactions.54-56

3.4.1.2 Synthesis of Dimethyl-H Ligand for NP1

MeHN

O O Ph3CS ( )9 ( O )3 OMs Ph3CS ( )9 ( O )3 N DIPEA, THF, 60 ºC Compound 1 Compound 2

Figure 3.4: Synthesis of compound 2 from compound 1.

To a solution of compound 1 (750 mg, 1.1 mmol) in THF (6.3 mL) was added a solution of N-methyl-benzylamine (389 mg, 3.2 mmol) and diisopropylamine (531 µL, 3.2 mmol). After being stirred at 60 °C for 2 days, EtOAc was added. The mixture was washed with sat. NaHCO3 and brine, and dried over Na2SO4. After concentration, the residue was purified by flash chromatography over silica gel with EtOAc–MeOH (1:0 to 9:1) to give the desired compound as

1 yellow oil (692 mg, 89 %). H-NMR (400 MHz, CDCl3) 1.18-1.44 (16H, m, -CH2-), 1.55-1.63

(2H, m, -CH2-), 2.15 (2H, t, J = 7.3 Hz, -SCH2-), 2.28 (3H, s, -NCH3), 2.65 (2H, t, J = 6.1 Hz, -

NCH2-), 3.45 (2H, t, J = 6.8 Hz, -OCH2-), 3.58-3.68 (16H, m, -OCH2-, -NCH2Ar-), 7.20-7.32

(14H, t, m, Ar), 7.42-7.44 (6H, m, Ar).

OTs O MeOTs Ph3CS ( )9 ( O )3 N O Ph3CS ( )9 ( O )3 N EtOH, CH2Cl2 Compound 2 40 ºC Compound 3

Figure 3.5: Synthesis of compound 3 from compound 2.

To a solution of compound 2 (300 mg, 0.41 mmol) in EtOH (4.1 mL) and CH2Cl2 (2.01 mL) was added methyl-p-toluenesulfonate (229 mg, 1.23 mmol). After being stirred at 40 °C for

15 h, the mixture was concentrated in vacuo. The residue was washed ten times with n-hexane– diethylether (3:1). The solvent was evaporated to give the desired compound as pale yellow oil

1 (390 mg, >99%). H-NMR (400 MHz, CDCl3) 1.14-1.42 (16H, m, -CH2-), 1.53-1.57 (2H, m, -

+ CH2-), 2.15 (2H, t, J = 7.3 Hz, -SCH2-), 2.36 (3H, s, Ar-CH3), 3.26 (6H, s, -N (CH3)2-), 3.41 (2H, t, J = 6.9 Hz, -OCH2-), 3.52-3.68 (12H, m, -OCH2-), 3.85 (2H, br, -OCH2-), 4.04 (2H, br, -

+ + N CH2-), 4.82 (2H, s, -N CH2Ar-), 7.16-7.49 (20H, m, Ar), 7.61 (2H, d, J = 8.0 Hz, Ar), 7.81

(2H, d, J = 8.1 Hz, Ar).

OTs OTs O TFA, (i-Pr)3SiH O Ph3CS ( )9 ( O )3 N HS ( )9 ( O )3 N CH2Cl2, rt Compound 3 Compound 4

Figure 3.6: Synthesis of compound 4 from compound 3.

To a solution of compound 3 (300 mg, 0.32 mmol) in CH2Cl2 (4.0 mL) was added TFA

(1.5 mL). After being stirred at rt for 5 min, triisopropylsilane (0.3 mL) was added. After being stirred at rt for 1 h, the mixture was concentrated in vacuo. The residue was washed six times with n-hexane. The solvent was evaporated to give the desired compound as pale yellow oil (210

1 mg, 98%). H-NMR (400 MHz, CDCl3) 1.28-1.44 (14H, m, -CH2-), 1.53-1.66 (4H, m, -CH2-),

+ 2.39 (3H, s, Ar-CH3), 2.54 (2H, q, J = 7.5 Hz, -SCH2-), 3.20 (6H, s, -N (CH3)2-), 3.46 (2H, t, J =

+ + 6.9 Hz, -OCH2-), 3.57-3.80 (14H, m, -OCH2-), 4.08 (2H, br, -N CH2-), 4.67 (2H, s, -N CH2Ar-),

7.20 (2H, d, J = 8.0 Hz, Ar), 7.42-7.56 (5H, m, Ar), 7.78 (2H, d, J = 8.0 Hz, Ar).

3.4.1.3 Synthesis of Dimethyl-t-Bu Ligand for NP2

MeHN t-Bu O O Ph3CS ( )9 ( O )3 OMs Ph3CS ( )9 ( O )3 N DIPEA, THF, 60 ºC Compound 1 Compound 2 t-Bu

Figure 3.7: Synthesis of compound 2 from compound 1.

To a solution of compound 1 (2.0 g, 2.85 mmol) in THF (17 mL) was added a solution of

N-methyl-4-(tert-butyl)-benzylamine (1.27 g, 7.1 mmol) and diisopropylamine (1.17 mL, 7.1

64 mmol). After being stirred at 60 °C for 2 days, the mixture was concentrated in vacuo. The residue was dissolved in EtOAc. The mixture was washed with sat. NaHCO3 and brine, and dried over Na2SO4. After concentration, the residue was purified by flash chromatography over silica gel with EtOAc–MeOH (1:0 to 95:5) to give the desired compound as yellow oil (1.6 g, 72 %).

H-NMR (400 MHz, CDCl3) 1.18-1.31 (14H, m, -CH2-), 1.33 (9H, m, -C(CH3)3), 1.37-1.44 (2H, m, -CH2-), 1.54-1.63 (2H, m, -CH2-), 2.15 (2H, t, J = 7.3 Hz, -SCH2-), 2.28 (3H, s, -NCH3), 2.64

(2H, t, J = 6.1 Hz, -NCH2-), 3.46 (2H, t, J = 6.8 Hz, -OCH2-), 3.54-3.67 (16H, m, -OCH2-, -

NCH2Ar-), 7.20-7.35 (13H, t, J = 8.7 Hz, Ar), 7.42-7.44 (6H, m, Ar). ESI-MS: m/z calculated for

+ C50H72NO4S [M+H] 782.5; found: 783.1.

OTs O MeOTs Ph3CS ( )9 ( O )3 N O Ph3CS ( )9 ( O )3 N EtOH, CH Cl Compound 2 t-Bu 2 2 Compound 3 40 ºC -t B u

Figure 3.8: Synthesis of compound 3 from compound 4.

To a solution of compound 2 (363 mg, 0.46 mmol) in EtOH (4.6 mL) and CH2Cl2 (2.3 mL) was added methyl-p-toluenesulfonate (259 mg, 1.39 mmol). After being stirred at 40 °C for

24 h, the mixture was concentrated in vacuo. The residue was washed ten times with n-hexane and five times with n-hexane–CH2Cl2 (95:5). After concentration, the residue was dissolved in

EtOAc. The mixture was washed with H2O and dried over Na2SO4. The solvent was evaporated

1 to give the desired compound as pale yellow oil (423 mg, 94%). H-NMR (400 MHz, CDCl3)

1.13-1.41 (25H, m, -CH2-, -C(CH3)3), 1.51-1.57 (2H, m, -CH2-), 2.14 (2H, t, J = 6.9 Hz, -SCH2-),

+ 2.35 (3H, s, Ar-CH3), 3.25 (6H, s, -N (CH3)2-), 3.36-3.64 (14H, m, -OCH2-), 3.84 (2H, br, -

+ + OCH2-), 4.01 (2H, br, -N CH2-), 4.76 (2H, s, -N CH2Ar-), 7.15-7.52 (21H, m, Ar), 7.82 (2H, d, J

+ = 7.4 Hz, Ar). ESI-MS: m/z calculated for C51H74NO4S [M] 796.5; found: 797.1.

OTs OTs O TFA, (i-Pr)3SiH O Ph3CS ( )9 ( O )3 N HS ( )9 ( O )3 N CH2Cl2, rt t-Bu t-Bu Compound 3 Compound 4

Figure 3.9: Synthesis of compound 4 from compound 3.

To a solution of compound 3 (371 mg, 0.40 mmol) in CH2Cl2 (5.0 mL) was added TFA

(2.0 mL). After being stirred at rt for 10 min, triisopropylsilane (3.0 mL) was added. After being stirred at rt for 6 h, the mixture was concentrated in vacuo. The residue was washed eight times with n-hexane. The solvent was evaporated to give the desired compound as pale yellow oil (255

1 mg, 99%). H-NMR (400 MHz, CDCl3) 1.27-1.43 (23H, m, -CH2-), 1.53-1.66 (4H, m, -CH2-),

+ 2.38 (3H, s, Ar-CH3), 2.54 (2H, q, J = 7.5 Hz, -SCH2-), 3.21 (6H, s, -N (CH3)2-), 3.44 (2H, t, J =

+ + 6.9 Hz, -OCH2-), 3.55-3.77 (14H, m, -OCH2-), 4.07 (2H, br, -N CH2-), 4.65 (2H, s, -N CH2Ar-),

7.20 (2H, d, J = 8.0 Hz, Ar), 7.45-7.50 (4H, m, Ar), 7.80 (2H, d, J = 8.0 Hz, Ar). ESI-MS: m/z

+ calculated for C32H60NO4S [M] 554.4; found: 555.2.

3.4.1.4 Synthesis of Dimethyl-CH2NH2 Ligand for NP3

Boc O Me2N LiAlH4 2 Me2N NHBoc CN THF CH2Cl2 50 ºC rt Compound 1 Compound 2

Figure 3.10: Synthesis of compound 2 from compound 1.

To a solution of compound 1 (500 mg, 3.1 mmol) in THF (9.4 mL) was added a 2.0 M solution of lithiumaluminiumhydride (4.68 mL, 3.0 mmol) at 0 °C. After being stirred at 0 °C for

15 min, the reaction mixture was warmed to rt. After being stirred at rt for 30 min, the reaction mixture was quenched by slow addition of H2O (0.4 mL), 2 M NaOH (0.4 mL), and H2O (1.2 mL). The resulting mixture was filtered through Celite pad. After concentrarion, the mixture was

66 dissolved in CH2Cl2 (15 mL). Boc2O (1.0 g, 4.7 mmol) were added to the reaction mixture. After being stirred at rt for 3 h, the mixture was concentrated in vacuo. The residue was purified by flash chromatography over silica gel with EtOAc–triethylamine (99:1) to give the desired

1 compound as colorless oil (308 mg, 74 %). H-NMR (400 MHz, CDCl3) 1.48 (9H, s, -C(CH3)3-),

2.25 (6H, m, -N(CH3)2-), 3.42 (2H, s, -NCH2Ar), 4.32 (2H, d, J = 5.5 Hz, -NHCH2Ar-), 4.90 (1H,

+ br s, -NH-), 7.23-7.29 (4H, m, Ar). MALDI-MS: m/z calculated for C15H25N2O2 [M+H] 265.191; found: 265.340.

Me2N NHBoc OTs O O Ph3CS ( )9 ( O )3 OTs Ph3CS ( )9 (O )3 N MeCN, 60 ºC NHBo Compound 3 Compound 4 c

Figure 3.11: Synthesis of compound 4 from compound 3.

To a solution of compound 3 (353 mg, 0.45 mmol) in MeCN (3.0 mL) was added a solution of compound 2 (240 mg, 0.91 mmol). After being stirred at 50 °C for 1 day and 65 °C for

2 days, the mixture was concentrated in vacuo. The residue was washed five times with n-hexane and twice with n-hexane–diethylether (3:1). The solvent was evaporated to give the desired

1 compound as pale yellow oil (471 mg, >99%). H-NMR (400 MHz, CDCl3) 1.13-1.33 (14H, m, -

CH2-), 1.34-1.44 (2H, m, -CH2-), 1.49 (9H, m, -C(CH3)3-), 1.53-1.59 (2H, m, -CH2-), 2.14 (2H, t,

+ J = 7.3 Hz, -SCH2-), 2.36 (3H, s, Ar-CH3), 3.25 (6H, s, -N (CH3)2-), 3.42 (2H, t, J = 6.9 Hz, -

+ OCH2-), 3.53-3.68 (12H, m, -OCH2-), 3.85 (2H, br, -OCH2-), 4.02 (2H, br, -N CH2-), 4.35 (2H, d,

+ -NHCH2-), 4.83 (2H, s, -N CH2Ar-), 5.09 (1H, br s, -NH-), 7.19-7.35 (13H, m, Ar), 7.41-7.44

(6H, m, Ar), 7.58 (2H, d, J = 8.0 Hz, Ar) , 7.83 (2H, d, J = 8.1 Hz, Ar). MALDI-MS: m/z

+ calculated for C53H77N2O6S [M] 869.550; found: 869.954.

OTs OTs O TFA, (i-Pr)3SiH O Ph3CS ( )9 ( O )3 N HS ( )9 ( O )3 N EDT, H2O NHBoc NH3 Otfa CH2Cl2, rt Compound 4 Compound 5

Figure 3.12: Synthesis of compound 5 from compound 4.

To a solution of compound 4 (400 mg, 0.38 mmol) in CH2Cl2 (0.5 mL) was added a mixture of TFA (18.5 mL), H2O (0.5 mL), 1,2-ethanedithiol (0.5 mL), and triisopropylsilane (0.5 mL) was added at 0 °C. After being stirred at rt for 1 h, the mixture was concentrated in vacuo.

The residue was washed five times with n-hexane, five times with n-hexane–diethylether (3:1), and ten times with diethylether. The solvent was evaporated to give the desired compound as pale

1 yellow oil (267 mg, 88%). H-NMR (400 MHz, CDCl3) 1.26-1.43 (14H, m, -CH2-), 1.53-1.65

+ (4H, m, -CH2-), 2.36 (3H, s, Ar-CH3), 2.53 (2H, q, J = 7.5 Hz, -SCH2-), 3.05 (6H, s, -N (CH3)2-),

+ 3.43 (2H, t, J = 6.8 Hz, -OCH2-), 3.53-3.60 (14H, m, -OCH2-), 3.83 (2H, br, -NH3 CH2-), 4.02

+ + (2H, br, -N CH2-), 4.67 (2H, s, -N CH2Ar-), 7.19 (2H, d, J = 7.9 Hz, Ar), 7.41 (4H, m, Ar), 7.74

+ (2H, d, J = 7.9 Hz, Ar). MALDI-MS: m/z calculated for C29H55N2O4S [M] 527.388; found:

527.417.

3.4.1.5 Synthesis of Dimethyl-Me Ligand for NP4

MeHN Me O O Ph3CS ( )9 ( O )3 OMs Ph3CS ( )9 ( O )3 N DIPEA, THF, 60 ºC Compound 1 Compound 2 Me

Figure 3.13: Synthesis of compound 2 from compound 1.

To a solution of compound 1 (701 mg, 1.0 mmol) in THF (3.1 mL) was added a solution of N-methyl-4-methylbenzylamine (338 mg, 2.5 mmol) and diisopropylamine (413 µL, 2.5 mmol) in THF (5.0 mL). After being stirred at 60 °C for 2 days, the mixture was concentrated in

68 vacuo. The residue was purified by flash chromatography over silica gel with EtOAc–MeOH (1:0 to 9:1) to give the desired compound as yellow oil (313.4 mg, 42 %). 1H-NMR (400 MHz,

CDCl3) 1.18-1.30 (14H, m, -CH2-), 1.30-1.62 (4H, m, -CH2-), 2.15 (2H, t, J = 7.3 Hz, -SCH2-),

2.27 (3H, s, -NCH3), 2.64 (2H, t, J = 6.1 Hz, -NCH2-), 3.45 (2H, t, J = 6.8 Hz, -OCH2-), 3.54-3.67

(16H, m, -OCH2-, -NCH2Ar-), 7.13 (2H, t, J = 7.8 Hz, Ar), 7.20-7.31 (11H, m, Ar), 7.41-7.44

+ (6H, m, Ar), ESI-MS: m/z calculated for C47H66NO4S [M+H] 740.5; found: 740.9.

OTs O MeOTs Ph3CS ( )9 ( O )3 N O Ph3CS ( )9 ( O )3 N Me EtOH, CH2Cl2 Compound 2 40 ºC Compound 3 Me

Figure 3.14: Synthesis of compound 3 from compound 2.

To a solution of compound 2 (300 mg, 0.41 mmol) in EtOH (4.0 mL) and CH2Cl2 (2.0 mL) was added methyl-p-toluenesulfonate (235 mg, 1.22 mmol). After being stirred at 40 °C for

14 h, the mixture was concentrated in vacuo. The residue was washed ten times with n-hexane– diethylether (1:1). The solvent was evaporated to give the desired compound as pale yellow oil

1 (384 mg, >99%). H-NMR (400 MHz, CDCl3) 1.17-1.32 (14H, m, -CH2-), 1.35-1.59 (4H, m, -

CH2-), 2.15 (2H, t, J = 7.3 Hz, -SCH2-), 2.36 (3H, s, Ar-CH3), 2.40 (3H, s, Ar-CH3), 3.23 (6H, s, -

+ N (CH3)2-), 3.41 (2H, t, J = 6.9 Hz, -OCH2-), 3.52-3.69 (12H, m, -OCH2-), 3.84 (2H, br, -OCH2-

+ + ), 4.03 (2H, br, -N CH2-), 4.74 (2H, s, -N CH2Ar-), 7.16-7.31 (13H, m, Ar), 7.41-7.47 (8H, m,

+ Ar), 7.80 (2H, d, J = 8.2 Hz, Ar). ESI-MS: m/z calculated for C48H68NO4S [M] 754.5; found:

755.0.

OTs OTs O TFA, (i-Pr)3SiH O Ph3CS ( )9 ( O )3 N HS ( )9 ( O )3 N CH2Cl2, rt Me Me Compound 3 Compound 4

Figure 3.15: Synthesis of compound 4 from compound 3.

To a solution of compound 3 (336 mg, 0.36 mmol) in CH2Cl2 (3.0 mL) was added TFA

(1.0 mL). After being stirred at rt for 10 min, triisopropylsilane (1.0 mL) was added. After being stirred at rt for 1 h, the mixture was concentrated in vacuo. The residue was washed ten times with n-hexane. The solvent was evaporated to give the desired compound as pale yellow oil (161

1 mg, 70%). H-NMR (400 MHz, CDCl3) 1.28-1.44 (14H, m, -CH2-), 1.53-1.66 (4H, m, -CH2-),

2.38 (3H, s, Ar-CH3), 2.42 (3H, s, Ar-CH3), 2.54 (2H, q, J = 7.5 Hz, -SCH2-), 3.20 (6H, s, -

+ + N (CH3)2-), 3.43 (2H, t, J = 6.9 Hz, -OCH2-), 3.54-3.78 (14H, m, -OCH2-), 4.07 (2H, br, -N CH2-

+ ), 4.65 (2H, s, -N CH2Ar-), 7.20 (2H, d, J = 8.0 Hz, Ar), 7.27 (2H, d, J = 8.0 Hz, Ar), 7.42 (2H, d,

+ J = 8.0 Hz, Ar), 7.79 (2H, d, J = 8.0 Hz, Ar). ESI-MS: m/z calculated for C29H54NO4S [M] 512.4; found: 512.5.

3.4.1.6 Synthesis of Dimethyl-F Ligand for NP6

MeHN F O O Ph3CS ( )9 ( O )3 OMs Ph3CS ( )9 ( O )3 N DIPEA, THF, 60 ºC F Compound 1 Compound 2

Figure 3.16: Synthesis of compound 2 from compound 1.

To a solution of compound 1 (701 mg, 1.0 mmol) in THF (5.0 mL) was added a solution of N-methyl-4-fluorobenzylamine (418 mg, 3.0 mmol) and diisopropylamine (496 µL, 3.0 mmol) in THF (5.0 mL). After being stirred at 60 °C for 2 days, the mixture was concentrated in vacuo.

The residue was purified by flash chromatography over silica gel with EtOAc–MeOH (1:0 to 9:1)

1 to give the desired compound as yellow oil (455 mg, 61 %). H-NMR (400 MHz, CDCl3) 1.17-

1.32 (14H, m, -CH2-), 1.35-1.61 (4H, m, -CH2-), 2.15 (2H, t, J = 7.4 Hz, -SCH2-), 2.26 (3H, s, -

NCH3), 2.63 (2H, t, J = 6.0 Hz, -NCH2-), 3.45 (2H, t, J = 6.8 Hz, -OCH2-), 3.51-3.78 (16H, m, -

OCH2-, -NCH2Ar-), 7.00 (2H, t, J = 8.7 Hz, Ar), 7.20-7.43 (17H, m, Ar). ESI-MS: m/z calculated

+ for C46H63FNO4S [M+H] 744.5; found: 745.0.

OTs O MeOTs Ph3CS ( )9 ( O )3 N O Ph3CS ( )9 ( O )3 N F EtOH, CH2Cl2 Compound 2 40 ºC Compound 3 F

Figure 3.17: Synthesis of compound 3 from compound 2.

To a solution of compound 2 (443 mg, 0.55 mmol) in EtOH (2.7 mL) and CH2Cl2 (1.4 mL) was added methyl-p-toluenesulfonate (305 mg, 1.65 mmol). After being stirred at 40 °C for

27 h, the mixture was concentrated in vacuo. The residue was washed seven times with diethylether. The solvent was evaporated to give the desired compound as pale yellow oil (370

1 mg, 61%). H-NMR (400 MHz, CDCl3) 1.17-1.32 (14H, m, -CH2-), 1.36-1.43 (2H, m, -CH2-),

1.51-1.58 (2H, m, -CH2-), 2.15 (2H, t, J = 7.3 Hz, -SCH2-), 2.36 (3H, s, Ar-CH3), 3.24 (6H, s, -

+ + N (CH3)2-), 3.41 (2H, t, J = 6.9 Hz, -OCH2-), 3.52-3.77 (14H, m, -OCH2-), 3.99 (2H, br, -N CH2-

+ ), 4.86 (2H, s, -N CH2Ar-), 7.10 (2H, t, J = 8.0 Hz, Ar), 7.16-7.31 (11H, m, Ar), 7.41-7.44 (6H, m, Ar), 7.65 (2H, dd, J = 8.0, 5.4 Hz, Ar) , 7.79 (2H, d, J = 8.1 Hz, Ar). ESI-MS: m/z calculated

+ for C47H65FNO4S [M] 758.5; found: 758.8.

OTs OTs O TFA, (i-Pr)3SiH O Ph3CS ( )9 ( O )3 N HS ( )9 ( O )3 N CH2Cl2, rt F F Compound 3 Compound 4

Figure 3.18: Synthesis of compound 4 from compound 3.

To a solution of compound 3 (371 mg, 0.40 mmol) in CH2Cl2 (5.0 mL) was added TFA

1 mg, 99%). H-NMR (400 MHz, CDCl3) 1.28-1.42 (14H, m, -CH2-), 1.52-1.67 (4H, m, -CH2-),

+ 2.38 (3H, s, Ar-CH3), 2.54 (2H, q, J = 7.3 Hz, -SCH2-), 3.24 (6H, s, -N (CH3)2-), 3.42 (2H, t, J =

+ + 6.8 Hz, -OCH2-), 3.59-3.78 (14H, m, -OCH2-), 4.05 (2H, br, -N CH2-), 4.79 (2H, s, -N CH2Ar-),

7.14-7.21 (4H, m, Ar), 7.63 (2H, dd, J = 8.5, 6.0 Hz, Ar), 7.79 (2H, d, J = 8.1 Hz, Ar). ESI-MS:

+ m/z calculated for C28H51FNO4S [M] 516.4; found: 516.4.

3.4.1.7 Synthesis of Dimethyl-CN Ligand for NP7

N OTs O CN O O Ph3CS ( )9 ( O )3 O S Ph3CS ( )9 ( O )3 N O CH3CN, reflux Compound 1 Compound 2 CN

Figure 3.19: Synthesis of compound 2 from compound 1.

To a solution of compound 1 (500 mg, 0.64 mmol) in CH3CN (10 mL) was added a solution of 4-(dimethylaminomethyl)benzonitrile (310 mg, 1.93 mmol) CH3CN (10 mL), and the mixture was stirred under reflux for 64 h. After concentration, the residue was washed three times with ethyl ether and seven times with n-hexane. The solvent was evaporated to give the desired

1 compound as pale yellow oil (596 mg, 99%). H-NMR (400 MHz, CDCl3) 1.10-1.31 (14H, m, -

CH2-), 1.37-1.44 (2H, m, -CH2-), 1.51-1.58 (2H, m, -CH2-), 2.15 (2H, t, J = 7.3 Hz, -SCH2-), 2.38

+ (3H, s, Ar-CH3) 3.32 (6H, s, -N (CH3)2-), 3.41 (2H, t, J = 6.9 Hz, -OCH2-), 3.52-3.70 (12H, m, -

+ + OCH2-), 3.85-3.87 (2H, m, -OCH2-), 4.04-4.06 (2H, m, -N CH2-), 5.06 (2H, s, -N CH2Ar-), 7.19-

7.32 (11H, m, Ar), 7.41-7.44 (6H, m, Ar), 7.74 (2H, d, J = 8.4 Hz, Ar), 7.82 (2H, d, J = 8.1 Hz,

+ Ar), 7.90 (2H, d, J = 8.4 Hz, Ar). ESI-MS: m/z calculated for C48H65N2O4S [M] 765.5; found:

766.0.

OTs OTs O TFA, (i-Pr)3SiH O Ph3CS ( )9 ( O )3 N HS ( )9 ( O )3 N CH2Cl2, rt CN C Compound 2 Compound 3 N

Figure 3.20: Synthesis of compound 3 from compound 2.

To a solution of compound 2 (320 mg, 0.64 mmol) in CH2Cl2 (10 mL) was added TFA (1 mL). After being stirred at rt for 10 min, triisopropylsilane (1 mL) was added. After being stirred at rt for 3 h, the mixture was concentrated in vacuo. The residue was washed four times with n- hexane and seven times with n-hexane–CH2Cl2. The solvent was evaporated to give the desired

1 compound as pale yellow oil (190.7 mg, 86%). H-NMR (400 MHz, CDCl3) 1.27-1.42 (14H, m, -

CH2-), 1.51-1.66 (4H, m, -CH2-), 2.38 (3H, s, Ar-CH3), 2.54 (2H, q, J = 7.5 Hz, -SCH2-), 3.26

+ (6H, s, -N (CH3)2-), 3.42 (2H, t, J = 6.9 Hz, -OCH2-), 3.53-3.75 (14H, m, -OCH2-), 4.03 (2H, br t,

+ + J = 4.4 Hz, -N CH2-), 4.89 (2H, s, -N CH2Ar-), 7.22 (2H, d, J = 7.9 Hz, Ar), 7.74-7.82 (6H, m,

+ Ar). ESI-MS: m/z calculated for C29H51N2O4S [M] 523.4; found: 523.5.

3.4.1.8 Synthesis of Monomethyl-t-Bu Ligand for NP9

H Otfa O TFA, (i-Pr)3SiH O Ph3CS ( )9 ( O )3 N HS ( )9 ( O )3 N CH2Cl2, rt t-Bu t-Bu Compound 1 Compound 2

Figure 3.21: Synthesis of compound 2 from compound 1.

To a solution of compound 1 (626 mg, 0.80 mmol) in CH2Cl2 (6.0 mL) was added TFA

(1.5 mL). After being stirred at rt for 10 min, triisopropylsilane (1.0 mL) was added. After being stirred at rt for 1 h, the mixture was concentrated in vacuo. The residue was washed eight times

73 with n-heptane and three times with n-hexane. The solvent was evaporated to give the desired

1 compound as pale yellow oil (430 mg, 82%). H-NMR (400 MHz, CDCl3) 1.27-1.42 (23H, m, -

CH2-), 1.53-1.66 (4H, m, -CH2-), 2.54 (2H, q, J = 7.5 Hz, -SCH2-), 2. 86 (3H, s, NCH3), 3.16-

3.22 (1H, m, -CH2N-), 3.42-3.47 (3H, m, -OCH2-, -CH2N-), 3.56-3.68 (12H, m, -OCH2-), 3.89-

3.92 (2H, m, -OCH2-), 4.28 (1H, d, J = 13.0 Hz, -NCH2Ar-), 4.39 (1H, d, J = 13.0 Hz, -NCH2Ar-

), 7.38 (2H, d, J = 8.4 Hz, Ar), 7.47 (2H, d, J = 8.7 Hz, Ar), 8.30 (1H, br, -N+H-). MALDI-MS:

+ m/z calculated for C31H58NO4S [M+H] 540.409; found: 540.645.

3.4.1.9 Synthesis of Monomethyl-Me Ligand for NP10

H Otfa O TFA, (i-Pr)3SiH O Ph3CS ( )9 ( O )3 N HS ( )9 ( O )3 N CH2Cl2, rt Me Me Compound 1 Compound 2

Figure 3.22: Synthesis of compound 2 from compound 1.

To a solution of compound 1 (350 mg, 0.47 mmol) in CH2Cl2 (4.0 mL) was added TFA

(1.0 mL). After being stirred at rt for 10 min, triisopropylsilane (0.8 mL) was added. After being stirred at rt for 1 h, the mixture was concentrated in vacuo. The residue was washed eight times with n-heptane and three times with n-hexane. The solvent was evaporated to give the desired

1 compound as pale yellow oil (213 mg, 74%). H-NMR (400 MHz, CDCl3) 1.28-1.42 (14H, m, -

CH2-), 1.53-1.66 (4H, m, -CH2-), 2. 39 (3H, s, -ArCH3), 2.54 (2H, q, J = 7.5 Hz, -SCH2-), 2.83

(3H, s, NCH3), 3.15-3.21 (1H, m, -CH2N-), 3.39-3.48 (3H, m, -OCH2-, -CH2N-), 3.56-3.67 (12H, m, -OCH2-), 3.89-3.92 (2H, m, -OCH2-), 4.26 (1H, d, J = 13.0 Hz, -NCH2Ar-), 4.38 (1H, d, J =

13.0 Hz, -NCH2Ar-), 7.25 (2H, d, J = 8.1 Hz, Ar), 7.34 (2H, d, J = 8.1 Hz, Ar). MALDI-MS: m/z

+ calculated for C28H52NO4S [M+H] 498.354; found: 498.563.

3.4.1.10 Synthesis of Monomethyl-OMe Ligand for NP11

H Otfa O TFA, (i-Pr)3SiH O Ph3CS ( )9 ( O )3 N HS ( )9 ( O )3 N CH2Cl2, rt OMe OM e Compound 1 Compound 2

Figure 3.23: Synthesis of compound 2 from compound 1.

To a solution of compound 1 (311 mg, 0.41 mmol) in CH2Cl2 (4.0 mL) was added TFA

(2.0 mL). After being stirred at rt for 10 min, triisopropylsilane (2.0 mL) was added. After being stirred at rt for 1.5 h, the mixture was concentrated in vacuo. The residue was washed four times with n-heptane and four times with n-hexane. The solvent was evaporated to give the desired

1 compound as pale yellow oil (265 mg, >99%). H-NMR (400 MHz, CDCl3) 1.28-1.41 (14H, m, -

CH2-), 1.53-1.66 (4H, m, -CH2-), 2.54 (2H, q, J = 7.5 Hz, -SCH2-), 2. 84 (3H, s, NCH3), 3.15-

3.21 (1H, m, -CH2N-), 3.38-3.48 (3H, m, -OCH2-, -CH2N-), 3.56-3.67 (12H, m, -OCH2-), 3.85

(3H, s, -OCH3), 3.90 (2H, m, -OCH2-), 4.25 (1H, d, J = 13.1 Hz, -NCH2Ar-), 4.38 (1H, d, J =

13.1 Hz, -NCH2Ar-), 6.96 (2H, d, J = 8.7 Hz, Ar), 7.38 (2H, d, J = 8.7 Hz, Ar), 8.50 (1H, br, -

+ + N H-). MALDI-MS: m/z calculated for C28H52NO5S [M+H] 514.357; found: 514.583.

3.4.1.11 Synthesis of Monomethyl-CN Ligand for NP12

MeHN CN O O Ph3CS ( )9 ( O )3 OMs Ph3CS ( )9 ( O )3 N DIPEA, THF, 60 ºC Compound 1 Compound 2 CN

Figure 3.24: Synthesis of compound 2 from compound 1.

To a solution of compound 1 (460 mg, 0.66 mmol) in THF (2.0 mL) was added a solution of N-methyl-4-cyanobenzylamine (289 mg, 2.0 mmol) and diisopropylamine (327 µL, 2.0 mmol) in THF (2.0 mL). After being stirred at 60 °C for 2 days, the mixture was concentrated in vacuo.

The residue was dissolved in EtOAc. The mixture was washed with sat. NaHCO3 and brine, and

75 dried over Na2SO4. After concentration, the residue was purified by flash chromatography over silica gel with EtOAc–MeOH (1:0 to 9:1) to give the desired compound as yellow oil (381 mg, 77

1 %). H-NMR (400 MHz, CDCl3) 1.15-1.45 (16H, m, -CH2-), 1.54-1.62 (2H, m, -CH2-), 2.15 (2H, t, J = 7.3 Hz, -SCH2-), 2.29 (3H, br s, -NCH3), 2.70 (2H, t, J = 7.3 Hz, -NCH2-), 3.44 (2H, t, J =

6.8 Hz, -OCH2-), 3.56-3.69 (16H, m, -OCH2-, -NCH2Ar-), 7.20-7.34 (11H, m, Ar), 7.41-7.44

+ (6H, m, Ar), 7.62-7.7.67 (2H, m, Ar). MALDI-MS: m/z calculated for C47H63N2O4S [M+H]

752.091; found: 751.813.

H Otfa O TFA, (i-Pr)3SiH O Ph3CS ( )9 ( O )3 N HS ( )9 ( O )3 N CH2Cl2, rt CN CN Compound 2 Compound 3

Figure 3.25: Synthesis of compound 3 from compound 2.

To a solution of compound 2 (293 mg, 0.39 mmol) in CH2Cl2 (3.0 mL) was added TFA

1 mg, quant). H-NMR (400 MHz, CDCl3) 1.27-1.42 (14H, m, -CH2-), 1.52-1.66 (4H, m, -CH2-),

2.54 (2H, q, J = 7.3 Hz, -SCH2-), 2.90 (3H, s, NCH3), 3.36 (2H, br s, -CH2N-), 3.46 (2H, t, J = 6.9

Hz, -OCH2-), 3.58-3.70 (12H, m, -OCH2-), 3.93 (2H, br s, -OCH2-), 4.43 (1H, br s, -NCH2Ar-),

4.54 (1H, br s, -NCH2Ar-), 7.72 (2H, d, J = 8.3 Hz, Ar), 7.77 (2H, d, J = 8.3 Hz, Ar). MALDI-

+ MS: m/z calculated for C28H49N2O4S [M+H] 509.341; found: 509.347.

3.4.1.12 Synthesis of Monomethyl-NO2 Ligand for NP13

MeHN NO2 O O Ph3CS ( )9 ( O )3 OMs Ph3CS ( )9 ( O )3 N DIPEA, THF, 60 ºC NO Compound1 Compound 2 2

Figure 3.26: Synthesis of compound 2 from compound 1.

To a solution of compound 1 (476 mg, 0.68 mmol) in THF (2.0 mL) was added a solution of N-methyl-4-nitrobenzylamine (338 mg, 2.0 mmol) and diisopropylamine (337 µL, 2.0 mmol) in THF (2.0 mL). After being stirred at 60 °C for 2 days, the mixture was concentrated in vacuo.

The residue was dissolved in EtOAc. The mixture was washed with sat. NaHCO3 and brine, and dried over Na2SO4. After concentration, the residue was purified by flash chromatography over silica gel with EtOAc–MeOH (1:0 to 9:1) to give the desired compound as yellow oil (416 mg, 79

1 %). H-NMR (400 MHz, CDCl3) 1.15-1.44 (16H, m, -CH2-), 1.56-1.62 (2H, m, -CH2-), 2.15 (2H, t, J = 7.3 Hz, -SCH2-), 2.29 (3H, br s, -NCH3), 2.70 (2H, t, J = 7.4 Hz, -NCH2-), 3.44 (2H, t, J =

6.8 Hz, -OCH2-), 3.56-3.69 (16H, m, -OCH2-, -NCH2Ar-), 7.20-7.34 (9H, m, Ar), 7.41-7.44 (6H,

+ m, Ar), 7.54 (2H, m, Ar), 8.21 (2H, m, Ar). MALDI-MS: m/z calculated for C46H63N2O6S [M+H]

771.441; found: 771.551.

H Otfa O TFA, (i-Pr)3SiH O Ph3CS ( )9 ( O )3 N HS ( )9 ( O )3 N CH2Cl2, rt Compound 2 NO2 Compound 3 NO2

Figure 3.27: Synthesis of compound 3 from compound 2.

To a solution of compound 2 (300 mg, 0.39 mmol) in CH2Cl2 (3.0 mL) was added TFA

(1.0 mL). After being stirred at rt for 10 min, triisopropylsilane (1.0 mL) was added. After being stirred at rt for 1 h, the mixture was concentrated in vacuo. The residue was washed ten times

77 with n-hexane. The solvent was evaporated to give the desired compound as pale yellow oil (241

1 mg, 99%). H-NMR (400 MHz, CDCl3) 1.26-1.42 (14H, m, -CH2-), 1.51-1.66 (4H, m, -CH2-),

2.54 (2H, q, J = 7.5 Hz, -SCH2-), 2.91 (3H, s, NCH3), 3.38 (2H, br s, -CH2N-), 3.45 (2H, t, J = 6.8

Hz, -OCH2-), 3.58-3.70 (12H, m, -OCH2-), 3.93 (2H, br t, J = 4.3 Hz, -OCH2-), 4.55 (1H, br s, -

NCH2Ar-), 7.79 (2H, d, J = 8.6 Hz, Ar), 8.32 (2H, d, J = 8.6 Hz, Ar). MALDI-MS: m/z calculated

+ for C27H49N2O6S [M+H] 529.331; found: 529.354.

3.4.1.13 Synthesis of DMBA Ligand

Me2N OMs Me O Me O ( O )3 OMs ( O )3 N EtOH, 40 ºC Me2 Compound 2 Compound 1

Figure 3.28: Synthesis of compound 2 from compound 1.

To a solution of compound 157 (450 mg, 1.57 mmol) in EtOH (1.57 mL) was added N,N- dimethylbenzylamine (705 µL). After being stirred at 45 °C for 1 day, the mixture was concentrated in vacuo. The residue was washed six times with diethylether. The solvent was evaporated to give the desired compound as colorless oil (670 mg, quant). 1H-NMR (400 MHz,

CDCl3) 2.83 (3H, s, -CH3), 3.26 (6H, s, -NCH3), 3.35 (3H, s, -CH3-), 3.50-3.69 (12H, m, -OCH2-

), 3.86 (2H, br s, -OCH2-), 4.04 (2H, br s, -CH2N-), 4.83 (2H, s, -NCH2Ar-), 7.45-7.65 (5H, m,

+ Ar). MALDI-MS: m/z calculated for C18H32NO4 [M] 326.233; found: 326.107.

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(51) Chinai, J. M.; Taylor, A. B.; Ryno, L. M.; Hargreaves, N. D.; Morris, C. A.; Hart, P. J.; Urbach, A. R. Molecular Recognition of Insulin by a Synthetic Receptor. J. Am. Chem. Soc. 2011, 133, 8810–8813.

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CHAPTER 4

MASS SPECTROMETRIC DETECTION OF NANOPARTICLE HOST-GUEST INTERACTIONS IN CELLS

4.1 Introduction

Host-guest chemistry using engineered molecular systems provides controllable platforms for biomedical applications such as cell targeting,1,2 biosensing,3 imaging,4 drug delivery,5-7 and cancer therapeutics.8 The reversibility of the association/dissociation process plays a vital role in these applications, allowing host systems to regulate the release of drug guests.9,10

Multifunctional nanomaterials provide particularly versatile scaffolds for these host-guest systems due to their biocompatibility and functional versatility.8-11 For example, the cytotoxicity of gold nanoparticles (AuNPs) can be triggered in cancer cells using competitive host-guest binding molecules, providing a new strategy for potential therapeutic applications.8

Effective use of nanomaterial-based supramolecular chemistry in biomedical applications requires the ability to monitor the association and dissociation of the non-covalent conjugates inside cells.12 Characterization of host-guest interactions is traditionally performed in simple solutions using techniques such as NMR13,14 and isothermal titration calorimetry (ITC).15,16 These methods, however, cannot be used to analyze host-guest interactions in biological systems due to the complex environments in cells and tissues. Fluorescence spectroscopy is an alternate strategy to detect host-guest complexes in complicated biological samples.17 The use of florescent probes, especially when additional labeling steps are required, can affect the biological behavior of original host-guest complexes due to the alteration of surface properties by the dye.18-20 Moreover, it is challenging for this method to simultaneously probe multiple host-guest complexes.

Mass spectrometry (MS) is an effective tool for characterizing host-guest interactions in solution.12,21-24 For example, electrospray ionization (ESI) MS25-27 and matrix assisted laser desoption/ionization (MALDI) MS28,29 have been utilized for the detection of host-guest

84 complexes. However, to the best of our knowledge, detecting host-guest interactions inside cells using MS has not been reported, due in large part to the large number of interfering ions generated from biological samples.

Scheme 4.1: (a) Schematic illustration of the MALDI-MS detection process of supramolecular complexes in cells. AuNP-CB[7] complexes are measured as complex ions between CB[7] and AuNP surface ligands, and these ions appear at m/z values above 1600. (b) Monitoring the selective dissociation of the supramolecular complexes after adding the competitive binding molecule ADA. The addition of ADA dissociates some AuNP-CB[7] complexes and also leads to a new ADA-CB[7] complex ion at m/z 1314.

In this chapter, we report here a direct method to monitor the association and dissociation of multiple NP-based host-guest complexes inside cells (Scheme 4.1 (a)) using a standard

MALDI mass spectrometer. Supramolecular complexes formed by the surface ligands of AuNPs and cucurbit[7]uril (CB[7]) serve as “mass barcodes” to indicate the presence of AuNP-CB[7] complexes inside cells. This method integrates NP-mediated laser desorption ionization (LDI-

MS)30-34 with MALDI using an organic matrix and acts to selectively desorb/ionize supramolecular complexes of the ligands, allowing observation of these species in the presence of other cellular materials. Using this method, the intracellular association and dissociation of

AuNP-CB[7] complexes were monitored, as well as competitive dissociation of these complexes using 1-adamantylamine (ADA) (Scheme 4.1 (b)).

4.2 Results and Discussion

4.2.1 Detection of Host-Guest Complexes in Aqueous Solution

We chose the cucurbituril (CB) supramolecular family for our studies. These host-guest complexes are particularly promising for biomedical applications due to their solubility in aqueous media, high affinity and non-toxicity.35-39 We probed intracellular CB interactions using

AuNPs with three types of surface functionalities (Figure 4.1 (a)). The AuNP-CB[7] complexes were formed by mixing AuNPs with excess CB[7] (molar ratio of AuNP:CB[7] = 1:200). The initial LDI/MALDI-MS detection of the AuNP-CB[7] host-guest complexes was first in simple aqueous solutions (Figure 4.2).

Figure 4.1: (a) Structures of the surface functionalities on the AuNPs used in this work. (b) The mass-to-charge (m/z) ratios of ligands and their corresponding supramolecular complexes monitored by MALDI-MS. Letter code key: molecular ions of the surface ligands (L ions), disulfide ions (D ions) formed by surface ligands and pentanethiol ligands, supramolecular complex ions (C ions) formed by surface ligand and CB[7], and disulfide ions formed by CB[7] and D ions (DC ions).

The molecular ion of the surface ligand (L1) of AuNP 1, which has a diaminohexyl ending group was readily detected using LDI/MALDI-MS (Figure 4.2 (a)). The disulfide ion

(D1), previously reported in LDI/MALDI-MS analysis of self-assembled monolayer surfaces,40-43 and the molecular ion (L1) confirmed the presence of AuNP 1. The supramolecular complex ions

C1 and DC1 (formed by the D1 ion and CB[7]) indicate the detection of the host-guest complexes

(Figure 4. 2 (a), see the inset for the enlarged region between m/z 1600 and 1800; ion identities are shown in Figure 1 (b)). This MALDI-MS method for monitoring the AuNP-CB[7] host-guest interactions is able to detect the intact supramolecular complexes without generating fragments of the gold clusters or ionizing the intact AuNPs, and thus provides information on the ligand-CB[7] interaction. We applied this method to supramolecular complexes formed by CB[7] and AuNPs with different surface functionalities, and analogous mass spectra were acquired using AuNP 2-

CB[7] and AuNP 3-CB[7] containing solutions (Figure 4.2 (b), (c)). The LDI/MALDI-MS characterization of the AuNP-CB[7] supramolecular structures can also be applied to NPs with a wide range of surface functionalities.

Figure 4.2: Monitoring AuNP-CB[7] interaction in solution using MALDI-MS. (a) AuNP 1- CB[7]. (b) AuNP 2-CB[7]. (c) AuNP 3-CB[7]. [AuNP]=2 µM, [CB[7]]=400 µM. See the Figure 1 caption for the identities of the L, D, C, and DC ions.

4.2.2 Detection of Host-Guest Complexes inside Cells

Building on the solution phase experiments, we next explored the ability of this method to selectively ionize and detect NP host-guest complexes in cells. HeLa cells were incubated with uncomplexed (250 nM) and complexed (250 nM AuNP, 200 equivalents of CB[7]) and washed with PBS three times to remove the AuNPs and AuNP-CB[7] complexes that were not taken up by the cells. After the cells were lysed, the resulting samples were transferred to centrifuge tubes, and the pellets containing AuNPs or AuNP-CB[7] complexes were collected after the centrifugation (Scheme 4.1 (a)). The high density of AuNPs relative to the biomolecules in the cells allows one to concentrate the AuNPs and AuNP-CB[7] complexes to some extent, minimizing interferences from biological molecules in the cell lysate. We then transferred the pellets to the MALDI-MS sample carrier and applied a thin layer of matrix on top of the pellets

(Scheme 4.1 (a)). Figures 4.3 (a) and 4.3 (b) show typical LDI/MALDI mass spectra that are

88 obtained. The surface ligand ions (L1 and D1) are observed, indicating the existence of AuNPs in the pellets (Figure 4.3 (a)). The supramolecular ions (C1 and DC1) are also readily observed, showing successful detection of host-guest complexes inside cells (Figure 4.3 (b)). The gold cores of the AuNPs and the added matrix seem to work together to enable the selective ionization of the surface ligands and complexes that are attached to the AuNPs. Interestingly, the relative intensities of the complexed and uncomplexed ligands in cells (Figure 4.3 (b)) are different than in solution (Figure 4.2 (a)); we are investigating the origins of this disparity.

Figure 4.3: Monitoring AuNP-CB[7] interactions using MALDI-MS. (a) Detection of AuNP 1 in cells after incubation with 250 nM AuNP. (b) Detection of AuNP 1-CB[7] ([AuNP]=250 nM, [CB[7]] = 50 µM) taken up by the cells. (c) The dissociation of the host-guest complex by adding ADA (4 µM) to the cells containing AuNP 1-CB[7].

4.2.3 Dissociation of Host-Guest Complexes Using a Competitive Guest Molecule

We next used LDI/MALDI to monitor the dissociation of host-guest complexes using

ADA, a strong binding competitor for CB[7]. Since a similar amount of particle was taken up with each of the ligands the same amount of ADA was added for each particle. The host-guest

89 complex “mass barcodes,” both C1 and DC1 ions, disappear after the cells containing AuNP-

CB[7] complexes are treated with ADA (Figure 4.3 (c)), indicating the dissociation of supramolecular complexes. Comparing the results in Figure 4.3 (a) and 4.3 (c), ADA treatment of cells incubated previously with AuNP 1-CB[7] complexes leads to very similar mass spectra as the cells treated with only AuNP 1. Figure 4.3 demonstrates the successful tracking of the association and dissociation of AuNP-CB[7] supramolecular complexes in cells by LDI/ MALDI-

MS.

4.2.4 Multiplexed Detection of Association and Dissociation of Host-Guest Complexes

Multiple supramolecular complexes can be followed simultaneously using MALDI-MS.30

This multiplexed detection could provide direct ratiometric measurements, significantly reducing the variability introduced from studying different supramolecular complexes in separate cell populations. Cells were incubated with three AuNP-CB[7] complexes (AuNP 1-CB[7], AuNP 2-

CB[7] and AuNP 3-CB[7]) to demonstrate this multiplexing capability. Ions corresponding to the surface ligands (L1, L2, and L3) of three AuNPs and the host-guest complexes (C1, C2, and C3) are readily detected (Figure 4.4 (a); however, the intensities of the complex ions detected by

MALDI-MS vary due to the different amounts and different ionization efficiencies of the supramolecular complexes.

As above, ADA was used to trigger the dissociation of the AuNP-CB[7] complexes inside the cells. In this study, 1.8 µM and 3.6 µM of ADA (total ADA amount: 0.9 nmol and 1.8 nmol, respectively) were added to the cells containing AuNP-CB[7] complexes. We used the intensity ratios of all the supramolecular complex ions (C and DC ions) and all the ligand related ions (L, D, C, and DC ions) to evaluate the ADA-triggered dissociation of the complexes. All the ion intensity ratios were then normalized (Figure 4.4 (b)) relative to cells without ADA treatment.

The decrease in the normalized ion intensity ratios shows the dissociation of these three supramolecular complexes are different. A more detailed examination using one-way ANOVA

90 reveals that AuNP 3-CB[7] complexes are much more stable to ADA treatment than the other two ligands. This observation of selectivity illustrates the utility of the of the LDI/MALDI-MS method to screen multiple host-guest interactions in cells.

Figure 4.4: Monitoring the dissociation of three AuNP-CB[7] complexes in cells. (a) Typical mass spectrum of cell samples incubated with a mixture of three AuNP-CB[7] complexes. (b) Normalized ion intensity ratios indicating the relative amount of the remaining supramolecular complexes after ADA treatments. n.s., no significant difference. **, 0.001

4.3 Summary and Future Outlook

In summary, we have demonstrated the use of LDI/MALDI-MS to detect AuNP-CB[7] complexes in cells, confirming that both formation and dissociation of host-guest interactions inside cells can be monitored. We predict that this method is adaptable for monitoring other host- guest systems with various types of NPs,30 with the inherent multiplex capabilities of the mass barcode approach facilitating high-throughput screening.

4.4 Experimental Section

4.4.1 Cell Culture Experiments

60k HeLa cells per well were plated into a 24 well plate 24 h before the experiment. Cells were incubated with AuNP-CB[7] complexes (250 nM, 500 uL) for 24 h in DEMEM media containing 10% FBS and 1% antibiotics and then washed 3 times with phosphate-buffered saline

(PBS) (500 uL each washing). Beta Gal lysis buffer (250 uL per well, 5 times diluted) was used to lyse the cell, with the cell culture plate kept at room temperature on a vibrator for 30 minutes.

4.4.2 Treatment of Competitive Guest Molecule, ADA

60k HeLa cells were treated with a single type of NP-CB[7] complex or a mixture of three NP-CB[7] complexes for 24 h. Then, they were washed 3 times with PBS (500 uL), and treated with ADA at a concentration of 1.8 µM and 3.6 µM for 1 h (total ADA amount: 0.9 nmol and 1.8 nmol, respectively). After that, cells were washed 3 times with PBS and lysed with Beta

Gal lysis buffer.

4.4.3 Cell Sample Preparation for MALDI-MS

The cell lysate samples were transferred from the 24-well cell culture plate to 1.5 ml centrifuge tubes. Then, they were centrifuged at 14000 rpm for 30 min. After removal of the supernatant containing the lysis buffer, the pellets were transferred to the stainless steel MALDI-

MS sample carrier. A saturated solution of the matrix α-cyano-4-hydroxycinnamic acid (α-

CHCA) solution was prepared in 70% acetonitrile and 30% water for the MALDI-MS analysis.

2.5 µL of the matrix solution was applied on top of each pellet. The samples were air-dried before

MALDI-MS analysis.

4.4.3.1 MALDI-MS Instrumentation and Analysis of Additional AuNP-CB7 Complexes

MALDI-MS experiments were carried out on a Bruker Autoflex III MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany), equipped with a Smartbeam 2 Nd:YAG laser. MALDI-MS operating conditions were as follows: ion source 1 = 19.00 kV, ion source 2 =

16.60 kV, lens voltage = 8.44 kV, reflector voltage = 20.00 kV, reflector voltage 2 = 9.69 kV, and positive reflectron mode in a mass range of m/z 400−3000. A total of 200 laser shots were fired per measurement. The laser energy was optimized to ~ 40 µJ/pulse. Data processing was performed using the Bruker flexAnalysis (version 3.3) software.

Figure 4.5: Various AuNPs which bind CB[7] that have been successfully characterized by MALDI-MS. The ones in the red box have been utilized for the selective dissociation study.

Figure 4.6: Mass spectra of the AuNP-CB[7] complexes. (a) AuNP 4-CB[7], (b) AuNP 5-CB[7], (c) AuNP 6-CB[7], (d) AuNP 7-CB[7].

Table 4.1: Detail p values for the comparison between different NP-CB[7] complexes.

p value 0.9 nmol ADA 1.8 nmol ADA AuNP 1 vs. AuNP 2 0.7330 0.2207 AuNP 1 vs. AuNP 3 0.0001 0.0019 AuNP 2 vs. AuNP 3 0.0002 0.0005

Table 4.2: The ion intensity ratios of MALDI-MS analysis of AuNP-CB[7] complexes in solutions. Ion Intensity Ratio Average SD AuNP 1 - CB[7] 0.76 0.08 AuNP 2 - CB[7] 0.41 0.08 AuNP 3 - CB[7] 0.025 0.010

4.4.4 Sample Preparation for ICP-MS and Analysis of the Cellular Uptake

After the cellular uptake, the cells were lysed and transferred to 15 mL centrifuge tubes.

0.5 mL of fresh aqua regia (highly corrosive and must be treated with extreme caution!) were added to each sample. Each sample was then diluted to 10 mL with de-ionized water. A series of gold standard solutions (0, 0.2, 0.5, 1, 2, 5, 10, and 20 ppb) were prepared for the ICP-MS measurements. Each standard solution also contained 5% aqua regia. The gold standard solutions and cellular uptake sample solutions were measured on a Perkin-Elmer NexION 300X ICP mass spectrometer. 197Au was measured under standard mode. Operating conditions are listed as below: nebulizer flow rate: 0.95-1 L/min; rf power: 1600 W; plasma Ar flow rate: 18 L/min; dwell time:

50 ms.

Figure 4.7: Cellular uptake amount per well of AuNP-CB[7] complexes measured by ICP-MS.

4.4.5 Synthesis of Ligands and AuNPs

4.4.5.1 Synthesis of Ligands

Scheme 4.2: Synthesis scheme of dimethyldiaminohexyl ligand (DMAH, L2).

Compound 1: 11-bromo-1-undecanol (8.22 g, 32.74 mmol) was dissolved in a solution of ethanol/toluene (1:1, 100 mL). Then, triphenylmethanethiol (10.86 g, 39.29 mmol) was also dissolved in a solution of ethanol/toluene (1:1, 50 mL) and added to the 11-bromo-1-undecanol mixture. NaOH (1.96 g, 49.11 mmol) in 3 mL of H2O was also added into the mixture. The reaction mixture was stirred for 24 h at 50oC. Once the reaction was completed (checked by

TLC), the reaction mixture was extracted with a saturated solution of NaHCO3 twice. The organic layer was separated and extracted with a saturated NaCl solution twice. Afterward the organic layer was separated, dried over Na2SO4, and concentrated using a rotavapor. The crude product was purified by column chromatography over silica gel using hexane/ethyl acetate (1:1, v/v) as an eluent. The solvent was removed in vacuum to obtain compound 1 as colorless oil (Yield 13.88 g,

95 %).

Figure 4.8: 400 MHz 1H NMR spectra of compound 1 in chloroform-D (D, 99.8%).

Compound 2: Compound 1(13.88 g, 31.1 mmol) was dissolved in dry DCM. Then, triethylamine (4.72g, 6.48 mL, 46.65 mmol) was added into this solution at 4 oC.

Methanesulfonyl chloride (3.92 g, 2.65mL, 34.21 mmol) was added dropwise to the solution kept in ice bath. After 30 minutes the reaction mixture was warmed up to room temperature and stirred overnight. After the reaction was completed (according to TLC), solvent was evaporated. The compound was again diluted with DCM and extracted with 0.1 M solution of HCl twice. Organic layer was collected and treated with a saturated solution of NaHCO3 and washed three times. The organic layer was separated and added into saturated NaCl solution and extracted three times.

After extraction, organic layer was separated, dried over Na2SO4 and concentrated at reduced pressure. The crude product was purified by column chromatography over silica gel using hexane/ethyl acetate (1:1, v/v) as an eluent. Solvent was removed in vacuum to obtain mesylated compound as light yellow oil (Yield 15 g, 92 %). To synthesize compound 2, NaOH (1.37 g,

34.3 mmol) dissolved in 1 mL of H2O was added to 99.24 mL of tetraethyleneglycol (111.15 g,

57.22 mmol) and stirred for 2 h at 80 °C. To this reaction mixture, 15 g of 11 (tritylthio)undecyl

97 methanesulfonate was added and stirred for 48 h at 80 °C. The reaction mixture was extracted by washing with a solution of hexane/ethyl acetate (4:1, v/v) six times. Then, the organic layer was separated and concentrated at reduced pressure. The crude product was purified by column chromatography over silica gel using ethyl acetate as an eluent. The solvent was removed in vacuum to obtain compound 2 as light yellow oil (Yield 15.28 g, 68%).

Figure 4.9: 400 MHz 1H NMR spectra of compound 2 in chloroform-D (D, 99.8%).

Compound 3: Compound 2 (10 g, 16.1 mmol) was dissolved in dry DCM at 4 °C.

Triethylamine (3.26g, 4.49 mL, 32.2 mmol) was added into the solution. Methanesulfonyl chloride (2.77 g, 1.87 mL, 24.1 mmol) was added drop by drop to the reaction mixture that was kept in ice-bath. After 30 minutes the reaction mixture was warmed up to room temperature and stirred overnight. Afterward, DCM solvent was evaporated under reduced pressure. The viscous compound was again diluted with DCM and was extracted with 0.1 M solution of HCl twice. The organic layer was poured into a saturate solution of NaHCO3 and washed three times. Organic layer was separated and added into saturated NaCl solution and also washed for three times.

Then, the organic layer was separated, dried over Na2SO4 and concentrated at reduced pressure.

The crude product was purified by column chromatography over silica gel using ethyl acetate as an eluent. Solvent was removed in vacuum to obtain compound 3 as a light yellow oil (Yield 10.7 g, 95 %).

Figure 4.10: 400 MHz 1H NMR spectra of compound 3 in chloroform-D (D, 99.8%).

Compound 4: Compound 3 (2 g, 2.85 mmol) was added to dimethylamine solution (25 ml, 2 M solution in THF, 57 mmol) in THF. The reaction mixture was stirred at 25oC for 24 h.

Crude product was checked by TLC and THF and excess of dimethylamine were eliminated at reduced pressure. The product formation was quantitative and their structure was confirmed by

NMR.

Figure 4.11: 400 MHz 1H NMR spectra of compound 4 in chloroform-D (D, 99.8%).

Compound 5: Compound 4 (0.6 g, 9.23 mmol) and compound 7 (0.8 g, 27.7 mmol) were dissolved in ethanol (5 mL) and stirred at 40 oC for 48 h. Crude product was checked by TLC and ethanol was eliminated at reduced pressure. The light yellow residue was purified by successive hexane (4 times) and diethylether (4 times) washings with support of sonication and then dried in a high vacuum system. The product formation was quantitative and their structure was confirmed by NMR.

100

Figure 4.12: 400 MHz 1H NMR spectra of benzyl ligand in chloroform-D (D, 99.8%).

Compound 6: Compound 5 (0.4 g, 0.47 mmol) was dissolved in dry dichloromethane

(DCM, 5 mL) and an excess of trifluoroacetic acid (TFA, 20 equivalents, 1.072 g, 0.72 mL, 9.41 mmol) was added. The color of the solution was turned to yellow upon addition of TFA. Then, triisopropylsilane (TIPS, 1.5 equivalents, 0.11 g, 0.15 mL, 0.705 mmol ) was added to the reaction mixture. The reaction mixture was stirred overnight under N2 at room temperature. The solvent and most TFA and TIPS were evaporated under reduced pressure. The yellow residue was purified by hexane washings (5 times) and dried in a high vacuum system. The product formation was quantitative and their structure was confirmed by NMR.

101

Figure 4.13: 400 MHz 1H NMR spectra of dimethyldiaminohexane ligand in chloroform-D (D, 99.8%).

Figure 4.14: 400 MHz 1H NMR spectra of benzyl ligand in chloroform-D (D, 99.8%).

102

Figure 4.15: 400 MHz 1H NMR spectra of diaminohexane ligand in chloroform-D (D, 99.8%).

Diaminohexyl (DAH, L1) and benzyl (BEN, L3) ligand were synthesized according to the procedure in literature.44,45

4.4.5.2 Synthesis of AuNPs

Brust-Schiffrin two-phase synthesis method was used to synthesize pentanethiol-coated

AuNPs with core diameter ~2 nm.46 Murray place-exchange method47 was followed to obtain the benzyl NPs. Pentanethiol conjugated AuNPs (20 mg) and thiol ligand (compound 6) (60 mg) was dissolved in mixture of dry DCM/methanol (4:1, 5 ml) and stirred under N2 atmosphere for 3 days at room temperature. The solvents were removed under reduced pressure and the resulting precipitate was washed with hexane three times and DCM three times. Then the precipitate was dissolved in distilled water and dialyzed for 3 days (membrane molecular cut-off =10000) to remove excess ligands and pentanethiol, acetic acid and other salts present in the nanoparticle solution. After dialysis, the particle was lyophilized to yield a solid brownish product. The

1 particles are then redispersed in deionized water. H NMR-spectra in D2O showed substantial broadening of the proton peaks with no sign of free ligands.

103

Figure 4.16: TEM image of pentanethiol (C5) coated AuNPs.

4.4.6 Complexation of AuNP with CB[7]

CB[7] solution in deionized water was added into AuNP solution and stirred for 24 h.

Ratio between CB[7]/AuNP was kept at 200:1. For example, 200 µL AuNP DAH solution (30

µM) was mixed with 200 µL CB[7] solution (6 mM).

104

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CHAPTER 5

SOLUBILIZATION OF HYDROPHOBIC CATALYSTS USING NANOPARTICLE HOSTS

5.1 Introduction

Transition metal-mediated catalysis (TMC) in aqueous media1 diminishes the dependence on expensive and environmentally unfriendly organic solvents.2,3 However, most TMC reactions are performed in organic solvents due to limited solubility,4 activity,5 and stability6 of the catalysts in water. Therefore, designing a general platform that solubilizes hydrophobic catalysts in water while preserving their activity and stability remains critical for environmental and economical benefits.

Covalent and non-covalent modifications to transition metal (TM) catalysts have been used to improve catalyst solubility in aqueous environments. Widely investigated approaches include covalent conjugation of the catalysts with hydrophilic ligands7 or anchoring charged substituents to the coordinating hydrophobic ligands.8 These covalent modifications demand specific ligand designs for each catalyst, which can affect the stability and reactivity of the native catalyst.9 Meanwhile, improving the solubility of TM catalysts does not inherently protect the catalysts from aqueous degradation. Covalent modification can change the steric and electronic environment of metal center, weakening the bond between the ligand and metal, resulting in poor initiation efficiency,6a decomposition of catalyst,10 and loss of activity.11 Non-covalent approaches such as host–guest assembly of catalysts with cyclodextrins,12 calixarenes,13 and cucurbiturils14 have been shown as effective methods to solubilize catalysts without the need to alter the structure of the catalysts.15 These supramolecular approaches, however, require the catalysts to possess suitable guest sites.16

Another emerging approach to address the challenges associated with catalyst solubility and stability is encapsulating catalysts into platforms such as micelles,17 dendrimers,18 polymeric

110 capsules,19 and hallow polymer shells.20 These constructs provide solubility to the catalyst and result in high yield under ambidient conditions due to the ability of confined nanocatalysis.21

However, issues such as the use of stabilizing agent and additives22/surfactants23 and their removal, multistep preparation procedures,24 lack of robustness due to the dynamic nature of these systems,25 and lack of complete template removal26 impede the effective use of the aforementioned platforms. Furthermore, these scaffolds are more suitable for the entrapment of catalyst made up from metal nanoparticles with size of larger than 10 nm.27 However, majority of homogeneous catalysts including TM catalysts are in the size range of 1-2 nm.

Nanoparticles (NPs) with their unique tunable physical and chemical properties are promising platforms to encapsulate/immobilize a variety of catalysts including small-sized TM catalysts.28 Performing catalysis using NP systems brings the advantages of both homogenous and heterogeneous catalysis into the same system.29 While efficient and diverse reactions are catalyzed within these NPs, reaction products are easily separated using simple separation methods such as filtration.30 In this work, we present a general method to solubilize and stabilize hydrophobic TM catalysts while preserving catalytic activity using a water soluble AuNP platform (Scheme 5.1). This water soluble NP host was designed bearing two crucial features: an aliphatic interior to create hydrophobic pockets for catalyst encapsulation and a water soluble exterior for aqueous solubility.[31] In our method, there is no need for covalent modification of the catalyst, and the supramolecular template supplies a protective niche for hydrophobic catalysts.

Using this platform we demonstrate the efficiency of two commercially important reactions.

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Scheme 5.1: a) The encapsulation process of hydrophobic catalysts in water soluble NP hosts. b) Schematic illustrating catalytic reaction within NP monolayer.

5.2 Results and Discussion

5.2.1 Encapsulation of Hoveyda-Grubbs 2nd Generation (HG2) Catalyst into AuNP Monolayer

Our initial catalysis studies focused on commercially available Hoveyda-Grubbs 2nd generation (HG2), widely used for ring opening metathesis polymerization (ROMP).

Hydrophobic (HG2) catalysts were encapsulated using a solvent displacement method.31 Water soluble NP hosts and hydrophobic HG2 catalysts molecules were dissolved in acetone/water mixtures and then the organic solvents were allowed to evaporate slowly. During the evaporation process, HG2 catalysts were encapsulated into the hydrophobic pockets in the monolayer of

AuNP, providing HG2 catalyst-encapsulated nanoreactors (AuNP-HG2), while excess hydrophobic catalyst precipitated out. Then filtrations using molecular weight cutoff filters were

112 performed to unbound catalysts followed by dialysis against water (Scheme 5.1 (a)) Other catalyst encapsulations were prepared in analogous fashion to obtain AuNP-cat nanoreactors.

To determine parameters for effective catalyst design, two different sized gold cores (2 and 7 nm), each with two different ligand structures (TTMA and TMA, Figure 2a) were investigated. Both TTMA and TMA ligands posses a hydrophobic chain, crucial for hydrophobic pocket formation and a charged ammonium head group for water solubility (Figure 5.1 (a)).

Figure 5.1: a) The structure of NP hosts (core 2 and 7 nm) and the structure of TTMA and TMA ligands. b) The encapsulation efficacy of various NP hosts that have different core sizes and monolayer structures for HG2.

Qualitative confirmation of catalyst presence in the monolayer of nanoreactor was

1 monitored by H-NMR in D2O after encapsulation. Peaks observed at ~6.8-7.8 ppm demonstrate that 2nd generation Hoveyda-Grubbs catalysts (HG2) were entrapped inside the nanoreactor

(Figure 5.2).

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Figure 5.2: a) Structure of the TTMA functionalized AuNP, b) NMR spectrum of HG2 catalyst encapsulated AuNP, c) Enlarged section of NMR spectrum in the range from 6 to 8 ppm.

The aggregation of NPs during the encapsulation and intense purification steps can be an issue for the stability of the nanoreactor. The size of the NP host was monitored before and after encapsulation of HG2 as well as after the purification process by transmission electron microscopy (TEM). TEM images showed no size change or aggregation of the NP host-HG2 compared to the NP host, displaying the stability of the nanoreactor (Figure 5.3).

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Figure 5.3: TEM images of HG2 catalyst encapsulated AuNPs.

Loaded catalyst amount in the monolayer of NP was calculated by tracking TM ions using KCN-induced decomposition experiments followed by inductively coupled plasma mass spectrometry (ICP-MS) measurements. The results indicate that 7 nm core NP hosts can hold more catalysts, however, 2 nm core NPs shows higher encapsulation yield according to weight ratio between catalyst and NP core (Figure 5.1 (b)). When same core size but different ligand structure was considered, NP bearing TEG groups showed a higher catalyst loading efficiency.

The surface ligand coverage was obtained using the laser desorption/ionization mass spectrometry32 to probe the effect of ligand coverage on encapsulation yield. The results revealed that the TTMA AuNPs possess more remaining pentane thiols after place exchange reaction than

TMA AuNPs do, indicating that the pentane thiols provide a larger space to encapsulate the catalysts (Figure 5.4). Based on ICP-MS results, 2nm core sized TTMA NP host is the most efficient one among other NP hosts; therefore, catalytic reactions were performed using 2 nm

TTMA NPs (Figure 5.1 (b)).

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Figure 5.4: Ion intensity ratio of TMA and TTMA coated 2 and 7 nm AuNPs.

5.2.2 Ring Opening Metathesis Polymerization in Aqueous Solution Using AuNP-HG2

Our initial catalysis studies focused on commercially available HG2 catalyst, one of the most used hydrophobic catalysts for ring opening metathesis polymerization (ROMP). In our system, ROMP was performed in deionized water by using 2 nm TTMA AuNP-HG2 catalyst and a positively charged norbornene monomer (Figure 5.5 (a)-(b)).33 After 24 hours, white suspension was observed as an indication of polymer formation. However, when the exact same conditions except no catalyst encapsulated in the AuNPs was used, polymer generation was not detected.

Effect of reaction time on polymerization was monitored by using gel permeation chromatography (GPC). Polymers collected at different time points were characterized in terms of molecular weight and PDI (polydispersity index) (Figure 5.5 (d)). Results show a typical linear trend of living polymerization that molecular weight increases with elevated reaction time.

Narrow PDI values were obtained as expected from a living ROMP reaction, indicating that our

AuNP-HG2 system supplies the fine molecular weight control (Figure 5.5 (c)-(d)).

Catalysts containing ruthenium carbene complexes in their structure generally decompose quickly in aqueous solvents34 and consequently, they are unable to catalyze ROMP reactions. In the literature, results indicated that instability originated from water coordination at ruthenium in

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35 the methylidene-propagating species. We have checked the stability of free HG2 catalyst and found that in the presence of monomer and free HG2 catalyst in 1:1 acetone/water mixture, the reaction failed to produce any polymer. Therefore, GPC could only detect the unreacted monomer at various reaction time points (Figure 5.5 (d), 5 (f)). However, HG2 catalyst embedded into the monolayer of AuNPs successfully catalyzed the ROMP reaction under same conditions (Figure

5.5 (d)-(e)). From these results, we deduce that our NP hosts can provide a protective environment blocking water molecules from coordinating to metal center and as a result prevent decomposition.

nd Figure 5.5: a) The structure of 2 generation Hoveyda-Grubbs catalyst (HG2). b) The reaction scheme of ROMP using NP host and water soluble monomer. c) The molecular weight and PDI values of polymers in terms of reaction time. d) While the free HG2 in acetone/water mixture failed to produce the polymers, the polymers formation was detected in the presence of the AuNP-HG2, showing increased molecular weight in terms of reaction time. e) GPC result of NP host-HG2-monomer mixture after 24 h. f) GPC result of free catalyst-monomer mixture after 24 h, no polymerization was observed.

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5.2.3 Cleavage of Allylcarbamate Bonds Using Ruthenium or Palladium Catalyst Embedded AuNP

The versatility of our encapsulation strategy was demonstrated by catalyzing the cleavage of allylcarbamate bonds using the hydrophobic [(pentamethylcyclopentadienyl) Ru(1,5- cyclooctadiene)Cl] catalyst (Cp*Ru(cod)Cl) (Figure 5.6 (a), (c)).36 We have quantified that there are 22.7 ± 1.9 Cp*Ru(cod)Cl catalysts per one NP host. Rhodamine 110 is a green fluorescent dye with amine functionality that can be protected with allylcarbamate. After caging, Rhodamine

110 becomes nonfluorescent, however, upon allycarbamate deprotection, strong green fluorescence is recovered as an indication of catalytic cleavage (Figure 5.6 (c)-(e)). Reaction activity was monitored using UV absorbance of the Rhodamine 110 dye at a wavelength of 500 nm wavelength. While absorbance increased linearly in the presence of substrate (Rhodamine 110 derivative) and AuNP-Cp*Ru(cod)Cl, it stayed constant when only AuNP and substrate were used.

Figure 5.6: The structure of a) Cp*Ru(cod)Cl and b) Pd(dppf)Cl2 catalyst. c) The reaction scheme of ruthenium-induced (or palladium-induced) allylcarbarmate cleavage using NP hosts and non-fluorescent Rhodamine 110 derivative. d) Catalytic activity of NP host- Cp*Ru(cod)Cl and only NP host. e) Photographs of the NP host reaction with and without ruthenium catalyst under UV-irradiation. f) Catalytic activity of NP host-Pd(dppf)Cl2 versus substrate and NP host. g) Photograph of the palladium catalysis under UV-irradiation with the control reactions.

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Similar to ruthenium catalyst, palladium catalysts are also known to cleave the allylcarbamate of bis-N,N′-allyloxycarbonyl rhodamine 110.37 We have encapsulated (1,1′- bis(diphenylphosphino) ferrocene)palladium(II)dichloride catalyst (Pd(dppf)Cl2) (Figure 5.6 (b)) in the hydrophobic pockets of the AuNP monolayer to provide AuNP-Pd(dppf)Cl2. The amount of palladium catalyst relative to AuNP was quantified using ICP-MS of 106Pd relative to 197Au. It was calculated that 12 ± 0.2 catalyst molecules were encapsulated per AuNP . Fluorescence started to increase immediately for AuNP-Pd(dppf)Cl2 after the addition of substrate, while no significant change in fluorescence was observed for only AuNP host (Figure 5.6 (f)-(g)). We checked the system after one week, results showed that fluorescence increased further for AuNP-

Pd(dppf)Cl2 while controls (only substrate and AuNP host + substrate) showed no fluorescence

(Figure 5.7 and 5.8).

Figure 5.7: Catalytic activity of NP host-Pd(dppf)Cl2 versus substrate and NP host after 24 h and 7 days.

Figure 5.8: Fluorescence increased further for AuNP-Pd(dppf)Cl2 while controls (only substrate and AuNP host + substrate without catalyst) showed no fluorescence after one week.

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5.2.4 Hydrogenation of Alkenes Using Wilkinson’s Catalyst Encapsulated AuNP

Another type of reaction we have studied for the AuNP host encapsulation process is the hydrogenation of alkenes. The complex RhCl(PPh3)3 (also known as Wilkinson’s catalyst ) is a hydrophobic, highly active catalyst for hydrogenation reactions. We encapsulated Wilkinson catalyst into the AuNP host and ICP-MS detected 29.7 ± 6.9 catalysts per NP host. 0.7 mole percent of NP-Wilkinson catalyst were reacted with sodium 4-vinylbenzenesulfonate in water under hydrogen (3 atm) at room temperature for various reaction times. Reactions were monitored by NMR; after hydrogenation, the alkene peaks coming between 5.2-6.8 ppm in the

NMR spectrum disappear while triplet and quartet peaks appear due to the formation of ethyl group. The integration of those peaks was used to calculate conversion percentage of the hydrogenation. Results show ~100% conversion after 24 hours of reaction (Figure 5. 9 (c) and

Figure 5.10). These hydrogenation experimental conditions prove the AuNP-cat system can effectively catalyze not only monophasic water solvent systems (ROMP reaction and allylcarbamate bond breakage) but also gas-water biphasic systems (hydrogenation reaction).

Figure 5.9: a) The structure of Wilkinson catalyst. b) The reaction scheme of hydrogenation using NP host and sodium 4-vinylbenzenesulfonate. c) Hydrogenation conversion profile in terms of reaction time for AuNP-Wilkinson. d) Reusability test of AuNP-Wilkinson system for five consecutive cycles.

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1 Figure 5.10: H-NMR spectra of the hydrogenation reactions in water at the different times (1, 3, 6, 24, and 48 h).

Catalytic reusability is an important factor in catalyzed reactions for industrial applications. We have examined the reusability of AuNP host-catalyst system for hydrogenation reaction. After the first reaction, a molecular weight cutoff filter (10 k) was used to separate

AuNP-Wilkinson catalyst and the product, 4-ethylbenzenesulfonate. AuNP-Wilkinson catalyst

121 was recovered after three times washing and reacted again with substrate sodium 4- vinylbenzenesulfonate under the same conditions (room temperature, 3 atm). This cycle repeated up to five times and 100% conversion was obtained as a proof of the recyclability of NP-catalyst template (Figure 5.9 (d) and Figure 5.11).

1 Figure 5.11: H-NMR spectra of hydrogenation reactions for recyclability test, yielding 4- ethylbenzenesulfonate as the final product. All of samples were measured after 24 h reaction.

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5.3 Summary and Outlook

In summary, we have developed a versatile AuNP-based platform for the encapsulation of hydrophobic catalysts through host-guest interactions. This supramolecular system eliminates the dependence on organic solvents and provides cost-effective and environmentally safe catalysis. Our NP platform is amenable to encapsulate a variety of catalysts and catalyze a multitude of reactions in aqueous environments without the need to specifically tailor the host’s structure for each hydrophobic catalyst. The functionalized monolayer of the NP host serves as a phase transfer agent for the catalyst while concurrently preventing catalyst ‘drowning’ through supramolecular stabilization. Future studies will explore the use of this platform in biological systems and as multifunctional catalytic reactors.

5.4 Experimental Section

5.4.1 Materials and Instruments

All chemicals and materials for the experiments were obtained from Aldrich or Fisher

Scientific. Reaction activities were monitored by a microplate reader (Molecular Device

SpectroMax M2) for fluorescent product (Rhodamine 110). NMR spectroscopy (Bruker

AVANCE 400 at 400 MHz) for hydrogenation and THF GPC (Agilent Technologies 1260

Infinity) for ROMP reactions were used.

5.4.2 Synthesis of TMA and TTMA Coated AuNPs

First, Brust-Schiffrin two-phase synthesis method was used to synthesize pentanethiol- coated AuNPs with core diameter ca. 2 nm38,39 and 7 nm40. Murray place-exchange method41 was followed to obtain the TTMA or TMA ligand protected AuNPs. Pentanethiol-conjugated AuNPs

(10 mg, 2 nm) or Dodecanethiol-conjugated AuNP (10 mg, 7 nm) and TTMA or TMA ligand (27 mg) were dissolved in dry DCM and stirred under N2 atmosphere for 3 days at room temperature.

The DCM was removed under reduced pressure and the resulting precipitate was washed with hexane three times and DCM twice. Then the precipitate was dissolved in distilled water and

123 dialyzed for three days (membrane molecular weight cut-off =10,000) to remove excess ligands, pentanethiol/dodecanetiol, acetic acid, and other salts present in the nanoparticle solution. After dialysis, the particle was lyophilized to yield a solid brownish product. The particles were then redispersed in deionized water.42

5.4.3 Encapsulation of the Catalysts

The catalysts (Grubbs, Cp*Ru(cod)Cl, (Pd(dppf)Cl2), or Wilkinson catalysts) and the

AuNPs were dissolved in an acetone/water or tetrahydrofuran/acetone/water mixture. In the case of the 2 nm sized AuNPs, 1 ml of 20 μM AuNP solutions and 1 mg of each hydrophobic catalyst in 1 ml of acetone or tetrahydrofuran/acetone (1:9, v/v) were mixed. The 0.5 ml of 400 nM solutions was used for 7 nm sized AuNPs. For the 7 nm AuNPs, 0.1 mg of Grubbs catalyst (HG2

) was used. Then the acetone and tetrahydrofuran was slowly removed by evaporation. During the evaporation, most of the catalysts were encapsulated in monolayers and the remainder of the hydrophobic catalysts precipitated. The precipitate was removed by filtration and the AuNPs were purified by multiple filtrations through a molecular-weight cutoff filter to remove free catalysts, followed by dialysis against water to remove free catalysts in water.43

5.4.4 Quantification of the Catalysts

The ICP-MS analyses were performed on a Perkin-Elmer NexION 300X ICP mass spectrometer. 197Au, 102Ru, 106Pd, and 103Rh were measured under the standard mode. Operating conditions are listed as below: nebulizer flow rate: 0.95 L/min; rf power: 1600 W; plasma Ar flow rate: 18 L/min; dwell time: 50 ms. Standard gold, ruthenium, palladium, and rhodium solutions (concentration: 0, 0.2, 0.5, 1, 2, 5, 10, and 20 ppb) were prepared for the quantification.

5.4.4.1 ICP-MS Sample Preparation for the Quantification of Gold, Ruthenium, Palladium, and Rhodium

0.5 mL of fresh aqua regia was added to the 10 uL sample solution and then the sample was diluted to 10 mL with de-ionized water.

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Table 5.1: Quantification of encapsulated catalysts in the 2 nm TTMA-AuNP.

5.4.5 Laser Desorption/Ionization Mass Spectrometry (LDI-MS) Instrumentation:

The surface ligand coverage comparison was done using the previous reported LDI-MS method.44 The mixed disulphide ions were formed by TTMA/TMA ligand and original alkanethiol ligand. Higher amount of remaining alkanethiol ligands result in higher ion intensity ratio between the mixed disulphide ion and the intensity sum of TMA/TTMA ligand and mixed disulphide ion.5 The quantification data was acquired on a Bruker Autoflex III MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) (Autoflex III). Operating conditions were as follows: ion source 1 = 19.00 kV, ion source 2 = 16.60 kV, lens voltage = 8.44 kV, reflector voltage = 20.00 kV, reflector voltage 2 = 9.69 kV, pulsed ion extraction time = 10 ns, suppression = 100 Da, and at positive reflectron mode. Molecular ion and mixed disulphide ion from each molecule has been chosen as quantification ion in the data analysis, for example:

TTMA (m/z 422), TMA (m/z 246), TTMA-C5 (m/z 524), and TMA-C5 (m/z 348). 40 mass spectra have been acquired for each sample, and each mass spectrum represents an average of 200 laser shots.

5.4.6 ROMP Reaction in Water by Using AuNP-HG2

520 nM of HG2 catalyst encapsulated 2 nm TTMA NPs was mixed with 6 mM of water soluble norbornene monomer in 3 ml water. After polymerization, water was lyophilized to yield a solid brownish product and THF (0.5 mL) was added to separate NP host and polymer from each other. NP host remained insoluble in THF while obtained polymers are transferred into THF

125 layer. Polymerization reaction was monitored for 1, 4, 6, and 24 h and THF GPC (Agilent

Technologies 1260 Infinity) was used to calculate molecular weights and PDI values of polymers.

Figure 5.12: Photos of the reaction mixtures including a) NP Host or b) NP Host-HG2 after 24 h reactions.

126

Figure 5.13: GPC spectra of the polymers that were synthesized by ROMP in water at different time intervals (1, 4, 6, and 24 h).

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Figure 5.14: GPC spectra of reactions that were performed using free catalysts in acetone/water mixture at different time intervals (1, 4, 6, and 24 h).

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5.4.7 Allyl Carbamate Bond Cleavage Reaction by Using AuNP-Cp*Ru(cod)Cl or AuNP- (Pd(dppf)Cl2)

Cp*Ru(cod)Cl or (Pd(dppf)Cl2) encapsulated 2 nm TTMA NPs (194 nM) was mixed with caged Rhodamine 110 (220 μM) in a final volume of 1 ml water. Green fluorescence of

Rhodamine 110 was observed as result of catalytic allycarbamate deprotection reaction. UV absorbance of the Rhodamine 110 at a wavelength of 500 nm wavelength was monitor for reaction activity. Fluoresence intensity of Rhodamine 110 was monitored at λex = 480 nm, λem

= 530 nm, and λcutoff= 515 nm.

5.4.8 Hydrogenation of Sodium 4-vinylbenzenesulfonate via AuNP-Wilkinson Catalyst

Wilkinson catalyst encapsulated 2 nm TTMA NPs (23 μM, 1.2 mL) was mixed with sodium 4-vinylbenzenesulfonate (2.3 mg, 0.8 mL) in water under 3 atm H2 at room temperature.

Reactions were monitored by NMR for various time intervals (1, 3, 6, 24, 48 h). For the reusability test, same conditions were applied.

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5.5 References

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CHAPTER 6

ENHANCING CATALYTIC ACTIVITY OF NANOZYME BY MONOLAYER ENGINEERING

6.1 Introduction

Transition metal catalysis (TMC) has received considerable attention for the potential abiotic reactions in biological system. Their bioorthogonality and high turnover rate can provide selective and efficient chemistries in living systems.1 Numerous types of TMCs have been demonstrated to be used for cell imaging,2 enzyme regulation,3 protein-,4 and cell-labeling5 strategies. Despite these significant advantages, hydrophobicity and low water solubility of TMCs result in nonspecific interactions with biomolecules, inducing protein aggregation and denaturation.6 Additionally, it has been recently reported that free TMC could inhibit the protein expression.7 As a result, several TMCs with hydrophilic ligands have been developed to improve their water solubility, but it adversely decreases cell membrane permeability, reducing the potential for intracellular chemistry.5 Therefore, the development of catalytic platform which prevents TMC from interaction with biological molecules is needed to improve their utility.

Nanomaterials have been utilized as highly dispersible and biocompatible scaffolds for broad range of bio-applications.8 These materials commonly possess the surface monolayer to improve their solubility and control the biological interactions,9 so such layer could provide

TMCs with a protective environment. Besides, this approach could design the nanomaterial-TMC complexes, so-called “nanozyme” with multiple catalytic centers10 which afford the higher local concentration of TMC inside cell than free TMC systems. Recently, the groups of Bradley and

Unciti-Broceta synthesized palladium (0) nanoparticle bound-polystyrene microsphere (0.5-150

µm) and demonstrated that palladium nanoparticle mediated reaction can induce the activation of fluorophore or prodrug.11 In the course of our researches, we have utilized gold nanoparticles

(AuNPs) (ca. 2 nm core size) with surface monolayer featuring two layers such as hydrophobic

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(green, Scheme 6.1), hydrophilic layers (blue) and found that hydrophobic layer of nanoparticle surface can capture the hydrophobic catalyst.12 The number of encapsulated catalyst dictates the catalytic activity, however, is insufficient, resulting in slow reaction rate. For the efficient amplification of catalytic process, the higher catalytic activity is preferred. Nevertheless, the structural information to improve the amount of TMC per particle and hence catalytic activity has been lacked. In this work, we present the development of five different AuNP-Cp*Ru(cod)Cl

(Cp* = pentamethylcyclopentadienyl, cod = cyclooctadiene) complexes to provide the structural information for an efficient catalytic activity. By examining the chemical structure of hydrophobic layer of nanoparticle surface, we have improved the catalyst loading per particle, increasing the catalytic efficiency for the activation of profluorogenic N,N’-bisallyloxycarbonyl rhodamine 110 in aqueous solution.

Scheme 6.1: Schematic showing the increase in the catalyst loading in three nanozymes used in the study.

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6.2 Results and Discussion

6.2.1 Design of AuNP Monolayer

Catalytic scaffold we utilized for previous research has 11-carbon chain which forms a hydrophobic layer on AuNP which drives the hydrophobic catalyst encapsulation. For the enhanced catalyst loading, we designed four different nanozyme monolayers: C16, Phenyl,

Naphthyl, and Pyrene AuNP (Figure 6.1). C16-AuNP is obtained by introducing additional 5- carbon to increase the volume of hydrophobic layer. In addition, we also introduced phenyl, naphthyl and pyrene moieties into hydrophobic layer to afford the favorable π-π stacking interactions between nanoparticle ligand and Cp*Ru(cod)Cl.

Figure 6.1: Monolayer structures of nanozymes used in the study.

6.2.2 Encapsulation of Hydrophobic Cp*Ru(cod)Cl Catalyst into AuNP

With newly developed AuNPs, we carried out the encapsulation of Cp*Ru(cod)Cl

(Figure 6.2). An aqueous solution of AuNPs was mixed with the solution of Cp*Ru(cod)Cl in acetone. The mixture was slowly evaporated in vacuo, and sonicated. After the filtration, we carried out the dialysis to remove unbound Ru catalyst. Purified AuNPs-Cp*Ru(cod)Cl

138 complexes were subjected to characterization such as dynamic light scattering and are highly soluble in aqueous solution.

Figure 6.2: Catalyst encapsulation into the monolayer of nanozyme.

In addition, inductively coupled plasma mass spectrometry (ICP-MS) determined the amount of Cp*Ru(cod)Cl encapsulated into each functionalized AuNPs and we detected around three times more catalyst in C16 and Phenyl AuNP and around five times more in Naphthyl and

Pyrene AuNP compared to C11 AuNP (Figure 6.3). These are because of increase of volume of hydrophobic pocket as well as the strong π-π interaction between catalyst and hydrophobic pocket as expected.

Figure 6.3: Amount of catalyst in nanozymes. All experiments were carried out in triplicate, and error bars represent standard deviation.

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6.2.3 Catalytic Activity of Nanozymes in Solution

Next, we investigated the catalytic activity of synthesized nanozymes. To examine the activity, we used N,N’-allyloxycarbonyl rhodamine 110 as a substrate which is non fluorescent molecule and can be activated to fluorescent molecule in the presence of Cp*Ru(cod)Cl catalyst

(Figure 6.4).

Figure 6.4: Activation of bisallyloxycarbonyl rhodamine 110 catalyzed by AuNP_Ru nanozyme.

Nanozymes (0.5 µM) are dissolved in the solution of N,N’-allyloxycarbonyl rhodamine

110 (110 µM) in 5 mM phosphate buffer (PB), and tracked the time dependent increase of fluorescent (Excitation 485nm, Emission 535 nm, Cutoff 515 nm). As a result, we observed enhanced catalytic activity of C16, Phenyl, Naphthyl, and Pyrene-AuNP compared to C11-AuNP.

C16 showed around 3 times higher catalytic activity than C11 counterpart presumably because of the enhancement of catalyst amount per particle as well as increase of surface area of hydrophobic pocket by the introduction of additional C5 chain. Naphthyl and Pyrene exhibited the highest activity among five nanozymes, showing around 7-8 times stronger activity than nanozyme bearing C11 chain (Figure 6.5). Notably, no reaction was observed with the AuNPs without catalyst formulation.

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Figure 6.5: Catalytic activity of nanozymes under ambient temperature. All experiments were carried out in triplicate, and error bars represent standard deviation.

6.2.4 Catalytic Activity inside Living Cells

Having characterized the activity of nanozyme catalysis in solution, we next studied the intracellular behavior of these nanozymes. We preformed the activation of prodrugs inside cells using nanozymes bearing five different monolayer designs. We protected amine of Doxorubicin

(Dox) using allylcarbamate moiety to convert toxic Dox into non-toxic prodrug form (Alloc-Dox)

(Figure 6.6). Deprotection of alloc groups by ruthenium catalyst embedded nanozymes yields the toxic Dox, resulting in cell death (Figure 6.7).

Figure 6.6: Activation of pro-Dox using ruthenium catalyst embedded nanozyme.

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Figure 6.7: Cell viability of Dox versus pro-Dox (Alloc-Dox).

Cell viability studies for prodrug system in the presence of nanozymes were carried out using HeLa cells in a 96 well plate. Cells were first incubated with nanozymes (C11, Phenyl,

Naphthyl, C16, and Pyrene-TTMA) at a concentration of 250 nM in serum-containing media for

24 h. Then, after multiple washings, cells were incubated with pro-Dox (5 µM) for another 24 h.

Cells were washed off with PBS buffer three times and 10% Alamar Blue in serum containing media was added to each well (220 µL) and further incubated at 37 °C for 4 h. The cell viability was then determined by measuring the fluorescence intensity at 570 nm using a SpectraMax M5 microplate spectrophotometer. Results showed that C11-TTMA nanozyme did not show much toxicity as the cell viability was around 90%. However, when nanozymes carrying higher amount of catalysts were used, a significant decrease in cell viability was observed (Figure 6.8). The highest cell death was obtained when cells were incubated with Pyrene-TTMA.

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Figure 6.8: Viability of cells incubated with nanozymes with different monolayer structures and pro-Dox.

6.3 Summary and Future Outlook

In summary, we have designed and synthesized three AuNP-Cp*Ru(cod)Cl complexes having the different functionalities in the hydrophobic layer of surface monolayer. The catalyst loading into those designed AuNPs as well as a catalytic activity of nanozyme can be tuned by the chemical structure of hydrophobic pocket, leading us to develop the nanozyme having higher catalytic activity for the cleavage reaction of allyloxycarbonyl group than previously reported nanozyme. This enhanced catalytic activity could greatly improve the potential of our nanozyme system for the future in vitro and in vivo applications.

6.4 Experimental Section

6.4.1 General

All the chemicals were purchased from Sigma-Aldrich or Fischer Scientific, unless otherwise specified. The chemicals were used as received. Dichloromethane (DCM) and tetrahydrofuran (THF) used as a solvent for chemical synthesis and dried according to standard procedures. The yields of the compounds reported here refer to the yields of spectroscopically pure compounds after purification. 1H NMR spectra were recorded at 400 MHz on a Bruker

AVANCE 400 machine.

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6.4.2 Synthesis of Ligands

Figure 6.9: Synthesis of compound S2 from compound S1.

2,2-Dimethyl-3,3-diphenyl-4,7,10,13-tetraoxa-3-silapentadecan-15-oicacid (S2). To a solution of compound S1 (5.9 g, 13.6 mmol) and TEMPO (213 mg, 1.36 mmol) in CH2Cl2 (45.8 mL) was added a solution of KBr (145 mg, 1.22 mmol), tetra-n-butyl-ammonium bromide (219 mg, 0.68 mmol) in sat. NaHCO3 (27.3 mL). After addition of a solution of NaOCl (5%, 65.9 mL,

54.4 mmol) in sat. NaHCO3 (15.9 mL) and brine (31 mL) at 0 °C, the mixture was stirred at 0 °C for 30 min. CHCl3, H2O, and KHSO4 (5.96 g, 43.5 mmol) were added, and the whole was extracted with CHCl3. The mixture was washed with brine (× 2), and dried over MgSO4. After concentration, the residue was purified by flash chromatography over silica gel with n-hexane–

EtOAc–AcOH (50:50:0 to 49:50:1) to give the desired compound S2 as pale yellow oil (5.45 g,

1 90%). H-NMR (400 MHz, CDCl3) 1.06 (9H, s, -CH3), 3.62-3.83 (12H, m, -CH2-), 4.15 (2H, s, -

CH2-), 7.39-7.41 (6H, m, Ar), 7.69-7.70 (4H, m, Ar). MALDI-MS: m/z calculated for

+ C24H34NaO6Si [M + Na] 469.202 ; found: 469.199

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Ligand with Pyrene (12)

Figure 6.10: Synthesis of pyrene ligand.

5,5'-(Pyrene-1,6-diyl)bis(pent-4-yn-1-ol) (2). To a suspension of 1,6-dibromopyrene

(10.7 g, 29.7 mmol), Pd(PPh3)2Cl2 (1.04 g, 1.5 mmol) and CuI (283 mg, 1.5 mmol) in anhydrous

THF (252 mL) and triethylamine (degassed, 252 mL) was added 1-pentyn-5-ol (10 g, 119 mmol) at rt. After being stirred at 80 °C for 5 h, the mixture was filtered through Celite pad and washed with CHCl3. After concentration, CHCl3 and 1M HCl were added, and the mixture was sonicated and solid was filtered through filter paper to give the desired compound 2 as pale yellow solid

(8.66 g, 80%). This compound was used for next step without further purification. 1H-NMR (400

6 MHz, DMSO-d ) 1.83-1.90 (4H, m, -CH2-), 2.73 (4H, t, J = 7.1 Hz, -CH2-), 3.64-3.68 (4H, m, -

CH2-), 4.67 (2H, t, J = 5.2 Hz, -OH), 8.12 (2H, d, J = 8.0 Hz, Ar), 8.27-8.29 (4H, m, Ar), 8.49

+ (2H, d, J = 9.0 Hz, Ar). MALDI-MS: m/z calculated for C26H22O2 [M] 366.162; found: 366.172.

5,5'-(Pyrene-1,6-diyl)bis(pentan-1-ol) (3). To a suspension of compound 2 (8.66 g, 23.6 mmol) in MeOH (92.5 mL) and CH2Cl2 (278 mL) was added 5% Pd-C (2.6 g, 1.18 mmol) at rt.

After being stirred at rt under H2 atmosphere for overnight, the mixture was filtered through

Celite pad. The solvent was evaporated in vacuo to give the desired compound 3 as pale yellow

145 solid (8.21 g, 93%). This compound was used for next step without further purification. 1H-NMR

(400 MHz, CDCl3) 1.51-1.72 (10H, m, -CH2-, -OH), 1.88-1.95 (4H, m, -CH2-), 3.37 (2H, t, J =

7.7 Hz, -CH2-), 3.69 (2H, t, J = 6.4 Hz, -OCH2-), 7.87 (2H, d, J = 7.8 Hz, Ar), 8.06-8.12 (4H, m,

+ Ar), 8.23 (2H, d, J = 9.2 Hz, Ar). MALDI-MS: m/z calculated for C26H30O2 [M] 374.225; found:

374.219.

Pyrene-1,6-diylbis(pentane-5,1-diyl)-dimethanesulfonate (4). To a suspension of compound 3 (8.21 g, 21.9 mmol) and triethylamine (7.9 mL, 54.8 mmol) in CH2Cl2 (62.3 mL) was added dropwise methanesulfonyl chloride (4.2 mL, 54.8 mmol) at 0 °C. After being stirred at

0 °C for 30 min, the mixture was washed with 0.1M HCl, sat. NaHCO3 and brine, and dried over

MgSO4. The solvent was evaporated in vacuo to give the desired compound 4 as pale yellow solid (11.6 g, 99%). This compound was used for next step without further purification. 1H-NMR

(400 MHz, CDCl3) 1.58-1.65 (4H, m, -CH2-), 1.82-1.97 (8H, m, -CH2-), 2.97 (6H, s, -CH3), 3.38

(4H, t, J = 7.6 Hz, -CH2-), 4.25 (4H, t, J = 6.5 Hz, -CH2-), 7.87 (2H, d, J = 7.8 Hz, Ar), 8.07-8.13

+ (4H, m, Ar), 8.22 (2H, d, J = 9.2 Hz, Ar). MALDI-MS: m/z calculated for C28H34O6S2 [M]

530.180; found: 530.175.

5-[6-(5-Azidopentyl)pyren-1-yl]pentyl-methanesulfonate (5). To a solution of compound 4 (11.6 g, 21.9 mmol) in DMF (200 mL) was added NaN3 (1.42 g 21.9 mmol) at 60

°C. After being stirred at 60 °C for 2 h, EtOAc was added. The mixture was washed with brine (×

6), and dried over MgSO4. After concentration, the residue was purified by flash chromatography over silica gel with n-hexane–CHCl3 (1:1 to 0:1) to give the desired compound 5 as pale yellow

1 solid (5.28 g, 51%). H-NMR (400 MHz, CDCl3) 1.54-1.74 (6H, m, -CH2-), 1.81-1.96 (6H, m, -

CH2-), 2.96 (3H, s, -CH3), 3.30 (2H, t, J = 6.8 Hz, -CH2-), 3.28-3.39 (4H, m, -CH2-), 4.24 (2H, t,

J = 6.5 Hz, -OCH2-), 7.85-7.88 (2H, m, Ar), 8.07-8.13 (4H, m, Ar), 8.20-8.23 (2H, m, Ar).

+ MALDI-MS: m/z calculated for C27H31N3O3S [M] 477.209; found: 477.215.

146

5-{6-[5-(Tritylthio)pentyl]pyren-1-yl}pentan-1-amine (7). To a solution of compound

5 (5.28 g, 11.1 mmol) in CHCl3 (40 mL) was added dropwise a mixture of triphenylmethyl mercaptan (6.7 g 24.3 mmol) and NaOEt in EtOH (21%, 7.16 mL, 22.1 mmol) at rt. After being stirred at 60 °C for 2.5 h, EtOAc was added. The mixture was washed with sat. NaHCO3 (× 2) and brine, and dried over MgSO4. After concentration, the residue was dissolved in THF (110 mL) and H2O (1.1 mL) and triphenylphosphine (5.8 g, 22.1 mmol) was added. After being stirred at 60 °C for 2.5 h, the solvent was evaporated in vacuo. The residue was purified by flash chromatography over silica gel with CHCl3–MeOH–NH4OH (100:0:0 to 89:10:1) to give the desired compound 7 as orange amorphous (4.14 g, 59%). 1.42-1.93 (14H, m, -CH2-), 2.19 (2H, t,

J = 6.9 Hz, -CH2-), 2.74 (2H, t, J = 6.6 Hz, -CH2-), 3.26 (2H, t, J = 7.7 Hz, -CH2-), 3.36 (2H, t, J

= 7.6 Hz, -CH2-), 7.19-7.47 (15H, m, Ar), 7.80 (1H, d, J = 7.8 Hz, Ar), 7.86 (1H, d, J = 7.8 Hz,

Ar), 8.03-8.11 (4H, m, Ar), 8.08 (1H, d, J = 9.2 Hz, Ar), 8.22 (1H, d, J = 9.2 Hz, Ar). MALDI-

+ MS: m/z calculated for C45H45NNaS [M + Na] 654.317; found: 654.340.

2,2-Dimethyl-3,3-diphenyl-N-(5-{6-[5-(tritylthio)pentyl]pyren-1-yl}pentyl)-4,7,10,13- tetraoxa-3-silapentadecan-15-amide (8). To a solution of compound 7 (1.5 g, 2.4 mmol), compound S2 (1.1 g, 2.4 mmol), diisopropylethylamine (1.3 mL, 7.2 mmol), and HOBt·H2O (368 mg, 2.4 mmol) in DMF (24 mL) was added EDC·HCl (920 mg, 4.8 mmol) at rt. After being stirred at rt for overnight, EtOAc was added. The mixture was washed with 0.1 M HCl (× 2), sat.

NaHCO3 (× 2) and brine (× 2), and dried over MgSO4. After concentration, the residue was purified by flash chromatography over silica gel with n-hexane–EtOAc (1:1 to 0:1) to give the

1 desired compound 8 as pale yellow oil (2.1 g, 81%). H-NMR (400 MHz, CDCl3) 1.05 (9H, s, -

CH3), 1.43-1.63 (8H, m, -CH2-), 1.71-1.92 (4H, m, -CH2-), 2.19 (2H, t, J = 6.8 Hz, -SCH2-), 3.24-

3.38 (6H, m, -CH2-), 3.56-3.64 (10H, m, -OCH2-), 3.80 (2H, t, J = 5.1 Hz, -OCH2-), 3.97 (2H, s, -

OCH2-), 6.98 (1H, br t, J = 5.7 Hz, -NH-), 7.19-7.45 (21H, m, Ar), 7.67-7.70 (4H, m, Ar), 7.80

(1H, d, J = 7.8 Hz, Ar), 7.84 (1H, d, J = 7.8 Hz, Ar), 8.02-8.09 (4H, m, Ar), 8.16 (1H, d, J = 9.2

147

Hz, Ar), 8.20 (1H, d, J = 9.2 Hz, Ar). MALDI-MS: m/z calculated for C69H77NNaO5SSi [M +

Na]+ 1082.519; found: 1082.597.

11-Oxo-17-{6-[5-(tritylthio)pentyl]pyren-1-yl}-3,6,9-trioxa-12-azaheptadecyl- methanesulfonate (10). To a solution of compound 8 (2.0 g, 1.9 mmol) in THF (37.6 mL) was added a solution of tetrabutylammonium fluoride in THF (1M, 3.76 mL, 3.8 mmol) at rt. After being stirred at rt for 2h, the reaction was quenched with sat. NH4Cl. After concentration, the residue was purified by flash chromatography over silica gel with EtOAc–MeOH (1:0 to 4:1). To a solution of intermediate and triethylamine (407 µL, 2.8 mmol) in CH2Cl2 (18.8 mL) was added dropwise methanesulfonyl chloride at 0 °C. After being stirred at 0 °C for 30 min, EtOAc was added. The mixture was washed with 0.1 M HCl, sat. NaHCO3 and brine, and dried over MgSO4.

After concentration, the residue was purified by flash chromatography over silica gel with -

EtOAc–MeOH (1:0 to 9:1) to give the desired compound 10 as pale yellow oil (1.55 g, 92%). 1H-

NMR (400 MHz, CDCl3) 1.43-1.78 (10H, m, -CH2-), 1.89-1.95 (2H, m, -CH2-), 2.19 (2H, t, J =

6.8 Hz, -SCH2-), 3.01 (3H, s, -CH3), 3.24-3.38 (6H, m, -CH2-), 3.57-3.70 (10H, m, -OCH2-), 3.98

(2H, s, -OCH2-), 4.30-4.32 (2H, m, -OCH2-), 6.87 (1H, br t, J = 5.2 Hz, -NH-), 7.19-7.30 (9H, m,

Ar), 7.41-7.44 (6H, m, Ar), 7.80 (1H, d, J = 7.8 Hz, Ar), 7.86 (1H, d, J = 7.8 Hz, Ar), 8.03-8.11

(4H, m, Ar), 8.16 (1H, d, J = 9.2 Hz, Ar), 8.21 (1H, d, J = 9.2 Hz, Ar). MALDI-MS: m/z

+ calculated for C54H61NNaO7S2 [M + Na] 922.379; found: 922.413.

17-[6-(5-Mercaptopentyl)pyren-1-yl]-N,N,N-trimethyl-11-oxo-3,6,9-trioxa-12- azaheptadecan-1-aminium-methanesulfonate (12). To a solution of compound 10 (500 mg,

0.56 mmol) in CHCl3 (0.84 mL) was added a solution of trimethylamine in EtOH (2.8 mL) at rt.

After being stirred at 50 °C for overnight, the mixture was concentrated in vacuo. To a solution of residue in CH2Cl2 (3.0 mL) were added trifluoroacetic acid (1.0 mL) and triisopropylsilane (0.25 mL). After being stirred at rt for 10 min, the mixture was concentrated in vacuo. The residue was suspended in n-hexane, and sonicated. After centrifugation, the supernatant was removed. This n-

148 hexane-washing step was repeated five times to give the desired 12 as pale yellow oil (395 mg,

1 99%). H-NMR (400 MHz, CDCl3) 1.47-1.77 (8H, m, -CH2-), 1.85-1.92 (4H, m, -CH2-), 2.57

(2H, q, J = 7.3 Hz, -SCH2-), 2.84 (3H, s, -CH3), 3.14 (9H, s, -CH3), 3.29-3.38 (6H, m, -CH2-),

+ 3.50-3.64 (10H, m, -OCH2-), 3.86 (2H, br s, -N CH2-), 4.04 (2H, s, -OCH2-), 6.99 (1H, br t, J =

5.7 Hz, -NH-), 7.86 (1H, d, J = 7.8 Hz, Ar), 7.87 (1H, d, J = 7.8 Hz, Ar), 8.07-8.13 (4H, m, Ar),

8.22 (1H, d, J = 9.2 Hz, Ar), 8.23 (1H, d, J = 9.2 Hz, Ar). MALDI-MS: m/z calculated for

+ C37H53N2O4S [M] 621.372; found: 621.403.

Ligand with aliphatic group (21):

Figure 6.11: Synthesis of C16 ligand.

16-(Tritylthio)hexadecyl-methanesulfonate (14). To a suspension of compound 13 (2.6 g, 5.0 mmol) and triethylamine (1.45 mL, 10 mmol) in CH2Cl2 (10 mL) was added dropwise methanesulfonyl chloride (483 µL, 6.3 mmol) at 0 °C. After being stirred at 0 °C for 10 min, the mixture was washed with 0.1M HCl, sat. NaHCO3 and brine, and dried over MgSO4. The solvent was evaporated in vacuo to give the desired compound 14 as colorless solid (2.33 g, 77%). This

1 compound was used for next step without further purification. H-NMR (400 MHz, CDCl3) 1.19-

1.45 (26H, m, -CH2-), 1.73-1.80 (2H, m, -CH2-), 2.15 (2H, t, J = 7.3 Hz, -SCH2-), 3.02 (3H, s, -

OCH3), 4.24 (2H, t, J = 6.6 Hz, -OCH2-), 7.21-7.32 (9H, m, Ar), 7.42-7.44 (6H, m, Ar).

(16-Azidohexadecyl)(trityl)sulfane (15). To a solution of compound 14 (2.3 g, 3.9 mmol) in DMF (19.4 mL) was added NaN3 (503 mg 7.8 mmol) at rt. After being stirred at 50 °C

149 for 2 h, EtOAc was added. The mixture was washed with brine (× 5), and dried over MgSO4. The solvent was evaporated in vacuo to give the desired compound 15 as colorless solid (1.81 g,

86%). This compound was used for next step without further purification. 1H-NMR (400 MHz,

CDCl3) 1.14-1.64 (28H, m, -CH2-), 2.15 (2H, t, J = 7.3 Hz, -SCH2-), 3.28 (2H, t, J = 7.0 Hz, -

OCH2-), 7.20-7.42 (9H, m, Ar), 7.42-7.45 (6H, m, Ar).

16-(Tritylthio)hexadecan-1-amine (16). To a solution of compound 15 (1.8 g, 3.3 mmol) in in THF (33 mL) and H2O (0.33 mL) was added triphenylphosphine (1.74 g, 6.6 mmol).

After being stirred at 40 °C for overnight, the solvent was evaporated in vacuo. The residue was purified by flash chromatography over silica gel with CHCl3–MeOH–NH4OH (100:0:0 to

80:19:1) to give the desired compound 16 as pale yellow solid (1.5 g, 88%). 1H-NMR (400 MHz,

CDCl3) 1.19-1.49 (30H, m, -CH2-, NH2), 2.16 (2H, t, J = 7.3 Hz, -SCH2-), 2.71 (2H, t, J = 7.0 Hz,

-OCH2-), 7.21-7.42 (9H, m, Ar), 7.43-7.45 (6H, m, Ar). MALDI-MS: m/z calculated for

+ C35H49NNaS [M + Na] 538.348; found: 538.432.

2,2-Dimethyl-3,3-diphenyl-N-[16-(tritylthio)hexadecyl]-4,7,10,13-tetraoxa-3- silapentadecan-15-amide (17). To a solution of compound 16 (1.5 g, 2.9 mmol), compound S2

(1.3 g, 2.9 mmol), diisopropylethylamine (1.6 mL, 8.7 mmol), and HOBt·H2O (444 mg, 2.9 mmol) in DMF (29 mL) was added EDC·HCl (1.11 g, 5.8 mmol) at rt. After being stirred at rt for overnight, EtOAc was added. The mixture was washed with 0.1 M HCl (× 2), sat. NaHCO3 (× 2) and brine (× 2), and dried over MgSO4. After concentration, the residue was purified by flash chromatography over silica gel with n-hexane–EtOAc (1:1 to 1:2) to give the desired compound

1 17 as pale yellow oil (2.6 g, 96%). H-NMR (400 MHz, CDCl3) 1.07 (9H, s, -CH3), 1.19-1.53

(28H, m, -CH2-), 2.15 (2H, t, J = 7.3 Hz, -SCH2-), 3.26 (2H, q, J = 6.8 Hz, -NCH2-), 3.61-3.68

(10H, m, -OCH2-), 3.83 (2H, t, J = 5.3 Hz, -OCH2-), 3.99 (2H, s, -OCH2-), 6.96 (1H, br s, -NH-),

7.21-7.46 (21H, m, Ar), 7.69-7.71 (4H, m, Ar). MALDI-MS: m/z calculated for C59H81NNaO5SSi

[M + Na]+ 966.550; found: 966.657.

150

20-Oxo-1,1,1-triphenyl-22,25,28-trioxa-2-thia-19-azatriacontan-30-yl- methanesulfonate (19). To a solution of compound 17 (2.6 g, 2.8 mmol) in THF (55 mL) was added a solution of tetrabutylammonium fluoride in THF (1M, 5.5 mL, 5.5 mmol) at rt. After being stirred at rt for 2h, the reaction was quenched with sat. NH4Cl. After concentration, the residue was purified by flash chromatography over silica gel with EtOAc–MeOH (1:0 to 4:1). To a solution of intermediate and triethylamine (596 µL, 4.12 mmol) in CH2Cl2 (27.5 mL) was added dropwise methanesulfonyl chloride (316 µL, 4.12 mmol) at 0 °C. After being stirred at 0 °C for

30 min, EtOAc was added. The mixture was washed with 0.1 M HCl, sat. NaHCO3 and brine, and dried over MgSO4. After concentration, the residue was purified by flash chromatography over silica gel with EtOAc–MeOH (1:0 to 9:1) to give the desired compound 19 as pale yellow oil

1 (2.11 g, 98%). H-NMR (400 MHz, CDCl3) 1.19-1.57 (28H, m, -CH2-), 2.15 (2H, t, J = 7.3 Hz, -

SCH2-), 3.08 (3H, s, -CH3), 3.30 (2H, q, J = 6.8 Hz, -NCH2-), 3.61-3.80 (10H, m, -OCH2-), 4.01

(2H, s, -OCH2-), 4.40 (2H, t, J = 4.7 Hz, -OCH2-), 6.85 (1H, br s, -NH-), 7.20-7.31 (9H, m, Ar),

+ 7.42-7.44 (6H, m, Ar). MALDI-MS: m/z calculated for C44H65NNaO7S2 [M + Na] 806.410; found: 806.487.

28-Mercapto-N,N,N-trimethyl-11-oxo-3,6,9-trioxa-12-azaoctacosan-1-aminium- methanesulfonate (21). To a solution of compound 19 (500 mg, 0.64 mmol) in CHCl3 (0.32 mL) was added a solution of trimethylamine in EtOH (3.2 mL) at rt. After being stirred at 50 °C for overnight, the mixture was concentrated in vacuo. To a solution of residue in CH2Cl2 (3.0 mL) were added trifluoroacetic acid (1.0 mL) and triisopropylsilane (0.25 mL). After being stirred at rt for 10 min, the mixture was concentrated in vacuo. The residue was suspended in n-hexane, and sonicated. After centrifugation, the supernatant was removed. This n-hexane-washing step was repeated five times to give the desired compound 21 as colorless oil (380 mg, 99%). 1H-NMR

(400 MHz, CDCl3) 1.26-1.63 (28H, m, -CH2-), 2.54 (2H, q, J = 7.1 Hz, -SCH2-), 2.86 (3H, s, -

+ CH3), 3.30 (9H, s, -CH3), 3.66-3.71 (10H, m, -OCH2-), 3.98 (2H, br s, -N CH2-), 4.11 (2H, s, -

151

+ OCH2-), 7.09 (1H, br s, -NH-). MALDI-MS: m/z calculated for C27H57N2O4S [M] 505.403; found: 505.407.

6.4.3 Synthesis of Gold Nanoparticles and Characterization

Pyrene-AuNP was prepared through place-exchange reaction of 1-pentanethiolprotected

2 nm gold nanoparticle (Au-C5) according to previously reported procedure.(3) Briefly, to the solution of Au-C5 (20 mg) in CH2Cl2 (1 mL) was added the solution of pyrene ligand 1 (72 mg,

0.1 mmol) in CH2Cl2:MeOH (4:1, 3 mL). After being stirred at rt for 24 h, the solvent was evaporated in vacuo. After nanoparticle residue was washed with EtOAc (10 mL×3), the nanoparticle was immediately dissolved in MilliQ water and the aqueous solution of the nanoparticle was purified by dialysis with distilled water using SnakeSkinTM Dialysis Tubing

(Thermo Scientific, 10,000 MWCO). The resulting AuNP was lyophilized and dissolved in

CH2Cl2 (1 mL) and MeOH (3 mL) and additional amount of ligand 1 (72 mg) was added. After being stirred at rt for 24 h, the solvent was evaporated in vacuo. After nanoparticle residue was washed with EtOAc (10 mL × 3), the nanoparticle was immediately dissolved in MilliQ water and the aqueous solution of the nanoparticle was purified by dialysis with distilled water using

SnakeSkinTM Dialysis Tubing (Thermo Scientific, 10,000 MWCO).; MALDI-MS: m/z calculated

+ for C37H53N2O4S [M] 621.372; found: 621.660. C16, Phenyl, and Naphthyl-TTMA were prepared by following the similar procedure to Pyrene-AuNP.

Size and zeta potential of AuNPs were recorded in 5 mM phosphate buffer (pH 7.4) at room temperature at a concentration of 1µM, using a Malvern Zetasizer Nano ZS.

152

Table 6.1: Size and zeta potential of nanozymes used in the study. Size (nm)Sd.(nm)a Size (nm)Sd.(nm)b Zeta potential(mV)

Sd.(mV)

Phenyl-TTMA 8.384.35 9.223.88 18.44.12

Naphthyl-TTMA 8.852.61 8.015.73 15.26.60

C16-TTMA 8.913.80 8.345.06 15.26.56

Pyrene-TTMA 8.532.85 7.612.87 18.34.34 a: Before catalyst encapsulation, b: After catalyst encapsulation.

153

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CHAPTER 7

SUPRAMOLECULAR REGULATION OF BIOORTHOGONAL CATALYSIS IN CELLS USING NANOPARTICLE-EMBEDDED TRANSITION METAL CATALYSTS

7.1 Introduction

Bioorthogonal chemistry1-3 is a promising strategy for the intracellular generation of molecules for therapeutic4 and imaging applications5,6 unattainable through naturally occurring bioprocesses.7,8 Transition metal catalysts (TMCs) are excellent candidates for use in bioorthogonal processes,9-11 rapidly catalyzing transformations that cannot be performed via enzymatic processes.12-15 However, the application of TMC-mediated reactions in living cells is challenging due to issues of biocompatibility, water solubility, catalyst stability, and rapid efflux of catalysts from living cells.12,13

Loading of TMCs into nanomaterial scaffolds can be used to provide water solubility and a protective environment for TMCs. Bradley et al.13 and Unciti-Broceta et al.16 used palladium- catalyst loaded polystyrene beads to catalyze reactions such as Suzuki-Miyaura coupling and alkylcarbamate / N-propargyl cleavage inside and outside cells, respectively. The particles used in these studies, however, were far larger than normal proteins, creating potential interference in cellular processes. Additionally, these particles did not provide the capability of mimicking allosteric regulation of enzymes, a key component in cellular homeostasis.17 The integration of biomimetic size and controlled response into a bioorthogonal catalysis platform would provide new avenues for both therapeutics and integrated biological/abiotic cellular systems.

In this work, we have developed a family of gold nanoparticles (AuNPs) based on ~2 nm core size18-20 that feature biomimetic size, possess diverse functional properties,21,22 and are efficiently transport of into cells.23,24 In this chapter, we report the use of this AuNP structural motif to encapsulate25 hydrophobic TMCs, providing NP_Ru (Figure 7.1) or NP_Pd. The

156 resulting nanozymes26-30 feature surface moieties that can be reversibly functionalized using host- guest chemistry31,32 to provide NP_Ru_CB[7] or NP_Pd_CB[7]. Complexation of the monolayer terminal functionalities by cucurbit[7]uril (CB[7])33-35 in this system blocks access to the catalytic site, resulting in essentially complete inhibition of catalytic activity. The gatekeeper molecules36,37 can then be released from the AuNPs using competitive guests,38 restoring catalytic activity (Figure 7.1). The efficacy of this system was demonstrated in solution and in cells through two applications: 1) the gated generation of a fluorophore through deallylation of a non- fluorescent precursor and 2) the gated activation of a prodrug by cleaving the propargyl functionality that has been introduced to block the active side of original drug. Such gated control of catalysis has not been demonstrated in cells to date and is important because it allows the potential for multiple useful capabilities, such as switching on therapeutics at target tissues and regulation of activity to maintain homeostasis for long-term therapeutics. To date, supramolecular machines based on gating strategy were designed to entrap the guest molecules in the pore reservoir of silica nanoparticles and their release was studied mostly in test tubes39 or intracellularly37 in a few studies however, no gated control of substrate activation was demonstrated inside cells.

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Figure 7.1: Bioorthogonal nanozyme design and supramolecular regulation of intracellular catalysis. a) AuNPs, catalyst embedded AuNPs, and CB[7] capped catalyst embedded AuNPs used in study. b) Endosomal uptake of nanozymes. c) Intracellular catalysis with NP_Ru converting substrate into product. d) CB[7] complexation with the ligand headgroup to provide NP_Ru_CB[7] inhibits catalyst activity. e) Nanozyme activity is restored through addition of the competitive guest 1-adamantylamine (ADA). f) Structures of the NP platform with the surface ligand bearing a dimethylbenzylammonium group to bind the CB[7] gatekeeper, CB[7] gatekeeper, and ADA, a competitive guest molecule for CB[7] binding. g) Structures of the non- fluorescent substrate (rhodamine 110 derivative), fluorescent product (rhodamine 110) obtained after catalysis, and embedded catalyst for allylcarbamate cleavage.

7.2 Results and Discussion

7.2.1 Design and Synthesis of Nanozymes

We used AuNPs with core diameters of ~2 nm as the scaffold for our catalysts, with the goal of creating protein-sized systems with functional monolayers. The monolayer coverage of the NP nanoreactor scaffold features three crucial components: 1) a hydrophobic alkane segment for catalyst encapsulation, 2) a tetra(ethylene glycol) unit to provide biocompatibility,40 and 3) a dimethylbenzylammonium group to impart water solubility and bind the non-toxic CB[7] gatekeeper (Figure 7.1 (f)).41,42

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We chose ruthenium-catalyzed deallylation43,44 as a model bioorthogonal process to regenerate the fluorescence from an allylcarbamate caged fluorophore in solution and inside cells.

To this end, we immobilized [Cp*Ru(cod)Cl] (Cp* = pentamethylcyclopentadienyl, cod = 1,5- cyclooctadiene) in the hydrophobic portion of the AuNP monolayer to provide NP_Ru.

Transmission electron microscopy (TEM) images of the AuNPs before and after encapsulation of the catalyst indicated that no aggregation or decomposition of AuNP structure occurred after encapsulation, a result that was confirmed by dynamic light scattering (Figure 7.2).

Figure 7.2: TEM images of AuNP with (a) without (b) encapsulation of the ruthenium catalysts. No size change or aggregation of nanoparticles was observed from TEM images, indicating no morphological change occurred during encapsulation process.

The amount of ruthenium catalyst relative to AuNP was quantified using inductively coupled plasma mass spectrometry (ICP-MS)45 of 101Ru relative to 197Au, with 42 ± 3 catalyst molecules encapsulated per AuNP.

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7.2.2 Catalytic Efficacy of NP_Ru Nanozymes in Solution

The catalytic efficacy of the nanozymes in solution was assessed using the allylcarbamate cleavage of bis-N,N’-allyloxycarbonyl rhodamine 110 (Figure 7.1 (g)). Fluorescence started to increase immediately for NP_Ru after the addition of substrate, while no significant change in fluorescence was observed for NP_Ru_CB[7] (Figure 7.3).

Figure 7.3: Bar graph of intensities of NP_Ru and NP_Ru_CB[7] at 5 different time points for 2 h.

After 24 h bright fluorescence was observed using the NP_Ru nanozymes (Figure 7.5

(a)). However, only minimal fluorescence was observed for the NP_Ru_CB[7], which originated from the background of caged fluorophore as observed at 0 h (Figure 7.4).

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Figure 7.4: Photo of the reaction mixtures in water with NP_Ru and NP_Ru_CB[7] under UV light at 0 h.

As expected no reaction occurred with the control particle NP that lacked embedded catalysts (Figure 7.5), as the [Cp*Ru(cod)Cl] was insoluble in water, preventing study of the catalyst alone. Additionally, fluorogenesis of NP_Ru and NP_Ru_CB[7] taken after 5 days confirmed the long term stability of the gated catalysis system in solution (Figure 7.6).

Figure 7.5: Reaction mixtures including a, the nanozyme and caged rhodamine 110 and b, AuNP and caged rhodamine 110 after 24 h.

Figure 7.6: Photo of the reaction mixtures in water with NP_Ru and NP_Ru_CB[7] under UV light after 5 days.

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7.2.3 Supramolecular Control of Catalysis

With catalytic efficacy of the nanozymes established, we next explored CB[7] complexation of the ligand headgroups to act as the gatekeepers in NP_Ru_CB[7]. Isothermal

46 titration calorimetry (ITC) indicated that 60 ± 5 CB[7] molecules bound per NP, with KD = 11.3

± 2.4 µM (Figure 7.7).

Figure 7.7: ITC titration of CB[7]s into the NP solution. The circles represent the integrated heat changes during complex formation and the lines represent the curve fit to the binding isotherm.

Complexation with CB[7] effectively shut down catalysis in NP_Ru_CB[7], as minimal fluorogenesis was observed with nanozymes after addition of CB[7] (Figure 7.8). Kinetic studies

(Figure 7.8 (a)) verified this observation, indicating essentially complete inhibition of catalysis by

CB[7] complexation. This inhibition was reversible: after the addition of 1-adamantylamine

(ADA), a competitive guest molecule for CB[7], catalytic activity was completely restored

(Figure 7.8 (b), (c)).

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Figure 7.8: Catalytic activity of nanozymes in solution. a) Fluorescence was generated by NP_Ru after the cleavage of profluorophore bis-Alloc-rhodamine 110, while NP_Ru_CB[7] showed no significant change. b) After adding ADA, catalytic activity of NP_Ru_CB[7] was restored and no significant effect was observed for the activity of NP_Ru. c) The reaction rates of NP_Ru and NP_Ru_CB[7] before and after adding ADA showing catalytic activity for NP_Ru_CB[7] was fully recovered after the addition of ADA. The reaction rate experiments were performed in triplicate. Error bars represent standard deviations of these measurements. d) Photo of the reaction mixtures in water with NP_Ru and NP_Ru_CB[7] under UV light.

Control studies of the free catalyst in an acetone/water solution showed that catalyst efficiency was unaffected by either CB[7] or ADA (Figure 7.9), demonstrating that particle-

CB[7] gating controls the catalytic process.

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Figure 7.9: Activity assay of allylcarbamate cleavage of the [Cp*Ru(cod)Cl] in acetone/water (1:1 v/v). No catalytic activity change was observed in presence of CB[7] or CB[7] + ADA, indicating CB[7] or ADA cannot affect the catalytic activity of the catalysts directly.

7.2.4 Kinetic Analysis of the NP_Ru Nanozymes Using Lineweaver–Burk Analysis

The analogy between controlled regulation of the nanozymes and their enzyme counterparts was explored through kinetic analysis of the nanozymes using Lineweaver–Burk analysis (LBA).47,48 These studies indicate that CB[7] complexation results in competitive inhibition of reactor activity (Figure 7.10 and Figure 7.11), presumably by blocking access to the

[Cp*Ru(cod)Cl] "active site".

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Figure 7.10: Lineweaver-Burk plot showing competitive binding of CB[7] to nanozyme. Kinetic studies of NP_Ru and NP_Ru_CB[7] in sodium phosphate buffer (5 mM, pH 7.4) indicate that CB[7] inhibits catalyst activity through a competitive inhibition mechanism, with CB[7] affinity and stoichiometry consistent with ITC binding studies. Numbers calculated on a per particle basis. Kinetic experiments with each nanozyme were repeated in triplicate. Error bars represent standard deviations of these measurements.

Figure 7.11: The dependence of reaction rates on the concentration of the CB[7] (a)0 μM; b) 4 μM; c) 16 μM; and d) 80 μM). e) The fitting curve of the reaction rate vs. concentration of the substrates.

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Little change in activity was observed for CB[7]:NPs ratios greater than ~60:1, consistent with the ITC results (Figure 7.12 and Figure 7.13). Likewise, the value of Ki from the LBA (11.4

± 4.3 µM) is essentially identical to the affinity obtained by ITC (11.3 ± 2.4 µM). Taken together, these studies demonstrate the direct correlation between CB[7]-AuNP equilibrium binding processes and kinetic behavior of the nanozyme.

Figure 7.12: The dependence of reaction rate on the concentration of CB[7].

Figure 7.13: The reaction curve for relative activity versus [CB[7]]/[NP].

7.2.5 Catalytic Efficacy of NP_Pd Nanozymes in Solution

To demonstrate the versatility of this catalysis platform, we chose a second catalyst: hydrophobic palladium catalyst: (1,1'- Bis(diphenylphosphino) ferrocene)palladium(II)dichloride.

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This palladium catalyst was encapsulated in the hydrophobic portion of the AuNP monolayer to provide NP_Pd. The amount of palladium catalyst relative to AuNP was quantified using ICP-MS of 106Pd relative to 197Au. It was calculated that 32 ± 1 catalyst molecules encapsulated per AuNP.

Similar to NP_Ru nanozyme, NP_Pd can also cleave the allylcarbamate of bis-N,N’- allyloxycarbonyl rhodamine 110.13 After 6 h, fluorescence was generated for the NP_Pd, however; NP_Pd_CB[7] showed only a slight background fluorescence of caged fluorophore

(Figure 7.14).

Figure 7.14: Photos of the reaction mixtures in water with NP_Pd and NP_Pd_CB[7] under UV light at a) 0 h and b) 6 h.

Kinetic studies for NP_Pd and NP_Pd_CB[7] indicated the inhibition of catalysis by

CB[7] complexation. Followed by the addition of ADA, the catalytic activity of nanozyme was restored (Figure 7.15). These findings supported that gated catalysis is efficiently working for different catalyst systems.

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Figure 7.15: Kinetics of palladium catalyst embedded nanozymes. Fluorescence generation by NP_Pd and NP_Pd_CB[7] a) before ADA addition and b) after ADA addition.

7.2.6 Catalytic Activity inside Living Cells

Having characterized the activity and gating of nanozyme catalysis in solution, we next studied the intracellular behavior of these nanozymes using HeLa cells. The cellular uptake of the nanozymes was quantified by tracking [197Au] using ICP-MS, with NP_Ru_CB[7] particles demonstrating a slightly more efficient (1.7-fold greater) uptake than uncomplexed NP-Ru

(Figure 7.16). Significantly, little to no toxicity was observed at the concentrations used for our studies with either NP or NP_Ru (Figure 7.17).

Figure 7.16: Nanozyme uptake assay by tracking gold amount through ICP-MS.

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Figure 7.17: Cytotoxicity of the NP_Ru and NP at various concentrations.

We then probed the catalytic activity of the nanozymes inside the living cells. HeLa cells were incubated with the nanozyme for 24 h in serum-containing media and then washed multiple times to remove adsorbed particles on the cell surface. Fresh media containing the substrate was added, followed by 24 h incubation and washing. As shown in Figure 7.18 (a), flow cytometry indicated that there was a significant increase in fluorescence with NP_Ru relative to the control

NP. Confocal microscopy (Figure 7.18 (d)) showed that the cells treated with NP_Ru had bright punctate fluorescence. This fluorescence co-localized with LysoTracker® (Figure 7.19), indicating that the deallylation reaction occurred in the endosomes and cleaved fluorophore stayed in the endosomes. This outcome is expected given the endosomal uptake pathway49,50 previously observed for similar nanoparticles, coupled with the limited membrane permeability of the cleaved dye.51

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Figure 7.18: Triggered allylcarbamate cleavage in living cells using gated nanozymes. a) Flow cytometry of NP_Ru, NP_Ru_CB[7], and controls (only cell and NP) revealing NP_Ru showed significant increase in fluorescence while NP_Ru_CB[7] was completely inhibited. b) Addition of ADA to NP_Ru_CB[7] treated cells recovered the catalysis and resulted in increase in fluorescence. c-f) Confocal microscopy images of HeLa cells showing the supramolecularly regulated intracellular chemical reactions, a punctate fluorescence was observed for NP_Ru and NP_Ru_CB[7] + ADA treated cells as the indication of catalysis while no fluorescence was obtained for only substrate and NP_Ru_CB[7] (scale bars = 10 μm).

We next investigated the intracellular gating of the nanozyme by CB[7] complexation.

Flow cytometry showed that the NP_Ru_CB[7] particles were completely inhibited, indicating intracellular stability of the complexes (Figure 7.18 (a), (e)). Treatment with ADA (400 µM) restored nanozyme activity, with even higher activity observed in the cells after treatment with

ADA than was observed with NP_Ru (Figure 7.18 (b)). This increased catalytic efficiency is echoed in the micrograph (Figure 7.18 (f)) and potentially arises from enhanced protection of the catalyst by the CB[7] coverage in serum and inside the cell.

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Figure 7.19: The confocal images of the cell treated with a) the nanozyme, substrate, and lysotracker; b) the nanozyme-bound-CB[7], substrate, ADA, and lysotracker.

We further tested the control of gated catalysis through the treatment of cells with free

CB[7]. First, HeLa cells were incubated with the NP_Ru for 24 h and then after multiple washings, free CB[7] in serum-containing media was treated to cells for 24 h before the addition of the substrate. A significant decrease in the fluorescence was observed indicating the complexation of CB[7] with NP_Ru and blocking access to the catalytic site (Figure 7.20).

Figure 7.20: Confocal microscopy images of HeLa cells incubated with only substrate (a), NP_Ru + substrate (b), and NP_Ru nanozyme + free CB[7] + substrate (c) (scale bars = 20 μm). Fluorescence intensities were obtained from confocal images using ImageJ program (d).

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Palladium catalyst embedded nanozymes were likewise used for intracellular controlled activation of the caged fluorophore. NP_Pd effectively performed the intracellular catalysis, while as expected the gated NP_Pd_CB[7] did not. ADA addition into cells that were previously incubated with NP_Pd_CB[7] resulted in the recovery of intracellular catalysis (Figure 7.21).

Figure 7.21: Intracellular gated-catalysis using NP_Pd/NP_Pd_CB[7] (scale bars = 10 μm).

7.2.7 NP_Pd Nanozymes for Prodrug Activation

Bioorthogonal activation of prodrugs using TMC-loaded nanomaterials was recently demonstrated by Unciti-Broceta and co-workers.16 Although prodrug activation was successfully achieved, no gated control over this activation was shown. We have bioorthogonally demonstrated gated-activation of a prodrug inside cells using NP_Pd/NP_Pd_CB[7]. 5- fluorouracil (5FU) is a chemotherapeutic drug used in cancer treatment including breast, stomach, pancreatic, and skin cancers. Although it has an established history in cancer treatment, it shows toxic side effects due to its limited safety profile.52 These side effects can be eliminated or at least minimized if a prodrug strategy coupled with gated-intracellular catalysis were to be available.

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By this gated catalysis, activation of prodrugs will be achieved on demand upon reaching to site of action thus eliminating the off target effects.

5FU can be converted into a prodrug via functionalization on its N1 position, as modification at this position will block the activity of 5FU and render it non-toxic.16 We introduced a propargyl moiety to turn 5FU into an inactive prodrug ‘pro-5FU’ (Figure 7.22 (a)).

First, through palladium mediated chemocatalysis propargyl masking unit is intracellularly cleaved to yield 5FU followed by the enzymatic reactions to convert 5FU into cytotoxic nucleotidic metabolites ‘fluorouridine monophosphate’ via functionalization on its N1 position.53

These active metabolites work through misincorporation into RNA and DNA molecules and irreversibly inhibit the nucleotide synthetic enzyme thymidylate synthase to disrupt cell functions and induce cytotoxicity. Overall, this process is an example of the integration of chemocatalysis and enzymatic biochemistry in a one-pot chemical sequence.54,55

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Figure 7.22: Prodrug activation in living cells using gated nanozymes. a) Structures of pro-5FU, 5FU and the palladium catalyst used for prodrug activation. b) Viability of cells treated with 5FU and pro-5FU at various concentrations, showing a nice therapeutic window was obtained between 5FU and pro-5FU. c) NP_Pd and ADA treated NP_Pd_CB[7] showed increasing intracellular toxicity as a result of more conversion of prodrug into 5FU drug at higher pro-5FU concentrations, while NP_Pd_CB[7] did not show any toxicity at any prodrug concentration used due to the blocking catalysis. Also, only prodrug, NP_Pd, NP_Pd_CB[7], and NP_Pd_CB[7] + ADA did not cause any toxicity into the system at zero prodrug concentration. Cell viability experiments were performed in triplicate. Error bars represent standard deviations of these measurements.

Cleavage of propargyl group on pro-5FU (1 mM) in the presence of NP_Pd (100 nM) was monitored using matrix-assisted laser desorption/ionization (MALDI)-MS. Most of the pro-

5FU was converted in 5FU after 48 h (Figure 7.23). After confirming the cleavage, the toxicity profile of 5-FU and pro-5FU were investigated performing a cell viability assay. Cells were treated with various concentrations from 10 nM to 1 mM. While 5-FU showed toxicity as concentration increased, pro-5FU retained high cellular viability at all concentrations studied.

(Figure 7.22 (b)).

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Figure 7.23: Cleavage of propargyl functionality of prodrug was monitored using MALDI-MS at (a) 15, (b) 48, and (c) 72 h.

Cell viability studies for prodrug system in the presence of nanozymes were carried out using HeLa cells in a 96 well plate. Cells were first incubated with NP_Pd or NP_Pd_CB[7] at a concentration of 100 nM in serum-containing media for 24 h. Then, after multiple washings, cells were incubated with different concentrations of pro-5FU (0, 0.05, 0.1, 0.25, 0.5 and 1 mM) while some of the cells that were incubated with NP_Pd_CB[7], were treated with pro-5FU and ADA at the same time. Cells that were incubated with NP_Pd and NP_Pd_CB[7] + ADA showed elevated toxicity at higher concentration of pro-5FU while NP_Pd_CB[7] retained ~100 % cell viability even at higher concentration of pro-5FU (Figure 7.22 (c)). As expected, pro-5FU was not toxic at

175 any concentrations used. Likewise, NP_Pd, NP_Pd_CB[7], and NP_Pd_CB[7] + ADA at zero pro-drug concentration were not toxic indicating toxicity was coming from the intracellular conversion of pro-5FU into 5-FU using gated-catalysis but not the nanozyme itself.

7.3 Summary and Future Outlook

In conclusion, we have described the fabrication of an AuNP based, bioorthogonal nanozyme that uses transition metal catalysis to effect transformations of imaging and therapeutic relevance without biological counterparts. These catalysts were built upon a platform featuring biomimetic size and surface functionality, making them attractive components for both in vitro and in vivo applications. The ability to control the activity of these nanozymes through host-guest interactions of CB[7] molecules with the benzyl headgroup of the AuNP ligands likewise provides an efficient and reversible means of regulating catalysis. This bioorthogonal catalysis can be employed not only in therapeutic applications such as the activation of prodrugs at the site of action but also in treating non-cancerous chronic diseases where the goal is not to kill the cell but to kill an infective agent or to restore a malfunctioning pathway. This platform enables to introduce an inactive reservoir of a bioorthogonal catalyst that would remain dormant between successive administrations of a bioorthogonal substrate, thus reducing potential interference of the catalyst with cell constituents and protecting the metal from poisoning, release, systemic distribution and/or clearance. Furthermore, our protein-sized system demonstrates biomimetic behavior and yet performs totally abiotic chemistry that can be controlled intracellularly through a very simple host-guest feature. In conclusion, this system integrates biomimetic and bioorthogonal design elements to provide a new platform for imaging and therapeutic applications as well as pharmacological treatments integrating biological activity with man-made synthetic tools.

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7.4. Synthesis of Materials and Experimental Sections

7.4.1 Synthesis of the Benzyl Ligand

Scheme 7.1: Synthetic scheme of the benzyl ligand for the functionalization of the nanoparticle.

Compound 2: 11-bromo-1-undecanol (1, 10 g, 39.8 mmol) was dissolved in 1:1 ethanol/toluene mixture (200 ml). Triphenylmethanethiol (13.2 g, 47.77 mmol) dissolved in 1:1 ethanol/toluene (50 ml) was added to 11-bromo-1-undecanol in solution. Then NaOH (2.38 g,

59.7 mmol) dissolved in 3 ml water was slowly added to the mixture. The reaction mixture was stirred for 24 h at 50°C. Upon completion, the reaction mixture was extracted with a saturated solution of NaHCO3 twice. The organic layer was extracted, dried over Na2SO4, and concentrated by evaporation of the solvent. The crude product was purified by column chromatography over silica gel using hexane/ethyl acetate (1:1, v/v) as the eluent. The solvent was removed in vacuum

1 to obtain compound 2 as colorless oil (Yield 16.42 g, 92%). H NMR (400 MHz, CDCl3, TMS) of

Compound 2 : δ 7.48-7.40 (m, 6H, HAr-), 7.37-7.27 (m, 6H, HAr-), 7.26-7.18 (m, 3H, HAr-),

3.65 (t, J = 6.7Hz, 2H,CH2OH), 2.16 (t, J = 7.2Hz, 2H,-CH2-), 1.66-1.52 (m, 2H, -SCH2CH2) ,

1.44-1.12 (m, 16H, -CH2CH2OH + -(CH2)8 CH2OH).

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1 Figure 7.24: H NMR spectrum (400 MHz) of compound 2 in CDCl3 (D, 99.8%).

Compound 3: Compound 2 (16.42 g, 36.76 mmol) in dry dichloromethane (DCM, 200 ml) was mixed with triethylamine (5.58 g, 7.66 mL, 55.14 mmol), followed by dropwise addition of methanesulfonyl chloride (4.64 g, 3.13 mL, 40.47 mmol) in ice bath. After 30 min the reaction mixture was warmed up to room temperature and stirred overnight. After the reaction was completed (according to thin layer chromatography; TLC), solvent was evaporated. The compound was again diluted with DCM and extracted with 0.1 M HCl twice. The organic layer was collected, treated with a saturated solution of NaHCO3, and washed three times. Following extraction, the organic layer was dried over Na2SO4 and concentrated at reduced pressure. The crude product was purified by column chromatography over silica gel using hexane/ethyl acetate

(1:1, v/v) as the eluent. Solvent was removed in vacuum to obtain mesylated compound as light yellow oil (Yield 17.4 g, 90%). To synthesize compound 3, NaOH (1.59 g, 39.8 mmol) solution

(1.5 mL) was added to 115 mL of tetraethyleneglycol (128.93 g, 66.37 mmol) and stirred for 2 h at 80°C. To this reaction mixture, 17.4 g of 11-(tritylthio)undecyl methanesulfonate (30.03 mmol) was added and stirred for 24 h at 100°C. The product was extracted in hexane/ethyl acetate (100

178 ml, 4:1, v/v) six times. Then, the organic layer was concentrated at reduced pressure and the crude product was purified by column chromatography over silica gel using ethyl acetate as the eluent. The solvent was removed in vacuum to obtain compound 3 as light yellow oil (Yield 11.6

1 g, 62%). H NMR (400 MHz, CDCl3, TMS) of Compound 3: δ 7.47-7.40 (m, 6H, HAr-), 7.34-

7.26 (m, 6H, HAr-), 7.25-7.19 (m, 3H, HAr-), 3.77-3.57 (m,16H, -CH2-(OCH2CH2)4-OH), 3.46 (t,

J = 6.8 Hz, 2H, -CH2-(OCH2CH2)4-OH), 2.95 (br, s, 1H, -TEG-OH), 2.15 (t, J = 7.2Hz, -SCH2-),

1.59 (p, J = 7.2Hz, 2H, -CH2CH2TEG-OH), 1.4 (p, J = 7.6Hz, 2H, -SCH2CH2-), 1.35-1.13(m,

14H, -(CH2)7 CH2CH2TEG-OH).

1 Figure 7.25: H NMR spectrum (400 MHz) of compound 3 in CDCl3 (D, 99.8%).

Compound 4: Triethylamine (2.6 g, 3.6 mL, 25.6 mmol) was added to compound 3 (8 g,

12.8 mmol) in dry DCM (80 ml) in an ice bath. Methanesulfonyl chloride (2.22 g, 1.49 mL, 19.28 mmol) was added dropwise to the reaction mixture in an ice bath. After 30 min the reaction mixture was warmed up to room temperature and stirred 12 h. The organic layer was extracted twice with a saturated solution of NaHCO3 (100 ml) and twice with 0.1 M HCl solution (100 ml).

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The extracted DCM layer was dried over Na2SO4 and concentrated at reduced pressure. The crude product was purified by column chromatography over silica gel using ethyl acetate as the eluent.

Solvent was removed in vacuum to obtain compound 4 as light yellow oil (Yield 8.2 g, 91 %). 1H

NMR (400 MHz, CDCl3, TMS) of Compound 4: δ 7.44-7.37 (m, 6H, HAr-), 7.31-7.23 (m, 6H,

HAr-), 7.22-7.16 (m, 3H, HAr-), 4.40-4.34 (m, 2H, -CH2OSO3CH3), 3.78-3.54 (m, 14H, CH2-

(OCH2CH2)3-CH2CH2OSO3CH3), 3.44 (t, J = 6.8Hz, 2H, CH2-CH2-(OCH2CH2)3-), 3.07 (s, 3H, -

OSO3CH3), 2.12 (t, J = 7.2Hz, 2H, -SCH2-), 1.56 (p, J = 7.2Hz, 2H, -CH2CH2TEG-N(CH3)2),

1.38 (p, J=7.6Hz, 2H, -SCH2CH2-), 1.32-1.11 (m, 14H, -(CH2)7CH2CH2TEGOSO3 CH3).

Figure 7.26: 1H NMR spectrum (400 MHz) of compound 4 in CDCl3 (D, 99.8%).

Compound 5: Compound 4 (1 g, 1.42 mmol) was added to dimethylbenzylamine (0.58 g, 0.65 ml, 4.28 mmol) in ethanol-DCM mixture (4:1,v/v, 5 ml). The reaction mixture was stirred at 40°C for 48 h. After evaporating ethanol at reduced pressure, the light yellow residue was purified by successive washings with hexane (four times) and hexane/diethylether (1:1 v/v, six times) and then dried under high vacuum. The product formation was around 100% and the

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1 product was confirmed by NMR. H NMR (400MHz, CDCl3, TMS) of Compound 5: δ 7.64-7.58

(m, 2H, HAr-), 7.38-7.32 (m, 9H, HAr-), 7.24-7.17 (m, 6H, HAr-), 7.16-7.09 (m, 3H, HAr-), 4.9

(s, 2H, -CH2-C6H5), 3.94 (s, br, 2H, -OCH2CH2N(CH3)2-), 3.8 (s, br, 2H, -OCH2CH2N(CH3)2-),

3.77-3.22 (m, 12H, -(OCH2CH2)3-CH2CH2N(CH3)2-), 3.33 (t, J = 6.8Hz, 2H, -CH2CH2O-), 3.23

(s, 6H, -N(CH3)2-), 2.06 (t, J = 7.2Hz, 2H, -SCH2-), 1.51-1.42 (p, J = 6.8Hz, 2H, -CH2CH2O-),

1.36-1.28 (p, J = 7.6Hz, 2H, -SCH2CH2-) 1.24-1.08 (m, 14H, -(CH2)7 CH2CH2O-).

1 Figure 7.27: H NMR spectrum (400 MHz) of compound 4 in CDCl3 (D, 99.8%).

Compound 6: An excess of trifluoroacetic acid (TFA, 20 equivalents, 3.07 g, 2.08 mL,

27 mmol) was added to compound 5 (1 g, 1.12 mmol) in dry DCM (5 ml). The color of the solution was turned to yellow-orange upon addition of TFA. Then, triisopropylsilane (TIPS, three equivalents, 0.64g, 0.83 mL, 4.05 mmol) was added to the reaction mixture and the color of the solution turned to clear. The reaction mixture was stirred 5 hr under N2 at room temperature. The solvent, most of TFA, and TIPS were evaporated under reduced pressure. The yellow residue was purified by hexane washings (four times) and dried under high vacuum. The final product

181 formation was around 100% and the structure of compound 6 was confirmed by NMR. 1H NMR

(400 MHz, CDCl3, TMS) of Compound 6: δ 7.57-7.47 (m, 5H), 4.61 (s, 2H, -CH2-C6H5), 4.01 (s, br, 2H, -OCH2CH2N(CH3)2-), 3.74-3.48 (m, 14H, -(OCH2CH2)3-CH2CH2N(CH3)2-), 3.41 (t, J =

6.8Hz, 2H, -CH2CH2O-), 3.14 (s, 6H, -N(CH3)2-), 2.52 (q, J = 7.2Hz, HSCH2-), 1.65-1.48 (m, 4H,

-CH2CH2O- + HSCH2CH2-), 1.43-1.20 (m, 15H, -(CH2)7 CH2CH2O- + HS-).

1 Figure 7.28: H NMR spectrum (400 MHz) of benzyl ligand in CDCl3 (D, 99.8%).

7.4.2 Synthesis of Benzyl-Ligand-Protected Gold Nanoparticle (AuNP)

First, Brust-Schiffrin two-phase synthesis method was used to synthesize pentanethiol- coated AuNPs with core diameter ca. 2 nm.56,57 Murray place-exchange method58 was followed to obtain the benzyl-ligand-protected AuNPs. Pentanethiol-conjugated AuNPs (10 mg) and thiol ligand (compound 5) (30 mg) were dissolved in a mixture of dry DCM (3 mL) and methanol (1 mL) and stirred under N2 atmosphere for 3 days at room temperature. The DCM was removed under reduced pressure and the resulting precipitate was washed with hexane (15 mL) three times and DCM (15 mL) twice. Then the precipitate was dissolved in distilled water (~ 5 mL) and

182 dialyzed for three days (membrane molecular weight cut-off =10,000, volume of the dialysis bucket is 5 L) to remove excess ligands, pentanethiol, acetic acid, and other salts present in the nanoparticle solution. After dialysis, the particle was lyophilized to yield a solid brownish

1 product. The particles were then re-dispersed in deionized water. H NMR-spectra in D2O showed substantial broadening of the proton peaks with no sign of free ligands. The presence of ligands was also confirmed by mass spectroscopy.

1 Figure 7.29: H NMR spectrum (400 MHz) of benzyl-AuNPs in D2O (D, 99.8%).

7.4.3 Mass Spectrometric Characterization of Ligand Composition

Matrix assisted laser desorption/ionization mass spectroscopy (MALDI-MS) has been performed to characterize the surface ligand on the Benzyl-AuNP.59 A saturated α-Cyano-4- hydroxycinnamic acid (α-CHCA) stock solution was prepared in 70% acetonitrile, 30% H2O, and

0.1% trifluoroacetic acid. An equal volume of 2 μM NP solution was added to the matrix stock solution. 2.5 μL of this mixture was applied to the sample carrier, and then the MALDI-MS analysis was performed on a Bruker Autoflex III mass spectrometer. The molecular ions (MH+,

183 m/z =498) was detected, and the disulfide ion formed by the benzyl ligand and the original pentanethiol was also detected at m/z 600.

Figure 7.30: MALDI-MS spectrum of benzyl-AuNP.

7.4.4 Catalyst Encapsulation into the Monolayer of AuNPs

3 mg of the catalyst, [Cp*Ru(cod)Cl] or (1,1'- Bis(diphenylphosphino) ferrocene)palladium(II)dichloride was dissolved in 5 ml acetone and the AuNP (20 μM, 0.5 mL) were diluted to a final concentration of 5 μM with DI water (2 ml). Then, the catalyst and the

AuNP solutions were mixed together and acetone was slowly removed by evaporation. During the evaporation hydrophobic catalyst was encapsulated in the particle monolayer to yield to

NP_Ru or NP_Pd. Excess catalysts which precipitated in water removed by filtration (Millex-GP filter; 25 mm PES, pore Size: 0.22µm). Further purifications were followed by multiple filtrations

(five times, Amicon® ultra 4, 10K) and dialysis (Snake Skin® dialysis tubing, 10K) against water

(5 L) for 24 h to remove free catalysts. The amount of encapsulated catalysts was measured by

ICP-MS by tracking 101Ru relative to 197Au for NP_Ru and 106Pd relative to 197Au for NP_Pd.

184

7.4.5 Transmission Electron Microscopy (TEM) Measurement of the Nanoparticle Before and After the Encapsulation of the Catalysts

TEM samples of AuNP and [Cp*Ru(cod)Cl] catalysts encapsulated AuNP were prepared by placing one drop of the desired solution (1 μM) on to a 300-mesh Cu grid-coated with carbon film. These samples were analyzed and photographed using JEOL CX-100 electron microscopy.

The average diameter of Au core is 2.5 ± 0.4 nm.

7.4.6 Size and Zeta Potential of the NP

Hydrodynamic diameter and zeta potential of the NPs were measured by dynamic light scattering (DLS) in DI water and 5 mM phosphate buffer (pH=7.4) respectively, using a Malvern

Zetasizer Nano ZS instrument. The measurement angle was 173° (backscatter). Data were analyzed by the “multiple narrow modes” (high resolution) based on non-negative-least-squares

(NNLS).

Figure 7.31: Characterization of the functionalized AuNP. a) Size (diameter) of AuNP was measured by DLS before and after catalyst encapsulation. DLS measurement shows that size of the NP after catalyst encapsulation stays same. b) Zeta potential of AuNP was measured by DLS. The overall charge of AuNP is measured as 25.5 ± 1 mV from three independent replicates.

185

7.4.7 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Instrumentation for Ruthenium Catalyst

The ICP-MS analyses were performed on a Perkin-Elmer NexION 300X ICP mass spectrometer. 197Au and 101Ru were measured under the standard mode. Operating conditions are listed as below: nebulizer flow rate: 0.95 L/min; rf power: 1600 W; plasma Ar flow rate: 18

L/min; dwell time: 50 ms. Standard gold and ruthenium solutions (concentration: 0, 0.2, 0.5, 1, 2,

5, 10, and 20 ppb) were prepared for the quantification.

7.4.7.1 ICP-MS Sample Preparation for the Quantification of Gold and Ruthenium

0.5 mL of fresh aqua regia was added to the 10 μL sample solution and then the sample was diluted to 10 mL with de-ionized water.

Table 7.1: Ruthenium amount in the nanozyme using ICP-MS measurement.

186

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