Synthesis of Polymer Nanoparticles Using

Home , Fluorocarbon, Trifluorotoluene

SYNTHESIS OF POLYMER NANOPARTICLES USING

INTRAMOLECULAR CHAIN COLLAPSE AND BENZOCYCLOBUTENE CHEMISTRY

A Dissertation

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

Of the Requirements for the Degree

Doctor of Philosophy

Ajay Ramesh Amrutkar

September 2016

SYNTHESIS OF POLYMER NANOPARTICLES USING

INTRAMOLECULAR CHAIN COLLAPSE AND BENZOCYCLOBUTENE CHEMISTRY

Ajay Ramesh Amrutkar

Dissertation

Approved: Accepted:

Advisor Department Chair Dr. Coleen Pugh Dr. Coleen Pugh

Committee Chair Dean of the College Dr. Li Jia Dr. Eric Amis

Committee Member Dean of the Graduate School Dr. Stephen Cheng

Committee Member Date Dr. Mesfin Tsige

Committee Member Dr. Alamgir Karim

ABSTRACT

Single chain polymer nanoparticle (SCPN) synthesis from different polymer precursors using benzocyclobutene (BCB) chemistry and intramolecular crosslinking was investigated in this study.

Synthesis of highly fluorinated SCPNs from highly fluorinated uni-block copolymer precursor utilizing

‘pseudo-high dilution continuous addition technique’ was investigated. GPC confirmed selective intramolecular crosslinking and TEM images supported formation of sub-20 nm spherical polymer nanoparticles. Amphiphilic SCPNs prepared via step-wise crosslinking of an amphiphilic di-block copolymer chain were investigated for their morphology using 4 different characterization techniques:

TEM, AFM, DLS and DOSY-NMR. TEM and AFM images showed presence of discreet SCPNs as loosely crosslinked coils that flatten out when deposited on the surface forming pancake like morphology with ≈

20 nm sizes. All the techniques showed presence of bimodal size distribution of these nanoparticles in solution. A smaller sized distribution represented discreet SCPNs whereas larger sized (>40 nm) distribution represented physical aggregates of SCPNs. These aggregates were broken down upon significantly diluting the solution of nanoparticles (≤50 ng/mL). AFM analysis and water contact angle studies on thin films of amphiphilic SCPNs and polystyrene homopolymer blends proved that nanoparticles possess Janus-type morphology. In order to aid synthesis of SCPNs from block copolymer precursors, a new 2-component room temperature polymer crosslinking based on 1-acetoxyBCB containing polymers and a nucleophile was developed. Development of this system involved developing synthesis of new monomer (1-acetoxy-4 and 5-vinylBCB), synthesis of polymers containing this new monomer followed by optimization of their crosslinking reaction. Mechanism of crosslinking and structure of crosslinking unit formed for this crosslinking system was also identified. SCPNs were synthesized at room temperature from uni-block copolymer precursors using this new crosslinking system. Crosslinking reaction was characterized using 1H NMR, GPC and LS-GPC. Symmetric ABA tri-block copolymer precursors are proposed as precursors for synthesis of SCPNs of different morphologies employing intramolecular crosslinking. Initial results on synthesis of ABA tri-block copolymer containing highly immiscible blocks using chain extension approach is also reported.

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DEDICATION

This thesis is dedicated to my parents, Mummy and Baba, for their sacrifices and unwavering support

And

In loving memory of my grandmother, Aai, her values and teachings remain very close to my heart

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor, Prof. Coleen Pugh, for giving me an opportunity to be a part of her research group despite my non-chemistry background. I would like to thank her for all the support, guidance and mentoring. I would also like to thank my committee members, Prof. Stephen Cheng, Prof. Li Jia, Prof. Mesfin Tsige and Prof. Alamgir

Karim, for their guidance and help. I would also like to thank Prof. Ali Dhinojwala for giving me an opportunity to be a part of Akron’s polymer program in the year 2010.

I would like to thank all the group members in the Pugh group for all the help and fun times in the lab. I was lucky to have an amazing mentor, Dr. Bill Storms, who taught me many new things. I would also like to thank Dr. James Baker, Isamu Ono and Liwen Xing for their help in BCB projects. Special thanks to Abhishek Banerjee, Gladys Rocío Montenegro-Galindo, Cesar

Lopez, Colin Wright, Brinda Mehta, Nicole Swanson, Carolyn Scherger, Abby Freedman, Tyler

Tommey and Dibyendu Debnath for their help.

During the course of this work, I had an opportunity to collaborate with various researchers. I would like to thank Jacob Scherger (AFM), Dr. Fadi Haso (DLS), Dr. Jessi

Baughmann (DOSY-NMR), Dr. Gary Leuty (Simulations) and Namrata Salunke (AFM on thin films). I have learnt many new things from each of them and their contribution has helped us gain more insight in our project.

I consider myself fortunate to have been blessed with constant support of my teachers and mentors from my undergraduate education. I would like to express my sincere gratitude to

Dr. Prakash Wadgaonkar (NCL, Pune), Dr. Shashank Mhaske (ICT, Mumbai) and Dr. Anagha

Sabnis (ICT, Mumbai) for their support and inspiration.

Life in Akron would have been difficult without my friends. I would like to thank Kaushik

Mishra for being an amazing roommate for 7 years. I would like to specially thank Kaushik

Mishra, Nishad Dhopatkar, Dharamdeep Jain and Dr. Murthy Maddipatla for their help during my injury in 2012. I would also like thank Emmanuel Anim-Danso, Attila Gergely and family and

Yeneneh Yimer and family for fond memories and lifelong friendships to cherish.

My journey so far would have been impossible without the constant support of my family members. My parents, Mummy and Baba, remain the backbone of my life. Their sacrifices, positive encouragement and values keep me going ahead. My younger brother,

Pavan, his maturity and understanding has been very crucial to my journey. My maternal grandparents, Aai and Bhau, remain a source of inspiration. My uncles and aunts have been very supportive during all my endeavors. I am extremely fortunate to have been born in a family where education is given utmost importance and support. I thank everyone from the bottom of my heart.

TABLE OF CONTENTS

Page

LIST OF TABLES………………………………………………………………………………………………………………………...... xi

LIST OF FIGURES…………………………………………………………………………………………………………………………………… xiii

LIST OF SCHEMES…………………………………………………………………………………………………………………………………. xxi

CHAPTER

I. INTRODUCTION……………………………………………………………………………………………………………………. 1

II. LITERATURE REVIEW……………………………………………………………………………………………………………. 4

2.1 Benzocyclobutene (BCB) Chemistry………………………………………………………………………. 4

2.2 Single Chain Polymer Nanoparticles…………………………………………………………………….. 12

2.3 Atom Transfer Radical Polymerization………………………………………………………………… 18

III. EXPERIMENTAL METHODS………………………………………………………………………………………………… 27

3.1 Introduction……………………………………………………………………………………………………….. 27

3.2 Materials……………………………………………………………………………………………………………. 27

3.3 Techniques…………………………………………………………………………………………………………. 28

3.4 Synthesis of and studies on small molecules……………………………………………………... 30

3.5 Synthesis of polymers………………………………………………………………………………………… 37

3.6 Intermolecular crosslinking of polymers………………………………………………………….… 42

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3.7 Intramolecular crosslinking of polymers to synthesize single chain

polymer nanoparticles……………………………………………………………………………………….. 44

IV. SYNTHESIS AND CHARACTERIZATION OF HIGHLY FLUORINATED SINGLE CHAIN POLYMER

NANOPARTICLES……………………………………………………………………...... 48

4.1 Introduction ………………………………………………………………………………………………………… 48

4.2 Parameters responsible for selective intramolecular crosslinking using pseudo-high

dilution continuous addition technique………………………………………………………………. 51

4.3 Synthesis of Highly Fluorinated Single Chain Polymer Nanoparticles…………………. 53

4.4 Characterization of Highly Fluorinated SCPNs using TEM……………………………………. 57

4.5 Conclusion …………………………………………………………………………………………………………. 60

V. CHARCATERIZATION OF AMPHIPHILIC NANOPARTICLES SYNTHESIZED VIA STEP-WISE

CROSSLINKING OF A SINGLE DI-BLOCK COPOLYMER CHAIN……………………………………………… 61

5.1 Introduction………………………………………………………………………………………………………… 61

5.2 Transmission Electron Microscopy (TEM)…………………………………………………………….. 68

5.3 Atomic Force Microscopy (AFM) analysis of individual nanoparticles…………………… 77

5.4 Dynamic Light Scattering (DLS)…………………………………………………………………………….. 90

5.5 Diffusion-Ordered Spectroscopy Nuclear Magnetic Resonance (DOSY-NMR) …….. 98

5.6 Atomic Force Microscopy analysis of nanoparticles and polystyrene homopolymer

blends………………………………………………………………………………………………………………… 104

5.7 Conclusions……………………………………………………………………………………………………….. 109

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VI. DEVELOPMENT OF A NEW ROOM TEMPERATURE POLYMER CROSSLINKING SYSTEM

BASED ON 1-FUNCTIONALIZED BENZOCYCLOBUTENE ……………………………………………….…… 111

6.1 Introduction ……………………………………………………………………………….…………………… 111

6.2 Problems with 1-ethoxy-4 and 5-vinylBCB……………………………………………….………… 113

6.3 Screening of potential 1-functionalized BCB-based molecules for room temperature

crosslinking………………………………………………………………………………………………..……… 114

6.4 Synthesis of a new monomer/crosslinker: 1-acetoxy-4- and 1-acetoxy-5-

vinylBCB……………………………………………………………………………………………..…………..…. 122

6.5 Synthesis of copolymers containing 1-acetoxyBCB………………………….….……………… 127

6.6 Intermolecular crosslinking of polymers containing 1-acetoxyBCB at room

temperature…………………………………………………………………………………………………….. 134

6.7 Intramolecular crosslinking of polymers containing 1-acetoxyBCB at room

temperature………………………………………………………………………………………………….…… 145

6.8 Conclusion………………………………………………………………………………………….……………… 152

VII. SYNTHESIS OF TRI-BLOCK COPOLYMERS CONTAINING HIGHLY IMMISCIBLE BLOCKS……… 153

7.1 Introduction …………………………………………………………………………………………..…………. 153

7.2 Proposed design of ABA tri-block copolymer precursors and possible morphologies

of their corresponding SCPNs………………………………………………………………………….…. 154

7.3 Preliminary results on the synthesis of ABA tri-block copolymer………………….…… 158

7.4 Conclusion …………………………………………………………………………….…………………….……. 167

VIII. SUMMARY AND FUTURE DIRECTIONS……………………………………………………………………………… 168

BIBLOGRAPHY………………………………………………………………….…………………………………………………………………… 176

LIST OF TABLES

Table Page

2.1 Effect of substituent on the ring-opening isomerization behavior…………………………………….. 7

4.1 Experimental conditions used in the synthesis of SCPNs from different linear precursors

and their characterization using GPC………………………………………………………………………………… 54

4.2 Comparison of molecular weights of linear polymers and their SCPN counterparts…………. 55

5.1 Solubility of PS and P[TF5FS] at room temperature in different solvents (conc. = 5 mg/mL)

and solubility observed over 5 minutes after addition of solvent……………………………………. 65

5.2 Summary of TEM analysis results performed on doubly crosslinked SCPNs. d = diameter

and r = radius. …………………………………………………………………………………………………………………. 76

5.3 Summary of AFM analysis results performed on doubly crosslinked SCPNs……………………… 89

5.4 Measured physical properties of different compositions of (THF + TFT) mixture……………… 95

5.5 Summary of results obtained from DLS analysis……………………………………………………………….. 97

1 6.1 Comparison of integration of methylene protons (Hc) in H NMR of 1-hydroxyBCB solution

(with 50 mol% 1,1,1,2-tetrachloroethane as internal standard in 1 mL DMSO-d6) with

respect to the integration of internal standard protons (methylene protons) at different

temperatures and 24 h time intervals………………………………………………………………… 117

1 6.2 Comparison of integration of methine proton (Hb) in H NMR of 1-acetoxyBCB solution

(10 mol% solution in bromopentafluorobenzene with 5 mol% 1,1,1,2-tetrachloroethane

as internal standard) with respect to the integration of internal standard protons

(methylene protons) at different temperatures and various time intervals……………………. 119

6.3 Table of different copolymers of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-

vinylBCB]) prepared using Free Radical Copolymerization and their characterization………. 127

6.4 Table of different copolymers of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-

vinylBCB]) prepared using ARGET ATRP and their characterization…………………………………… 130

6.5 Table of different copolymers of poly(methyl methacrylate-co-[1-acetoxy-4-vinylBCB]-co-

[1-acetoxy-5-vinylBCB]) prepared using AGET ATRP at 70 0C and their characterization…… 133

6.6 Results of crosslinking 1-acetoxyBCB-containing polystyrene copolymers using NaOMe.

*BCB+ ≈ 25 - 50 mM for all entries……………………………………………………………………………………… 140

6.7 Table showing different parameters and their effect on the crosslinking process as

observed during the optimization of the pseudo-high dilution continuous addition

method……………………………………………………………………………………………………………………………… 146

6.8 Comparison of the intramolecular crosslinking performed using NaOMe and BuLi as the

nucleophile………………………………………………………………………………………………………………………… 151

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LIST OF FIGURES

Figure Page

2.1 Schematic showing intramolecular chain collapse yielding nanoparticle and linear-

nanoparticle architecture…………………………………………………………………………………………………. 13

2.2 GPC analysis of linear polymer and intramolecularly crosslinked nanoparticle as reported

by Hawker and coworkers………………………………………………………………………………………………… 14

2.3 A low temperature cross-linker based on BCB as developed by Harth……………………………… 15

2.4 A schematic showing the synthesis of a single chain poly(acrylic acid) nanoparticle as a

potential MRI contrast agent………………………………………………………………………………………….. 16

2.5 Schematic showing work by Meijer and coworkers on orthogonal folding of block

copolymers……………………………………………………………………………………………………………………… 17

2.6 Schematic depicting synthesis of SCPN by Lutz and coworkers……………………………………….. 17

2.7 Schematic of di-block copolymer and its conversion to amphiphilic SCPNs via stepwise

and selective intramolecular crosslinking…………………………………………………………………………. 18

2.8 1H NMR analysis of the chain end groups in polystyrene prepared using ATRP

0 (styrene/MeBP/CuBr/dNbipy = 100/1/1/2; 110 C; 3.5 h; Mn = 12.0 kDa; Ɖ = 1.12)…………. 25

4.1 DSC thermogram of polystyrene copolymer containing 1-ethoxyBCB showing maximum

exotherm around 140 0C………………………………………………………………………………………………….. 52

4.2 Representative setup used for synthesis of SCPNs from (3.3) using ‘pseudo-high dilution

continuous addition technique’………………………………………………………………………………………… 53

4.3 A representative 1H NMR spectra of linear copolymer (top) and crosslinked nanoparticle

(bottom; zoomed in) [Table 4.1, Entry 1] showing loss of resonances characteristic to BCB

ring (e.g. methylene protons between 3.0 - 3.5 ppm)…………………………………………………… 54

4.4 Representative GPC chromatograms of linear copolymers and their corresponding

SCPNs. Top: Table 4.1, Entry 1 and bottom: Table 4.1, Entry 3……………………………………… 56

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4.5 TEM images showing SCPNs prepared from linear copolymer (PFS/VEBCB = 91/9; Entry 1,

Table 4.1). Scale bars: 100 nm (left) and 50 nm (right). Size as analyzed using ImageJ = 17

± 3 nm. SCPNs stained with RuO4 for 15 min…………………………………………………………………….. 58

4.6 TEM images showing SCPNs prepared from linear copolymer (PFS/VEBCB = 95/5; Entry 3,

Table 4.1). Scale bars: 100 nm (left) and 50 nm (right). Size as analyzed using ImageJ = 17

± 3 nm (diameter). SCPNs stained with RuO4 for 15 min…………………………………………………… 59

5.1 Stacked 1H NMR of linear di-block copolymer (top) and doubly crosslinked amphiphilic

polymer nanoparticle (bottom) as analyzed by Dr. Bill Storms indicating disappearance of

resonances belonging to BCBs in both the blocks……………………………………………………………… 63

5.2 Stacked chromatograms of different morphologies obtained during the process of step-

wise intramolecular crosslinking of a single di-block copolymer chain indicating

sequential reduction in hydrodynamic volume (as analyzed by Dr. Bill Storms)……………….. 64

5.3 Stacked chromatograms of different morphologies obtained during the process of step-

wise intramolecular crosslinking of a single di-block copolymer chain (performed in a

solvent bad for the block undergoing crosslinking)…………………………………………………………… 67

5.4 TEM images of samples prepared with THF solution of doubly crosslinked nanoparticles

with 20-25 mg/mL concentration and 15 min of staining. Scale bars: (a), (b) and (e) 100

nm; (c) and (d) 50 nm……………………………………………………………………………………………………….. 71

5.5 TEM images of samples prepared with THF solution of doubly crosslinked nanoparticles

with 15 mg/mL concentration and 15 min of staining. Scale bars: (a) 50 nm, (b) 100 nm…. 73

5.6 TEM images of samples prepared with THF solution of doubly crosslinked nanoparticles

with 5-7 mg/mL concentration and 15 min of staining. Scale bars: (a) 100 nm. (b) 20 nm… 73

5.7 TEM images of samples prepared with THF solution of doubly crosslinked nanoparticles

with 2-3 mg/mL concentration and 15 min of staining. Scale bars: (a) and (b) 50 nm………. 74

5.8 TEM images of samples prepared with THF solution of doubly crosslinked nanoparticles

with 5-7 mg/mL concentration and 5 min of staining. Scale bars: (a) and (b) 50 nm………… 75

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5.9 TEM images of samples prepared with THF solution of doubly crosslinked nanoparticles

with 5 μg/mL concentration and 10 min of staining. Scale bars: (a) 500 nm, (b) 100 nm…… 75

5.10 AFM images of highly fluorinated SCPNs. (a) height image and (b) adhesion image…………. 78

5.11 AFM images of samples prepared by deposition of THF solution of doubly crosslinked

nanoparticles with 1 mg/mL concentration on mica using spin-casting. Scan size: (a) and

(b) 1 μm 1 μm…………………………………………………………………………………………………………… 79

5.12 AFM images of samples prepared by deposition of THF solution of doubly crosslinked

nanoparticles with 25 μg/mL concentration on mica using drop-casting. Scan size: (a) 0.5

μm 0.5 μm, (b) 1 μm 1 μm. (c) height linecut for one of the discreet SCPNs …………. 81

5.13 AFM images of samples prepared by deposition of THF solution of doubly crosslinked

nanoparticles with 5 μg/mL concentration on mica using drop-casting. Scan size: (a) and

(b) 1 μm 1 μm. (a) height image and (b) adhesion image…………………………………………….. 82

5.14 AFM images of samples prepared by deposition of THF solution of doubly crosslinked

nanoparticles with 5 μg/mL concentration on mica using drop-casting. Scan size: (a) 1 μm

1 μm. (a) 3D view height image shown in Figure 5.14 (a). (b) Ring-like structures

reported by Hawker and coworkers in Macromolecules 2005, 38, 2674-2685………………. 83

5.15 AFM images of samples prepared by deposition of THF solution of doubly crosslinked

nanoparticles with (a), (b) 5 μg/mL and (c) 0.5 μg/mL concentration on mica using drop-

casting. ……………………………………………………………………………………………………………………….. 84

5.16 AFM images of samples prepared by deposition of THF solution of doubly crosslinked

nanoparticles with 0.1 μg/mL concentration on mica using drop-casting. Scan size: 1 μm

1 μm. (a) height image and (b) adhesion image………………………………………………………… 86

5.17 AFM images of samples prepared by deposition of THF solution of doubly crosslinked

nanoparticles with 0.05 μg/mL concentration on mica using drop-casting. Scan size: 0.5

μm 0.5 μm. (a) height image and (b) adhesion image……………………………………………….. 87

5.18 AFM images of samples prepared by deposition of THF solution of doubly crosslinked

nanoparticles with (a), (b) 0.1 μg/mL and (c), (d) 0.05 μg/mL concentration on silicon

wafer using drop-casting. Scan size: 1 μm 1 μm. (a), (c) height image and (b), (d)

adhesion image…………………………………………………………………………………………………………… 88

5.19 CONTIN analyzed DLS intensity plot for THF solution of doubly crosslinked nanoparticles

with 1 mg/mL concentration. Solution prepared by direct addition of THF to

nanoparticles. Data collected at different angles……………………………………………………………... 91

5.20 CONTIN analyzed DLS intensity plot for THF solution of doubly crosslinked nanoparticles

with 0.5 mg/mL (top) and 0.125 mg/mL (bottom) concentration. Solutions prepared by

direct addition of THF to nanoparticles. Data collected at different angles………………………. 93

5.21 CONTIN analyzed DLS intensity plot for solutions of doubly crosslinked nanoparticles

prepared in different compositions of (THF + TFT) mixture with 1.0 mg/mL concentration.

Solutions prepared by direct addition of solvent to nanoparticles. Data collected at

different angles………………………………………………………………………………………………………………. 96

5.22 DOSY-NMR spectra reported by Darcos and coworkers for the determination of polymer

nanoparticle morphology. Linear tri-block copolymer (left) and micelles (right) show two

different diffusion coefficients corresponding two distinct morphologies and sizes……….. 99

5.23 CONTIN analyzed DLS intensity plot for d8-THF solution of doubly crosslinked

nanoparticles with 1 mg/mL concentration. Solutions prepared by direct addition of THF

to nanoparticles. Data collected at 900. Average diffusion coefficient values were

calculated using Stokes-Einstein equation where, T = 303.15 K (30 0C), η = 0.48 * 10-3

kg/(m2. s)…………………………………………………………………………………………………………………………. 100

5.24 DOSY-NMR spectrum of doubly crosslinked nanoparticles in d8-THF (1 mg/mL) acquired

at 30 0C. An average value corresponding to diffusion coefficient is reported…………………. 103

5.25 Optical microscopy images of flow coated films cast from THF solution of blend of doubly

crosslinked nanoparticles and PS homopolymer………………………………………………………………. 105

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5.26 AFM images of annealed films cast from THF solution of blend of doubly crosslinked

nanoparticles and PS homopolymer…………………………………………………………………………………. 106

5.27 Representative schematic showing the arrangement of nanoparticles at the air-film

interface wherein the fluorinated part has phase separated to the surface while PS part is

miscible with the film……………………………………………………………………………………………………….. 107

5.28 Analysis of all the nanostructures observed at the air-film interface in this AFM image

(same image shown in Figure 5.25 (a); with all the particles selected) showing their

narrow height distribution………………………………………………………………………………………………… 107

5.29 Contact angles for different liquids measured on neat PS film (left) and film containing

nanoparticle/homopolymer blend (right)…………………………………………………………………………. 108

6.1 Stacked 1H NMR spectra of 1-hydroxyBCB solution (with 1,1,1,2-tetrachloroethane as

internal standard in 1 mL DMSO-d6) heated to 60 0C and NMR collected at different time

intervals: (a) 0 h (top) and (b) 24 h (bottom)……………………………………………………………………. 116

6.2 Stacked 1H NMR spectra of 10 mol% solution of 1-acetoxyBCB in

bromopentafluorobenzene with 5 mole% 1,1,1,2-tetrachloroethane as internal standard

heated to 100 0C at different time intervals: 0 h (top), 24 h (center) and 48 h………………….. 118

6.3 1H NMR spectrum of crude reaction mixture showing the presence of dimerized lactol as

the major reaction product obtained after deprotection of 1-acetoxyBCB by NaOMe in

the absence of a dienophile……………………………………………………………………………………………… 121

6.4 1H NMR spectrum of the major isomer, 1-acetoxy-5-iodoBCB…………………………………………. 124

6.5 1H NMR spectrum of a mixture of 1-acetoxy-4-vinylBCB and 1-acetoxy-5-vinylBCB. Entire

spectrum shown on top and expanded regions on bottom……………………………………………… 126

6.6 1H NMR spectrum of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB])

(Entry 2, Table 6.4). For this example, the molar composition based on the aromatic

protons: St/(6.6) = 96.5/3.5…………………………………………………………………………………………….. 129

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6.7 An example GPC chromatogram (RI detector) of poly(styrene-co-[1-acetoxy-4-vinylBCB]-

co-[1-acetoxy-5-vinylBCB]) (Entry 3 in Table 6.4)…………………………………………………………….. 130

6.8 An example 1H NMR spectrum of poly(methyl methacrylate-co-[1-acetoxy-4-vinylBCB]-

co-[1-acetoxy-5-vinylBCB]) (Entry 5 in Table 6.5). For this example, molar composition

based on methine proton of BCB and methoxy protons of MMA is: MMA/(6.6) = 86/14… 132

6.9 An example GPC chromatogram (RI detector) of poly(methyl methacrylate-co-[1-acetoxy-

4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) (Entry 5 in Table 6.5)………………………………………… 134

6.10 GPC chromatogram of starting copolymer and the soluble extract from crosslinked

polymer showing intermolecular crosslinking using BuLi as nucleophile………………………. 135

6.11 1H NMR spectrum of crosslinked polymer obtained using BuLi as nucleophile. No BCB

resonances are observed at 5.80 ppm. New resonances belonging to o-tolualdehyde

units are seen at around 10 ppm…………………………………………………………………………………. 136

6.12 1H NMR spectrum of the partially crosslinked polystyrene using NaOMe as nucleophile at

room temperature. No BCB resonances are observed at 5.80 ppm. New resonances

belonging to o-tolualdehyde units are seen at around 10 ppm………………………………………. 138

6.13 GPC chromatogram of starting copolymer and soluble fraction of crosslinked polymer

obtained using NaOMe as nucleophile showing intermolecular crosslinking (Entry 3 in

Table 6.6)………………………………………………………………………………………………………………………… 139

6.14 GPC chromatogram (RI detector) of starting copolymer and soluble fraction of the

crosslinked polymer obtained using NaOMe as nucleophile indicating intermolecular

crosslinking…………………………………………………………………………………………………………………… 141

6.15 1H NMR spectrum of the partially crosslinked methacrylate copolymer using NaOMe as

nucleophile at room temperature. No BCB resonances are observed…………………………….. 142

6.16 GPC chromatogram (UV detector) of starting copolymer and partially crosslinked

polymer showing intermolecular crosslinking at 0 0C using NaOMe as nucleophile……….. 143

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6.17 1H NMR spectrum of the partially crosslinked polystyrene (expanded region at bottom)

using NaOMe as nucleophile at 0 0C. Resonances belonging to o-tolualdehyde indicate

partial deprotection of 1-acetoxyBCB units in the backbone…………………………………………….. 144

6.18 GPC chromatograms of linear and crosslinked polymer showing intramolecular

crosslinking………………………………………………………………………………………………………………………. 147

6.19 Plot to determine the dn/dc value of linear copolymer (composition: St/VAcOxyBCB =

65/35)……………………………………………………………………………………………………………………………… 148

6.20 Stacked chromatograms of linear copolymer (blue) and crosslinked nanoparticles (red)

measured using LS-GPC………………………………………………………………………………………………….. 149

6.21 1H NMR spectrum of partially crosslinked polymer nanoparticle showing complete

absence of resonances belonging to 1-acetoxyBCB (5.80 ppm) and appearance of

resonances belonging to o-tolualdehyde (10.00 ppm)………………………………………………………. 150

7.1 Proposed morphologies likely to form upon intramolecular crosslinking of tri-block

copolymer precursor……………………………………………………………………………………………………… 155

7.2 Snapshots from simulations predicting that, when a tri-block copolymer chain (M-1) [left]

is subjected to intramolecular crosslinking in a solvent bad for the central block, it is

likely to form (M-2)………………………………………………………………………………………………………….. 156

7.3 Snapshots from simulations predicting that, when a tri-block copolymer chain (M-1) [left]

is subjected to intramolecular crosslinking in a solvent bad for the end blocks, it is likely

to form (M-4) [right]………………………………………………………………………………………………………… 157

7.4 Snapshots from simulations predicting that, when a SCPN (M-4) [left] is subjected to

intramolecular crosslinking in a solvent that is bad for end blocks to start with and then

slowly becoming bad for the central block, it is likely to form (M-9) [right]…………………….. 157

7.5 Snapshots from simulations predicting that, when a SCPN (M-2) [left] is subjected to

intramolecular crosslinking in a solvent that is bad for the central block to start with and

then slowly becoming bad for the end blocks, it is likely to form (M-5) [right]…………………. 158

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7.6 Stacked 1H NMR spectra of poly(styrene) synthesized using ATRP (top) and ATRP-ATRC

(bottom) approach. Inset shows zoomed in region corresponding to polymer chain ends.. 161

7.7 GPC traces of poly(styrene) synthesized using ATRP (left) and ATRP-ATRC (right)

approach…………………………………………………………………………………………………………………………… 162

7.8 GPCPS,UV traces of poly(styrene) [left], poly(S-b-TF5FS) [center] and poly(S-b-TF5FS-b-S)

*right+ synthesized using ARGET ATRP and chain extension technique……………………………… 164

7.9 Stacked 1H NMR of poly(S-b-TF5FS) [top] and poly(S-b-TF5FS-b-S) [bottom] prepared

using ARGET ATRP and chain extension technique……………………………………………………………. 165

8.1 TEM images (top) and AFM images (bottom; height and adhesion) showing discreet

SCPNs as observed by individual techniques…………………………………………………………………….. 169

8.2 AFM images of annealed films cast from THF solution of blend of doubly crosslinked

nanoparticles and PS homopolymer…………………………………………………………………………………. 171

LIST OF SCHEMES

Scheme Page

2.1 Benzocyclobutene (BCB) and its thermal isomerization to o-QDM………………………………… 4

2.2 Proposed reaction mechanism of self Diels-Alder reaction of oQDM to produce

cyclooctadiene dimers and poly(o-xylene)………………………………………………………………………. 5

2.3 Different precursors used in the generation of oQDM……………………………………………………. 6

2.4 Numbering convention in the nomenclature of BCBs……………………………………………………… 6

2.5 Synthesis of BCB and its subsequent transformation to 1-bromoBCB…………………………….. 8

2.6 Various organic transformations on 1-bromoBCB…………………………………………………………… 8

2.7 Synthesis of benzocyclobutene-1-ol and 1-ketoBCB……………………………………………………….. 9

2.8 Synthesis of methyl- and methylene-substituted BCB…………………………………………………….. 9

2.9 Synthesis of 1-ethoxy and 1-acetoxyBCB by cycloaddition reaction between benzyne

generated via thermal decomposition of benzenediazonium carboxylate and olefin……… 10

2.10 Synthesis of 1-substituted BCBs via reaction of benzyne and olefin……………………………….. 10

2.11 Synthesis of poly(aryl ether ketone) using 2,5-disubstituted benzocyclobutene

monomer………………………………………………………………………………………………………………………… 12

2.12 General reactions involved in atom transfer radical polymerization (ATRP)……………………. 19

2.13 Reactions involved in atom transfer radical coupling (ATRC)…………………………………………… 21

2.14 Β-H elimination involved in ATRP of styrene…………………………………………………………………… 24

2.15 Thermal self-initiation involved in styrene polymerization……………………………………………… 24

3.1 Synthesis of 1-acetoxyBCB………………………………………………………………………………………………. 30

3.2 Synthesis of 1-hydroxyBCB……………………………………………………………………………………………… 31

3.3 Synthesis of 3-(2-Methylphenyl)-1-isochromanol via deprotection of 1-acetoxyBCB using

a nucleophile in the absence of a dienophile………………………………………………….……………. 33

3.4 Synthesis of 2,3,5,6-etrafluoro-4-(2,2,3,3,3-pentafluoropropoxy)-styrene……………………… 36

xxi

4.1 Synthesis of poly([2,3,4,5,6-pentafluorostyrene]-co-[1-ethoxy-4-and 1-ethoxy-5-

vinylBCB+)……………………………………………………………………………………………………………………….. 50

4.2 Synthesis of highly fluorinated SCPNs from highly fluorinated copolymer containing 1-

ethoxyBCB…………………………………………………………………………………………………………………….... 51

5.1 Schematic representation of di-block copolymer and its conversion to Janus-type

amphiphilic polymer nanoparticle via stepwise and selective crosslinking……………………… 62

5.2 Schematic representation of a di-block copolymer and its conversion to a Janus-type

amphiphilic polymer nanoparticle via stepwise and selective intramolecular crosslinking

in a solvent poor for the block undergoing crosslinking…………………………………………………… 66

5.3 Possible morphologies resulting from intramolecular crosslinking of a di-block

copolymer precursor………………………………………………………………………………………………………. 69

6.1 Schematic representation of the proposed di-block copolymer and its conversion to

Janus amphiphilic polymer nanoparticle via stepwise and selective intramolecular

crosslinking……………………………………………………………………………………………………………………… 112

6.2 New monomer (1-ethoxy-4- and 1-ethoxy-5-vinylBCB) synthesized by the Pugh group

and maximum temperature that can be used for its (co)polymerization without

premature ring-opening………………………………………………………………………………………………… 113

6.3 Schematic representation of deprotection of 1-acetoxyBCB using nucleophiles and

subsequent reactions as reported by Choy and coworkers……………………………………………… 115

6.4 Reaction showing unusual behavior associated with alkoxide o-QDM species………………… 120

6.5 Synthesis of new monomer/crosslinker, 1-acetoxy-4- and 1-acetoxy-5-vinylBCB……………. 123

6.6 Synthesis of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) using

ARGET ATRP……………………………………………………………………………………………………………………. 128

6.7 Synthesis and characterization of poly(MMA-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-

vinylBCB+) copolymers using AGET ATRP…………………………………………………………………………. 131

xxii

6.8 Crosslinking poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) using

BuLi at room temperature………………………………………………………………………………………………. 134

6.9 Crosslinking of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB] using

NaOMe at room temperature…………………………………………………………………………………………. 137

6.10 Crosslinking of poly(methyl methacrylate-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-

vinylBCB]) using NaOMe at room temperature……………………………………………………………… 141

6.11 Crosslinking of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) using

NaOMe at sub-ambient temperature……………………………………………………………………………… 142

7.1 Representative design of tri-block copolymer precursor in the Pugh group. Each of the

immiscible blocks contains BCB that would crosslink at distinctly different temperature

compared to BCB in other block……………………………………………………………………………………… 154

7.2 Schematic depicting synthesis of symmetric ABA tri-block copolymer using ATRP-ATRC

approach…………………………………………………………………………………………………………………………. 159

7.3 Synthesis of poly(styrene) using ATRP-ATRC approach……………………………………………………. 160

7.4 Synthesis of poly(styrene-b-[2,3,5,6-tetrafluoro-4-(2,2,3,3,3-pentafluoropropoxy)-

styrene]-b-styrene) using ARGET ATRP and chain extension approach……………………………. 164

8.1 Schematic showing structure of new copolymer poly(styrene-co-[1-acetoxy-4-vinylBCB]-

co-[1-acetoxy-5-vinylBCB]) and its subsequent crosslinking using NaOMe at room

temperature……………………………………………………………………………………………………………………. 173

8.2 AFM images of annealed films cast from THF solution of blend of doubly crosslinked

nanoparticles and PS homopolymer……………………………………………………………………………….. 174

xxiii

CHAPTER I

INTRODUCTION

The overall goal of my project involved synthesis and characterization of single chain polymer nanoparticles (SCPNs) prepared from different polymer precursors using benzocyclobutene (BCB) chemistry and intramolecular crosslinking. Successful SCPN synthesis from block copolymers also required development of a new low temperature crosslinker based on BCB. This thesis reports: (a) synthesis of highly fluorinated SCPNs from uni-block copolymer precursors, (b) characterization of amphiphilic SCPNs prepared from di-block copolymer precursors, (c) development of a new room temperature crosslinking system based on BCBs and (d) preliminary results on SCNP synthesis using ABA tri-block copolymer precursors.

BCB-based molecules present a very unique set of polymer crosslinkers. These molecules contain a strained 4-membered ring attached to an aromatic ring. When heated to an appropriate temperature, these molecules undergo ring-opening isomerization to form highly reactive intermediate, o- quinodimethane (o-QDM). In the absence of any other dienophile, o-QDM units react with each other forming dibenzocyclooctadiene type thermally stable structures via C-C bond formation. Most of these

BCBs can be activated by pure thermal stimulus and do not require any catalyst. o-QDMs generated from

BCBs undergo cycloaddition reactions without generation of any other byproducts, making it attractive even for industrial applications. BCB-based molecules also provide an opportunity to tune the temperature at which the formation of o-QDM and subsequent dimerization/crosslinking occurs.

Depending upon the substituent on the benzylic position of BCB, the crosslinking temperature can be varied from as low as room temperature to as high as more than 250 0C. The Pugh group developed a new low temperature crosslinker/monomer as 1-ethoxy-4 and 1-ethoxy-5-vinylBCB that was found to undergo polymer crosslinking in the range of 100-150 0C. This thesis reports the crosslinking of polymers containing

1-ethoxy-4- and 1-ethoxy-5-vinylBCB in their backbone.

This thesis also reports the development of a new BCB-based monomer, 1-acetoxy-4 and 1- acetoxy-5-vinylBCB that contains an ester group on the benzylic position. Unlike other 1-substituted BCBs that undergo pure thermal ring-opening isomerization to form o-QDM, this new crosslinker was found to form o-QDM in the presence of a suitable nucleophile at room temperature. Surprising results that demonstrate a different mechanism of dimerization/crosslinking for 1-acetoxyBCB-based crosslinking system are also reported. This crosslinker was developed as a low temperature crosslinker to aid in the selective crosslinking of block copolymers.

Crosslinking is one of the most widely used chemical reactions in the polymer and allied industries. The most commonly utilized mode of crosslinking is intermolecular crosslinking, where the crosslinkable groups on different polymer chains react with each other forming a crosslinked polymer network. In my project, the intramolecular mode of crosslinking was employed, where the crosslinkable groups on the same polymer chain react with each other forming crosslinked single chain polymer nanoparticles (SCPNs). Intramolecular crosslinking affords the synthesis of polymer nanoparticles in the sub-20 nm size range, which is otherwise difficult to obtain by other methods of polymer nanoparticle synthesis. Another unique feature of this technique is that, the characteristic properties of polymer precursor are also carried in the final crosslinked nanoparticle. We took advantage of this unique feature and prepared highly fluorinated SCPNs from highly fluorinated polymer precursor (synthesized by Dr.

James Baker, a former graduate student in our group).

Most of the work in the field of SCPN synthesis has used uni-block copolymer precursors that contain at least two monomers and a crosslinkable unit. The Pugh group designed an amphiphilic di-block copolymer containing two immiscible blocks and two different BCBs in each block that can undergo selective intramolecular crosslinking to prepare SCPNs with unique morphologies. Since the di-block copolymer used was amphiphilic in nature and consisted two immiscible blocks, it was expected that the final crosslinked nanoparticle would also be amphiphilic in nature and would adopt phase-segregated morphology. Dr. Bill Storms synthesized the di-block copolymer and subsequent SCPNs. This thesis reports results on the characterization of these new amphiphilic SCPNs using TEM, AFM, DLS and DOSY-NMR. We

2 were mainly interested in evaluating whether these nanoparticles have phase-segregated morphology (or

Janus) or not.

In an effort to further increase the complexity in the polymer precursor used for SCPN synthesis, this thesis proposes ABA tri-block copolymer as an interesting polymer precursor for intramolecular crosslinking. Different morphologies that are likely to form, upon multiple and selective intramolecular crosslinking of ABA tri-block copolymer, are also proposed.

CHAPTER II

LITERATURE REVIEW

This chapter presents review of literature relevant to my research. This review covers three topics: (a) Benzocyclobutene (BCB) chemistry, (b) Single Chain Polymer Nanoparticles (SCPNs) and (c)

Atom Transfer Radical Polymerization (ATRP).

2.1 Benzocyclobutene (BCB) Chemistry

Bicyclo[4.2.0]octa-1,3,5-triene, often referred to as benzocyclobutene (BCB), and its derivatives are useful building blocks in organic and polymer chemistry. The molecular structure of BCB provides a unique compromise between the thermodynamic stability associated with the benzenoid aromatic system and the kinetic reactivity of a strained cyclobutane ring not observed in other similar compounds. E.g., benzocyclopropenes, the next lower homologue of BCBs, are too reactive with the 3-membered ring, whereas, indan, a higher five-membered analogue, is too unreactive toward ring opening.1 On the other hand, strained 4-membered cyclobutane ring attached to the benzene ring in BCB is relatively stable but undergoes thermal isomerization at elevated temperatures to o-quinodimethane (or o-xylylene) (oQDM), a highly reactive intermediate (Scheme 2.1).2,3

Scheme 2.1 Benzocyclobutene (BCB) and its thermal isomerization to o-quinodimethane (oQDM)2,3

The ability of oQDM to undergo facile intra- and inter-molecular Diels-Alder reactions with various dienophiles has been widely utilized in the synthesis of complex polycyclic compounds.4 In the absence of any external dienophile, this o-quinodimethane undergoes Diels Alder reactions with itself to produce an intermediate spirodimer. This spirodimer then fragments to give benzylic diradical species.

These species can react in two ways: either undergo intramolecular coupling to give dibenzocyclootadiene or oligomerize to give poly(o-xylene) (Scheme 2.2). This thermal activation of BCB and subsequent reactions of oQDM do not produce any condensation by-products.5,6

Scheme 2.2 Proposed reaction mechanism for self Diels-Alder reaction of oQDM to produce cyclooctadiene dimers and poly (o-xylene).5

This widely used and useful oQDM intermediate can be generated from various precursors and by utilizing various promoters. As shown in Scheme 2.3, these can be classified broadly in five categories.2

Path A: Thermolysis of benzocyclobutenes,

Path B: 1,4-elimination of α,α’-substituted o-xylenes,

Path C, D: reverse Diels-Alder reaction of benzo-fused heterocyclics,

Path E: Photoenolization and Photorearrangement of o-methylbenzaldehydes and o-methylstyrenes,

Path F: o-xylylene – metal complexes.

Between these paths, generation of oQDM by thermolysis of BCBs by far remains the most widely used method, mainly due to the ability to vary the cyclobutane ring opening temperature depending upon the substituent on 4-membered ring. In polymer chemistry, BCBs are predominantly used as precursors for oQDM mainly due to their stability towards polymerization conditions and the lack

5 of condensation by-products.3 Nevertheless, there are reports of other precursors used in polymer chemistry.7,8,9

Scheme 2.3 Different precursors used in the generation of oQDM. 3

2.1.1 Functionalized Benzocyclobutene:

Benzocyclobutene was first reported by Finkelstein et. al. in 1909.10 This field remained unnoticed until 1956 when Cava and co-workers repeated the first synthesis.11 These authors also established numbering convention for the nomenclature of BCBs. As shown in the scheme below, benzylic carbons are designated as 1 & 2 position carbons with the most substituted carbon being position 1.12 A different numbering convention has been followed in exceptional reports where aromatic carbons are referred to as 1 to 6 and benzylic positions denoted as 7 and 8.13

Scheme 2.4 Numbering convention as established by Cava et. al. in the nomenclature of BCBs.

According to this numbering convention, benzocyclobutenes functionalized at 1 and/or 2 positions have been reported in the literature from the first report11 itself by Cava and coworkers. Placing substituents at these benzylic carbons gives control over the temperature and the rate of ring opening.

Table 2.1 reports the approximate reaction temperatures for the ring opening of 1-substituted BCBs compiled from different reports in the literature.2,14,15,16,17,1819

For unsubstituted BCB, ring opening occurs at around 200 0C. Placing an electron donating substituent at the 1-position leads to a decrease in the reaction temperature because activation energy for the ring opening is lowered. Endo and co-workers studied substituent effects using semi-empirical calculations and found that the activation energy for the ring-opening isomerization decreased with increasing electron donating ability of the substituent at 1-position.16 In another study by the same authors, BCBs with electron-withdrawing substituents at the 1-position, such as 1-cyanoBCB, underwent ring-opening isomerization at ~ 160 0C.19 These functionalized BCBs have been widely used as precursors for the generation of functional oQDMs for different organic syntheses.

Table 2.1: Effect of substituent on the ring-opening isomerization behavior

Substituent (R) Isomerization Temperature (0C) -O-Mt+ <25 14 2 -NH2 25* 15 -OCH=CH2 60 -OH 80* 2 -OR 100 - 150 16,17,18 -NHCOR’ 110* 2 2 -O(CH2)nNHCOR' 150 -C=O 150* 2 -CN 160 19 -(CH ) H 180* 2 2 n -H 200* 2

(* The reactions were carried out at these temperatures for 18 h)

Though there are many reports in the literature concerning the synthesis of functionalized BCBs

2,20, we were mainly interested in the synthesis of vinyl BCBs substituted at 1-position. Cava and co-

7 workers were the first to report the synthesis of BCB and were successful in reporting different functional group substituted benzocyclobutenes using various organic transformations. They reduced 1,2- diiodobenzocyclobutene with Pd/C in the presence of sodium ethoxide to yield a colorless oil of benzocyclobutene. This was further brominated using N-bromosuccinimide (NBS) to produce 1-bromoBCB

(Scheme 2.5).21

Scheme 2.5 Synthesis of BCB and its subsequent transformation to 1-bromoBCB.21

Cava also treated this 1-bromoBCB with sodium cyanide in warm DMSO to produce 1-cyanoBCB in 93% yield. The nitrile group on this compound was further oxidized using alkaline hydrogen peroxide to produce benzocyclobutene-1-carboxyamide. Benzocyclobutene-1-carboxylic acid was prepared either by alkaline hydrolysis of the amide or by direct hydrolysis of the 1-cyanoBCB (Scheme 2.6).22

Scheme 2.6 Various organic transformations on 1-bromoBCB.22

In another paper by the same group23, 1-bromoBCB was treated with silver trifluoroacetate to produce corresponding ester, which was further hydrolyzed to benzocyclobutenol. Oxidation of this alcohol yielded 1-ketoBCB, but the same alcohol was converted to o-tolualdehyde in the presence of a base (Scheme 2.7).

Scheme 2.7 Synthesis of benzocyclobutene-1-ol and 1-ketoBCB. 23

Utilizing benzocyclobutene-1-carboxylic acid, methyl and methylene-substituted benzocyclobutene were synthesized by a series of organic transformations as shown in Scheme 2.8.

Reduction of the acid with lithium aluminium hydride produced the benzocyclobutenol and its subsequent esterification with tosyl chloride produced the tosyl ester. This ester on reduction with lithium aluminium hydride produced alkyl BCB and with t-butoxide it produced 1-methyleneBCB.24

Scheme 2.8 Synthesis of methyl- and methylene-substituted BCB.24

1-functionalized BCBs have been successfully prepared by [2+2] cycloaddition between benzyne and a dienophile. This route enables the synthesis of BCBs with different substituents at the 1-position by choosing a suitable dienophile. For example, Wasserman and Solodar prepared 1-ethoxy and 1-acetoxy

BCB by reacting benzyne with ethyl vinyl ether and vinyl acetate, respectively. They generated benzyne in situ by thermally decomposing benzenediazonium-2-carboxylate.25

Scheme 2.9 Synthesis of 1-ethoxy and 1-acetoxyBCB by cycloaddition reaction between benzyne generated via thermal decomposition of benzenediazonium carboxylate and olefin.25

Using the same strategy, other groups have reported this 1-step synthesis for 1-functional BCB with other substituents. Reacting benzyne generated in situ with acrylonitrile produced 1-cyanoBCB in

20% yield, whereas reaction with ethyl acrylate resulted in poor yield (<10%) of benzocyclobutene-1- carboxylate. A successful [2+2] cycloaddition between benzyne and vinyl bromide resulted in the synthesis of 1-bromoBCB in 40% yield.26,27

Scheme 2.10 Synthesis of 1-substituted BCBs via reaction of benzyne and olefin. 26,27

2.1.2 Benzocyclobutenes in Polymers:

Utilization of BCBs in polymers can be classified into two categories: 1) BCBs as crosslinkable moieties for the preparation of either thermosets or copolymers and 2) use of BCBs as precursors to highly reactive oQDM monomer and synthesis of polymers from thereof.

Thermal isomerization of BCB to oQDM and its subsequent Diels Alder reaction with itself or another dienophile has been used to great advantage to prepare thermoset resins and copolymers. This was achieved through incorporation of BCB in the monomer units. Polymerization of these BCB-containing monomers should keep the 4-membered ring intact for further use as a cross-linker. Researchers at Shell

Oil Company were the first to report the synthesis of 4-vinylbenzocyclobutene (VBCB) and its subsequent copolymerization with styrene by anionic and Ziegler-Natta polymerizations.28 These copolymers were cross-linked at elevated temperatures (> 200 0C) to obtain cross-linked polystyrenes with improved solvent resistance and higher Tg. Homopolymerization and copolymerization of 4-vinylBCB with styrene has also been well studied by other groups using anionic29 and controlled radical polymerizations.30 Wong and co-workers at Shell Oil Company also synthesized styrene-butadiene block copolymers with selective incorporation of BCB monomer in the styrene block and crosslinked it at elevated temperature to obtain better mechanical and physical properties.31 In another study, they hydrogenated the butadiene block of these BCB-containing block copolymers to produce copolymers with better mechanical properties after crosslinking the BCB units.32 These researchers also developed a versatile procedure for the incorporation of pendant BCB units into commercially available engineering thermoplastics. They synthesized 4- chloromethylBCB and alkylated/acylated it using Lewis acid catalysts to produce polymers with different chemical backbones such as polyethersulfone (UDEL), polyarylester (ARDEL) and polycarbonate

(MERLON). Post-polymerization modification and subsequent cross-linking of these polymers provided crosslinked polymers with improved solvent resistance while retaining toughness and thermoformability.33

Moore and coworkers synthesized a 2,5-disubstituted benzocyclobutene monomer and polymerized it to prepare poly(aryl ether ketone).34 On studying the thermal behavior of these polymers using differential scanning calorimetry (DSC), they observed an exotherm between 300-370 0C arising from the ring-opening reaction of pendant BCB units to oQDM and their subsequent reaction (Scheme

2.11).

Scheme 2.11 Synthesis of poly(aryl ether ketone) using 2,5-disubstituted benzocyclobutene monomer.34

This higher reaction temperature for cross-linking of BCB-based compounds was attributed to the steric encumbrance provided by substitution at the 2- and 5- positions of the aromatic ring. This behavior was found true in other BCBs with different substituents at the 2 and 5 positions.13

2.2 Single Chain Polymer Nanoparticles (SCPNs)

There is great interest in preparation of organic nanoparticles owing to their interesting properties.35 Polymer nanoparticles and dendrimers are important examples of organic nanoparticles that are particularly interesting because of their uses in variety of applications.36 Dendrimers often give the 3D spherical nanoparticles in the size range 1-10 nm, but their utility is limited by the complexity involved in their synthesis. Polymeric precursors to prepare polymer nanoparticles on the other hand are relatively easy to synthesize. Moreover, the recent developments in the controlled radical polymerizations have made it possible to synthesize them from many different monomers and with distinct architectures.37

Conventional methods of synthesizing nanoparticles from polymers have included miniemulsion, microemulsion, and self-assembly of block copolymers into micelles followed by cross-linking. These

12 methods produce particles that contain multiple polymer chains within a discreet nanoparticle and are typically obtained in the size range of 20-100 nm.38

Recently, a new method for the synthesis of polymer nanoparticles has been developed that enables these nanoparticles to be obtained in 5-20 nm size range which is difficult to obtain by the conventional methods mentioned above. Developed by Hawker and coworkers,30 this method relies on the collapse and intramolecular crosslinking of a single polymer chain to produce discreet single chain polymer nanoparticles (SCPNs). The preparation of SCPNs typically involves synthesis of a copolymer with crosslinkable groups incorporated along the polymer backbone followed by selective intramolecular crosslinking of crosslinkable groups under dilute reaction conditions. Hawker and coworkers used unsubstituted benzocyclobutene (BCB) as crosslinkable group that undergoes thermal ring opening followed by irreversible crosslinking at elevated temperatures of 250 0C. They were successful in synthesizing a polystyrene SCPN from linear polystyrene copolymer and a linear-nanoparticle hybrid SCPN via selective collapse of one block of a block copolymer as shown in Figure 2.1.

Figure 2.1 Schematic showing intramolecular chain collapse yielding nanoparticle and linear-nanoparticle architecture.30

Since these nanoparticles are synthesized via intramolecular chain collapse of linear precursors, they undergo reduction in their hydrodynamic volume/size as they go from random coil to crosslinked nanoparticle (globule) conformation. This change in conformation of a polymer chain is easily detectable via Gel Permeation Chromatography (GPC) where molecules are separated based on size. Moreover, it was shown that the sizes of the nanoparticles can be tuned by changing either the crosslink density of the crosslinkable groups or molecular weight of the linear precursor (Figure 2). Same authors used other characterization techniques to confirm the formation of SCPNs. For example, dynamic light scattering

(DLS) studies were performed to monitor the collapse process where size of the linear chain decreased as it was converted to a more compact structure of crosslinked nanoparticle. Also, 1H NMR characterization was used to substantiate the crosslinking process since the resonances belonging to benzylic protons disappeared following the thermal ring opening and subsequent cycloaddition reactions.

Figure 2.2 GPC analysis of linear polymer and intramolecularly crosslinked nanoparticle as reported by

Hawker and coworkers.30

Utilizing the advantages offered by BCB chemistry, Harth and coworkers17 developed a 1- functionalized BCB based crosslinker that underwent thermal ring opening and subsequent crosslinking

14 around 150 0C, less than the BCB crosslinker reported by Hawker and coworkers (Figure 2.3). Their approach involved synthesizing a BCB-based compound and attaching it to the polymer backbone in a post-polymerization modification manner followed by intramolecular crosslinking.

Figure 2.3. A low temperature cross-linker based on BCB as developed by Harth.17

After the successful attempt of synthesizing SCPNs by Hawker and coworkers, many researchers have developed new chemistries and approaches to synthesize SCPNs. For example, thiol-disulfide chemistry has been utilized to prepare SCPNs that change their conformation reversibly from random coil to crosslinked SCPN.39 Chain collapse via intramolecular hydrogen bonding has been reported by Hawker40 and Meijer.41 ‘Click’ chemistries that employ mild reaction conditions and highly efficient couplings have also been utilized for the purposes of synthesizing SCPNs. For example, Loinaz42 used Cu-catalyzed alkyne- azide cycloaddition between alkyne and azide groups pendant to the polymer backbone, whereas

Hawker43 reported the use of isocyanate-amine chemistry at room temperature to produce SCPNs. Berda has recently reviewed the synthesis and applications of SCPNs covering all the reports extensively.44

Functionalizing/decorating sub-20 nm SCPNs to impart useful properties is also gaining attention since; these smaller nanoparticles will be potential candidates for wide variety of applications. For example, Thayumanavan and coworkers developed polystyrene nanoparticles having pendant amine functional groups via 2, 2-azo-bis-isobutyronitrile (AIBN) induced radical crosslinking of pendant styrene groups.45 Similar chemistry and approach with pendant acrylate functional copolymers was used before to

15 make unimolecular cross-linked nanoparticle from copolymers of aliphatic esters, such as poly (ε- caprolactone).46 In another approach, Pomposo and coworkers synthesized SCPNs with pendant 4- chloromethyl styrene units.47 The free chlorine group available on the surface of these nanoparticles was used for grafting polymer via ATRP. The free chlorine was also converted to azide group which in turn was used to attach molecules that imparted useful functionalities to these nanoparticles. In another report by same authors, linear polymer with excess azide groups was synthesized and azide groups were functionalized after intramolecular crosslinking.42 Odriozola and coworkers reported a novel crosslinker that not only facilitated intramolecular collapse but also provided a site for non-covalent functionalization on the surface of a SCPN.48 The crosslinker, diethylenetriaminepentaacetic acid with two terminal alkynes, induced chain collapse on reacting with azide groups on the polymer backbone and also provided chelation site for gadolinium(III) cation. This system helped to put more number of gadolinium centres per nanoparticle and improved its performance as a MRI contrast agent. The schematic below depicts their synthesis (Figure 2.4).

Figure 2.4 A schematic showing the synthesis of a single chain poly(acrylic acid) nanoparticle as a potential

MRI contrast agent.48 Black dots represent gadolinium ions in the nanoparticle.

There is growing interest in controlled/selective intramolecular crosslinking of more complex precursors such as block copolymers. Hawker and coworkers were the first ones to report the selective collapse of one block of diblock copolymer using BCB chemistry (Figure 2.1). Liu and coworkers reported

16 the preparation of tadpole molecules from diblock copolymers using photo crosslinking in a solvent mixture bad for the crosslinking block.49 In another case, Liu and coworkers prepared similar type of structures and studied their self-assembly behavior.50 Recently, Meijer and coworkers reported synthesis of an all methacrylate ABA tri-block copolymer having two different crosslinkable moieties in each block that underwent crosslinking in an orthogonal fashion. In other words, the two blocks could be crosslinked in a stepwise and selective manner to produce SCPN (Figure 2.6).51

Figure 2.5 Schematic showing work by Meijer and coworkers on orthogonal folding of block copolymers.51

Lutz and coworkers also reported multiple, stepwise and selective intramolecular crosslinking of

ABA tri-block copolymer chain to prepare SCPNs with crosslinked subdomains that were separated by a large polystyrene spacer (Figure 2.6).52 In this case, the polymer backbone contained styrene and N- substituted maleimide in the backbone that resulted in the formation of SCPN with uniform surface chemistry.

Figure 2.6 Schematic depicting synthesis of SCPN by Lutz and coworkers.52

Figure 2.7 Schematic of di-block copolymer and its conversion to amphiphilic SCPNs via stepwise and selective intramolecular crosslinking.

Dr. Bill Storms (former student in Pugh group) has synthesized SCPNs via multiple, stepwise and selective crosslinking of an amphiphilic di-block copolymer chain containing immiscible blocks and two different BCBs in each block (Figure 2.7).53 The polymer precursor used in this case was more complex as compared to work by Meijer and Lutz mentioned above. Also, since the amphiphilic block copolymer precursor contained immiscible blocks, the resulting SCPNs were expected to have amphiphilic surface chemistry.

2.3 Atom Transfer Radical Polymerization (ATRP)54,55

ATRP, one of the most versatile of all controlled radical polymerization (CRP) techniques, is a transition metal catalyzed radical polymerization. Any CRP relies on a dynamic equilibrium between active and dormant species in order to access control over the polymerization reaction. In ATRP, this dynamic equilibrium is established with the help of a rapid and reversible (pseudo)halogen transfer between the growing radicals and the transition metal (pseudo)halide-ligand complex.

Scheme 2.12 General reactions involved in atom transfer radical polymerization (ATRP).

Here,

Pn-X represents the dormant species (can be composed of any number of repeating units),

n th Mt -Y is a transition metal in the n oxidation state and Y may be another counter ion (generally it is another halogen such as Br or Cl).

The ligand can be any organic ligand that ligates the transition-metal, increasing its electron density and thus lowering its oxidation potential.

n Mt -Y/ligand is also called an activator,

Pn● is a radical formed in the halogen transfer step, to which monomer units are added with a rate constant for propagation of kp,

ka is the rate constant of activation,

kda is the rate constant of deactivation,

kt is the rate constant for termination,

n+1 th X-Mt -Y is a transition metal in the (n+1) oxidation state due to the redox reaction between the dormant species and the transition metal-ligand complex in the lower oxidation state.

n+1 X-Mt -Y/ligand is also called a deactivator.

A typical ATRP consists of monomer, initiator, transition-metal halide and ligand. In ATRP, alkyl halides (R-X) are typically used as initiators. The halogen atom of the initiator is reduced by the transition-

n metal-ligand complex in a lower oxidation state (Mt -Y) through a one electron reaction with a rate constant ka of activation. Transition-metal thus gets oxidized forming a transition-metal ligand complex in

n+1 a higher oxidation state (X-Mt -Y) and a radical (P●). Monomer units add to this radical with a rate constant kp for propagation till the radical is quenched by reversible halogen transfer from the transition

n+1 metal-halide complex (X-Mt -Y) through a deactivation reaction (kda). This deactivation reaction produces the dormant species again, though with the addition of a few monomer units. These activation- deactivation cycles are very important in any CRP since the frequency of these cycles determine the polydispersity. Ideally, kda >> ka , which insures that deactivation is favored and at any time, the number of dormant species are much higher than the number of growing radicals. This suppression in the number of instantaneous growing radicals leads to reduced termination (< 5%) relative to propagation and thus produces polymers with predetermined molecular weights and narrow molecular weight distribution.

As with conventional radical polymerization, there are a number of vinyl monomers that can be polymerized using ATRP. When X is bromine or chlorine in alkyl halide (R-X) initiators, the molecular weight control is the best. Other pseudo halogens, such as thiocarbamates56 and thioesters,57 have also been used successfully.

The choice of the transition metal is important for a successful ATRP. Many different transition metals have been used for this purpose, such as molybdenum, manganese, rhenium, ruthenium, iron, cobalt, rhodium, nickel, palladium and copper. Though all of these have been successful to some extent for different reaction conditions, copper-based catalysts remain the best. The choice of ligand also influences the characteristics of an ATRP. Amongst all, nitrogen-based ligands exhibit better control compared to other classes of ligands, such as phosphorus-based ligands.

ATRP can be conducted in bulk, in solution, or in heterogeneous systems (e.g., emulsion, suspension).

2.3.1 Atom Transfer Radical Coupling (ATRC)

Atom transfer radical coupling (ATRC), as the name suggests, is a coupling reaction between two radicals under atom transfer conditions.58 These reactions are an important tool for coupling reactions of halide-terminated polystyrenes prepared via ATRP. In a typical ATRC, the reaction conditions are the same as those of ATRP, but a halide-terminated polymer is used instead of monomer and an excess of reducing agent (such as a transition metal in lower oxidation state) is introduced in the system from the start.

Mechanistically, a halide chain end of a polymer chain (Pn-X) is abstracted by a transition metal-ligand complex (e.g. Cu(I)/Ligand), converting the transition metal to a higher oxidation state (e.g. X-

Cu(II)/Ligand). From the previous section, we know that this key step of rapid halogen transfer between polymer radical and dormant chain is responsible for keeping the concentration of growing radicals low at any time in the reaction system and thus reducing termination.59

In ATRC, the addition of excess reducing agent (Cu(0)) ensures that any Cu(II) formed in the system is reduced back to Cu(I), favoring the halogen abstraction. This leads to exactly the opposite situation as that of ATRP. In ATRC, the concentration of polymer radicals is purposefully increased and is kept high at any point in the reaction. This promotes the reaction between polymer radicals. In the case of polystyrenes, radical-radical coupling reaction is preferred over disproportionation,60,61 and thus leads to polymers with double molecular weight.

Scheme 2.13 Reactions involved in atom transfer radical coupling (ATRC),

ATRC was first investigated by Fukuda in order to investigate the behavior of polymer radicals in a monomer-free ATRP system.62 ATRC was first successfully employed for the synthesis of α,ω-telechelic polystyrenes by Yagci and coworkers,63 who reported hydroxyl, aldehyde, carboxylic acid and dimethylamino telechelic polystyrenes using functional initiators. A detailed study of the effect of different parameters of ATRC was reported by Matyjaszewski and coworkers.59 They reported the effect of different reducing agents such as Cu(0), ascorbic acid and tin octanoate. Their application of ATRC to the α,ω-dibrominated polystyrene prepared using a bifunctional initiator resulted in polystyrenes with multimodal molecular weight distributions.

After these initial reports, several papers studied this system further. Boutevin reported ATRC without Cu(I) and using only Cu(0) to synthesize telechelic polystyrenes with different functionalities.64

ATRC employing metal-free coupling approach (silane radical atom abstraction approach) has also been reported.65 Newer methods of ATRP employing lower concentrations of copper catalysts have also been studied for ATRC and are called activators generated by electron transfer – atom transfer radical coupling

(AGET-ATRC).66

ATRC has also been reported for the synthesis of telechelic polystyrenes containing functional groups that are important for biological applications.67 In a new approach, Yagci reported synthesis of poly(p-xylylene) using ATRC.68

Most of the reports on ATRC have focused on polystyrenes, since, unlike poly(methacrylate) radicals, they preferentially terminate by combination rather than disproportionation. However, the concentration of methacrylate radicals formed in the ATRC equilibrium is not sufficient enough to favor reactions between two poly(methacrylate) chains. Matyjaszewski and coworkers reported the use of styryl radical chain ends of a poly(methyl methylacrylate) to couple poly(methacrylate) radicals.69

Recently, this concept has been extended to develop an efficient low temperature ATRC called ‘Styrene

Assisted Atom Transfer Radical Coupling’. The same authors further exploited this concept to couple poly(benzamides) and produce ABA type tri-block copolymers of poly(benzamides).70

2.3.2 Synthesis of Highly Functional Polymers

ATRC works only with polymers having activated halogen at their chain end. Polymers without

halogen at the chain termini will not be activated to produce radicals. Thus, preservation of the chain end

functionality (CEF) in precursor polymers is extremely important for these reactions.

Living polymerization was defined by Szwarc71 as a chain growth process without any chain

breaking reactions such as termination and transfer. Any CRP, be it ATRP, nitroxide mediated

polymerization (NMP) or reversible addition-fragmentation chain transfer (RAFT) polymerization, are not

‘living’ polymerizations as defined by Szwarc, since not all chains are end-capped by a desired functional

group. That is, in any CRP, termination or other side reactions of radicals are not totally suppressed; thus

polymers with 100% CEF are not accessible.

Specifically in ATRP, termination reactions occur at the start of the reaction when not enough

Cu(II) is generated to deactivate all chains efficiently. Also, early in the reaction, the chain lengths are

relatively small due to the low conversion, which favors the termination process since termination is a

chain length dependent process.72 This termination produces dead polymer chains of short length.

Termination in this case might lead to coupled or disproportionated polymer chains. Other type of chain

breaking reactions, transfer reactions, has same dependence on instantaneous radical concentration as

propagation reactions in radical polymerizations and hence may be suppressed by controlling the species

(their reactivities and concentration) with which radical is likely to undergo transfer reactions.

In ATRP, side reactions that hamper the % CEF include: a) β-H elimination: These elimination reactions catalyzed by Cu(II) are particularly troublesome in ATRP of

styrene-type monomers. The two possible mechanisms are shown below. In both cases, HBr is

eliminated when propagating radicals react with Cu(II) to yield unsaturated or cyclic chain ends and

Cu(I).73

Scheme 2.14 β-H elimination involved in ATRP of styrene b) Reaction between growing radicals and copper catalyst: These side reactions are based on outer sphere

electron transfer (OSET) processes in which the radical is either oxidized in the presence of Cu(II) to

produce a carbenium ion or is reduced in the presence of Cu(I) to produce a carbanion.74,75 c) Thermal Self-initiation of Styrene: Styrene undergoes thermal initiation above 700C to self-initiate

thermal polymerization. The rate of this initiation increases as temperature increases further, though the

minimum is at 70 0C.76 Scheme 2.15 gives an idea about thermal self-initiation reaction in which a

benzylic radical is formed that can initiate the polymerization of styrene.

Scheme 2.15 Thermal self-initiation involved in styrene polymerization.

Thus, in order to prepare highly functional (high %CEF) polymers, occurrence of these reactions should be reduced.

Matyjaszewski and coworkers monitored the evolution of chain end functionality (CEF) in ATRP of styrene using 1H nuclear magnetic resonance (NMR; 600 MHz) and reported the conditions suitable for the synthesis of highly functional, yet narrow polydispersity polystyrenes.59 Their findings suggest that highly functional polymers can be prepared using ATRP by stopping the reactions at low conversions, carrying them out in bulk rather than solvent, and carrying them out at low enough temperatures (such as

70 0C or 80 0C, where thermal self-initiation is low). In a typical system reported there, where styrene is polymerized using CuBr/dNbipy (dNbipy: 4,4’-Di-(5-nonyl)-2,2’-bipyridine) system and methyl 2- bromopropionate (MeBP) as initiator, they quantified the %CEF using 600 MHz 1H NMR by considering the

1 ratios of methoxy protons of initiator (Ha in the H NMR spectrum below) and methine proton next to

bromine (Hf).

Figure 2.8 1H NMR analysis of the chain end groups in polystyrene prepared using ATRP

0 59 (styrene/MeBP/CuBr/dNbipy = 100/1/1/2; 110 C; 3.5 h; Mn = 12.0 kDa; Ɖ = 1.12).

Polystyrene with > 90% CEF was reported. An extension to this study showed that adding some amount of Cu(II) at the start of reaction (5% w.r.t. to all metal centers) successfully reduced the early- stage termination.59

Though above studies were successful in giving highly functional polymers, it wasn’t able to eliminate the catalyst induced reactions, such as β-H elimination and OSET reactions. Logically, in order to reduce the occurrence of these reactions, we should reduce the amount of catalyst used the system.

Matyjaszewski reported an improved ATRP procedure called, activators regenerated by electron transfer-

ATRP (ARGET-ATRP), which allows a decrease in the amount of catalyst from 10000 ppm to 10 ppm or less, making it more environmentally and industry-friendly.77 The same research group reported their studies on the application of this technique for the synthesis of polystyrene homopolymer with improved

%CEF. They reported conditions for the synthesis of polystyrene with high %CEF (as high as 87%) and somewhat high molecular weight (Mn, GPC = 9600 g/mol). They attribute this to the decreased frequency of side reactions due to lowered catalyst concentration. Since side reactions are reduced, it should lead to high molecular weight homopolymer of polystyrene. This hypothesis was confirmed in their studies reporting polystyrene as high as 185000 g/mol in molecular weight (GPC) and PDI of 1.35.78

CHAPTER III

EXPERIMENTAL METHODS

3.1 Introduction

The contents in this chapter pertain to the materials, techniques and procedures used in the synthesis of various molecules and nanoparticles. All of the syntheses are divided into three sections: small molecules, polymers and crosslinking reactions on polymers.

3.2 Materials

2-Aminobenzoic acid (anthranilic acid) (Alfa Aesar, 98+%), ammonium iodide (Alfa Aesar, 98+%), ascorbic acid (Fisher Sci.; 99+%), bromopentafluorobenzene (Oakwood, 99%), n-butyl lithium (Acros

Organics, 2.2 M solution in cyclohexane), copper(0) powder (Aldrich, 99%), copper(II) bromide (Aldrich,

99%), ethyl-2-bromoisobutyrate (Aldrich, 98%), glacial acetic acid (Fisher Chemical), hydrogen peroxide

(Acros Organics, 35% (w/w) aqueous solution), isoamyl nitrite (Alfa Aesar, 97%), palladium (II) dichloride

(Artcraft Chemicals, 99.9+%), 1H,1H-pentafluoropropane-1-ol (Oakwood, 95%), 2,3,4,5,6- pentafluorostyrene (Oakwood, 98%), sodium acetate (J.T. Baker, anhydrous), sodium hydride (Aldrich,

95%), sodium methoxide (Aldrich, 25 wt% solution in methanol), n-tetrabutyl ammonium chloride (Fluka,

99%), p-toluene sulfonic acid (Malinckrodt Inc., 99+%), α,α,α-trifluorotoluene (Aldrich, 99+%), vinyltrimethyl silane (Alfa Aesar, 97%) were used as received. Copper(I) bromide (Alfa Aesar, 99%) was stirred over glacial acetic acid, collected in a glass frit, washed with ethanol,79 dried under vacuum and stored in a desiccator. Potassium fluoride dihydrate (J.T. Baker, 100%) was dried by heating at 80 0C under vacuum and stored in a desiccator. Palladium(0) bis(dibenzylideneacetone) was synthesized according to a

80 literature procedure. Tris[2-(dimethylamino)ethyl]amine (Me6TREN) was synthesized according to a literature procedure.81 N,N,N’,N”N”-Pentamethyldiethylenetriamine (Aldrich, 99%) was stored over KOH.

Styrene (Aldrich, 99%) was passed through basic alumina and distilled from CaH2 prior to use. Methyl

27 methacrylate (Acros, 99%) was passed through basic alumina just prior to use. Methanol (Sigma Aldrich,

99.8%), dichloromethane (Sigma Aldrich), acetone (Sigma Aldrich; 99.5+%), hexanes (Fisher Chemical), diethyl ether (EMD Millipore), chloroform-d (Cambridge Isotope Laboratories, D-99.8%), dimethyl sulfoxide-d6 (Cambridge Isotope Laboratories, D-99.9%), tetrahydrofuran-d8 (Cambridge Isotope

Laboratories, D-99.5%) were used as received. 1,1,1,2-Tetrachloroethane (Aldrich, 99%) was distilled from

CaCl2. Reagent grade Tetrahydrofuran (THF) and toluene were distilled from purple sodium benzophenone ketyl. Reagent grade xylenes were distilled at 134 0C (760 mm Hg). Mesitylene (Aldrich;

98%), o-dichlorobenzene (Sigma Aldrich; 99%) and Mineral oil (USP grade; purchased at a pharmacy store) were used as received.

3.3 Techniques

All reactions were performed under a N2 atmosphere using a schlenk line unless otherwise noted. All 1-D Nuclear Magnetic Resonance (NMR) spectra were recorded with either a Mercury 300 MHz spectrometer or a Varian 500 MHz spectrometer. All NMR spectra were referenced to TMS (δ = 0.0 ppm) unless otherwise noted. Number-average (Mn) and weight-average (Mw) molecular weights relative to linear polystyrene standards and dispersities (Ɖ = Mw/Mn) were determined by gel permeation

0 chromatography (GPC) from calibration curves of log Mn vs elution volume at 35 C using THF as solvent

(1.0 mL/min), a guard column and a set of 50, 100, 500, and 104 Å, as well as linear (50 – 104 Å) Styragel

5 μm columns, a waters 486 tunable UV/Vis detector set at 254 nm, a waters 410 differential refractometer, and Millenium Empower 3 software. All samples were filtered through a 450 nm PTFE filter before injecting into the GPC. Absolute molecular weights were determined by GPC with a light scattering detector (LS-GPC) at 35 0C using THF as solvent (1.0 mL/min), a set of 50, 100, 500, 104 Å and linear (50-106 Å) 5 μm columns, a Wyatt Technology miniDAWN TREOS three-angle (46.60, 90.00, 133.40) light scattering detector equipped with a Ga-As laser (659 nm, 50 mW), with the concentration at each elution volume determined using a Wyatt Optilab T-rEX differential refractometer (658 nm). The molecular weight data were calculated using Astra 6.0.3.16 software (Wyatt Technology) and a Zimm fit.

Refractive index (RI) increments (dn/dc) were measured off-line in THF at room temperature at 658 nm

28 using the Optilab T-rEX differential refractometer. All samples were filtered through a 0.45 μm PTFE filter prior to analysis.

Transmission Electron Microscopy (TEM) was recorded on JEOL 1200 EXII with an accelerating voltage of 120 kV. THF solution of the crosslinked polymer nanoparticles was deposited onto carbon coated copper grid. Excess solution was absorbed by a piece of filter paper. The deposited sample was vapor stained using an aqueous solution of staining agent and then dried under ambient conditions. TEM images were recorded on a digital CCD camera and processed with the accessory digital imaging system.

Atomic Force Microscopy: (A) For studies with individual nanoparticles, samples were prepared by either drop-casting or spin-casting from a dilute solution in THF on freshly cleaved mica and silicon wafer substrates. Concentration of polymer nanoparticles in THF was varied over a long range [1 mg/ml to

50 ng/ml]. Topographic images of nanoparticles were collected using a multimode Dimension Icon Atomic

Force Microscopy (AFM) with a NanoScope V controller (Bruker) in PeakForce Quantitative

Nanomechanical Mapping (QNM) mode. Silicon tips on a nitride cantilever (Bruker: Scanasyst-Air) with a tip diameter of 4-24 nm (average 10 nm), a resonance frequency of 50-90 kHz, and a spring constant of

~0.4 N/m were used. All images were analyzed using Nanoscope Analysis V. 1.20 software. (B) For studies with films of nanoparticle homopolymer blend, samples were prepared by flow coating THF solution of nanoparticle homopolymer blend onto freshly cleaned silicon wafer (using UV ozone exposure). Blend consisted of 3% nanoparticles and 2% polystyrene homopolymer (w/w) in THF solution. Topographic images were collected using AFM (Bruker AXS). Silicon tips on nitride cantilever with a tip radius of ≤ 10 nm and a spring constant of 7.8 N/m were used.

Dynamic Light Scattering (DLS) measures the intensity−intensity time correlation function by means of a BI-9000AT multichannel digital correlator. The field correlation function |g (1)(τ)| was analyzed by the constrained regularized CONTIN method to yield information on the distribution of the characteristic line width Γ from |g (1)(τ)| = ∫ G(Γ)e−Γτ dΓ. The normalized distribution function of the characteristic line width, G(Γ), so obtained, can be used to determine an average apparent translational

2 diffusion coefficient, Dapp = Γ/q . The hydrodynamic radius (Rh) is related to D via the Stokes− Einstein equation: Rh = kT/(6πηD), where k is the Boltzmann constant and η the viscosity of the solvent at temperature T. From DLS measurements, we can obtain the particle-size distribution in solution from a plot of ΓG(Γ) versus Rh. DLS measurements were performed at 298 K.

DOSY-NMR: 1H nuclear magnetic resonance (NMR) and 1H detected diffusion ordered spectroscopy (DOSY) experiments were performed at 298 K on a Varian 500 NMR spectrometer equipped with a Bruker multinuclear z-gradient inverse probehead.

For the intramolecular crosslinking experiment, syringe pump used was from New Era Pump

Systems (Model: NE-1000).

Cycloaddition reactions were performed in a Parr reactor to handle benzenediazonium-2- carboxylate salt very carefully.

3.4 Synthesis of and studies on small molecules

3.4.1 Synthesis of 1-acetoxybenzocyclobutene

Scheme 3.1: Synthesis of 1-acetoxyBCB

In a beaker with a stir bar, anthranilic acid (10 g, 73 mmol) and trichloroacetic acid (105 mg, 0.73 mmol) were dissolved in 50 mL THF and stirred in an ice bath for 10 min. Iso-amyl nitrite (10.7 g, 91.2 mmol) was added to the cooled solution batch wise over 5 min and the contents were further stirred in an ice bath for 30 min. The beaker was taken out of an ice bath and stirred at room temperature for 3 h after which, the contents had turned into tan-colored precipitate suspended in red solution. The precipitate, benzenediazonium-2-carboxylate salt,82 was collected on a glass frit and washed with dichloromethane to

30 displace THF till the filtrate is colorless. The slurry of benzenediazonium-2-carboxylate salt in dichloromethane was transferred to a Parr reactor containing vinyl acetate (38 g, 438 mmol). Parr reactor was assembled and programmed to heat to 95 0C in 5 min and to hold at 95 0C for 30 min. After 30 min, the heating was stopped and the contents were cooled to room temperature overnight. The contents from Parr reactor were then concentrated to remove excess of the solvent and the vinyl acetate. The crude obtained was then dissolved in 10 mL of acetone and added batch wise over 10 min to a refluxing mixture of 10 g of silica gel and 100 mL of hexanes to boil off the acetone and precipitate the impurities.

The slurry obtained was then collected on a glass frit, the solids disposed of and the filtrate concentrated on rotovap. The crude obtained was purified by vacuum distillation to collect yellow oil at 35 0C – 37 0C (at

1 1 mm Hg) as the product. Total weight = 3.01 g (% Yield = 26 %). H NMR (300 MHz): 2.11 (3H, s, O=C-CH3),

3.22 (1H, dd, 2J = 14.5, 3J = 1.9 Hz, PhCHH), 3.66 (1H, dd, 2J = 14.5, 3J = 4.5 Hz, PhCHH), 5.91 (1H, dd, 3J =

4.5, 3J = 1.9 Hz, PhCHO-AC), 7.15 (1H, d, J=7.3 Hz, aromatic H), 7.23 - 7.37 (3H, m, 3 aromatic H’s).

3.4.2 Synthesis of 1-hyroxybenzocyclobutene

Scheme 3.2 Synthesis of 1-hydroxyBCB

In a 50 mL round bottom flask equipped with a stir-bar, 1-acetoxyBCB (748 mg, 4.62 mmol), p- toluene sulfonic acid (87.5 mg, 0.46 mmol) were dissolved in 2 mL methanol. The contents were heated to

30 0C for 22 h after which the excess of methanol was removed using rotary evaporation. The crude obtained was diluted with 5 mL of water and extracted with diethyl ether (3 mL; 1 mL 3 times each) to obtain white crystalline material. This material developed brownish-color over its edges when stored in refrigerator overnight. The crude was dissolved in 15 mL of hexanes at 60 0C and then cooled slowly to room temperature on bench top to obtain white crystals. These crystals were then collected on a glass frit, washed with hexanes and dried overnight under vacuum. Total weight = 320 mg (% Yield = 61%).

Melting Point = 48 0C – 50 0C. 1H NMR (300 MHz): 1.79 (1H, br, CH-OH), 3.04 (1H, dd, 2J = 14.3, 3J = 1.5 Hz,

PhCHH), 3.63 (1H, dd, 2J = 14.3, 3J = 4.4 Hz, PhCHH), 5.30 (1H, dd, 3J = 4.4, 3J = 2.0 Hz, PhCHOH), 7.15 (1H, d, J = 7 Hz, aromatic H), 7.22 - 7.34 (3H, m, 3 aromatic H’s).

3.4.3 Thermal decomposition studies of 1-hydroxybenzocyclobutene and 1-acetoxybenzocyclobutene

(a) 1-hydroxyBCB

A stock solution of 1-hydroxyBCB (50 mg, 0.42 mmol) with 1,1,1,2-tetrachloroethane (35 mg,

0.21 mmol) as internal standard in 3 mL of DMSO-d6 was prepared. This solution was split equally into 3

NMR tubes and each of the tube was heated at different temperatures (60 0C, 80 0C, 100 0C) for various times (0 h and 24 h). The decomposition of 1-hydroxyBCB was monitored via loss of methylene proton resonances of 1-hydroxyBCB (3.04 ppm and 3.63 ppm) compared to internal standard (1,1,1,2- tetrachloroethane; 4.78 ppm). Also, the amount of o-tolualdehyde formed via thermal rearrangement of

1-hydroxyBCB was calculated by comparing the aldehydic resonance (10.23 ppm, 1H, s) with internal standard.

(b) 1-acetoxyBCB

A 10 mol% solution of 1-acetoxyBCB in bromopentafluorobenzene with 5 mol% 1,1,1,2- tetrachloroethane as internal standard was heated to different temperatures (60 0C, 80 0C, 100 0C). A 10 mol% solution in fluorinated solvent was used to mimic the possible polymerization conditions for the preparation of poly(pentafluorostyrene-co-1-acetoxy-4,5-vinylBCB) with 90:10 as the comonomer feed ratio. Any solvent with a boiling point higher than 100 0C can be used for this NMR-Temperature study.

Aliquots were taken at different times (0 h, 24 h and 48 h) and analyzed by 1H NMR spectroscopy using

CDCl3 as deuterated solvent. The decomposition of 1-acetoxyBCB was monitored by comparing the integration of benzylic methine proton in 1-acetoxyBCB (5.91 ppm) to the integration of internal standard

(1,1,1,2-tetrachloroethane; 4.30 ppm).

3.4.4 Deprotection of 1-acetoxybenzocyclobutene using a nucleophile in the absence of a dienophile

[Synthesis of 3-(2-Methylphenyl)-1-isochromanol]

Scheme 3.3 Synthesis of 3-(2-Methylphenyl)-1-isochromanol] via deprotection of 1-acetoxyBCB using a nucleophile in the absence of a dienophile

A solution of NaOMe in MeOH (25 wt% in MeOH) (270 mg, 1.24 mmol) was added at once to 1 mL THF solution of 1-acetoxyBCB (100 mg, 0.62 mmol) under nitrogen atmosphere at room temperature.

On addition of NaOMe, the color of the contents instantaneously changed from light yellow to dark orange and the flask became warm. The contents were quenched with 1 mL of saturated aqueous NH4Cl after 10 min and transferred to a separating funnel. The organic phase was extracted using 6 mL of Et2O (2 mL each 3 times). All the organic phases were combined, dried over MgSO4 and concentrated on rotary evaporator. The crude reaction mixture was analyzed by 1H NMR spectroscopy and the resonances belonging to the dimerized lactol were observed along with other side-products. The crude reaction mixture was not isolated since the purpose of this experiment was to check the formation of lactol and

1 83 1 compare the H NMR resonances to the literature reports. H NMR (300 MHz): 2.38 (3H, s, PhCH3), 2.88

(1H, d, J = 2.9 Hz, PhCHHCH), 3.00 (1H, d, J = 11.4 Hz, PhCHHCH), 5.45 (1H, dd, 3J = 11.4, 3J = 2.9 Hz,

PhCH2CH-O-), 6.14 (1H, s, PhCH-OH), 7.10-7.60 (8H, m, aromatic Hs).

3.4.5 Synthesis of 5-iodo-2-aminobenzoic acid

In a 600 mL beaker with a stir bar, anthranilic acid (20.4 g, 146 mmol) and ammonium iodide

(22.0 g, 153 mmol) were dissolved in 100 mL glacial acetic acid and cooled in an ice bath for 10 min.

Hydrogen peroxide (35% w/w in H2O) (14.96 g of aqueous solution, 153.4 mmol) was added to cooled solution batch wise over 10 min and stirred in an ice bath for further 30 min. The contents were removed from ice bath and stirred at room temperature for 90 min after which they had turned into a slurry of

33 precipitated tan-colored powder suspended in the solvent. This precipitate was collected on a glass frit, washed with plenty of water until the filtrate was colorless. The contents on the frit were dried in vacuum oven overnight at 80 0C to obtain tan-colored powder with melting point = 218 0C – 220 0C. %Yield =

96.9%.1H NMR (300 MHz) [DMSO-d6]: 6.62 (1H, d, J = 8.5 Hz, aromatic H ortho to iodine and meta to amine), 7.46 (1H, d, J = 8.5 Hz, aromatic H ortho to amine and meta to iodine), 7.93 (1H, br, aromatic H ortho to iodine and carboxylic acid), 8.75 (2H, br, ArNH2).

3.4.6 Synthesis of 1-acetoxy-4 and 5-iodobenzocyclobutene

In a beaker with a stir bar, 5-iodo-2-aminobenzoic acid (10 g, 38 mmol) and trichloroacetic acid

(63 mg, 0.38 mmol) were dissolved in 50 mL THF and stirred in an ice bath for 10 min. Iso-amyl nitrite (5.6 g, 47 mmol) was added batch wise over 15 min to the cooled solution. On complete addition, the contents had turned orange in color and were further stirred in an ice bath for 30 min. The beaker was taken out of ice bath and stirred at room temperature for 90 min after which the contents had turned into dark grey precipitate suspended in the solvent. The precipitate, iodobenzenediazonium-2-carboxylate salt,4 was collected on a glass frit and washed with dichloromethane to displace THF till the filtrate is colorless. The slurry of iodobenzenediazonium-2-carboxylate salt in dichloromethane was transferred to a Parr reactor containing vinyl acetate (20.0 g, 228 mmol). The Parr reactor was immersed in an oil bath preheated to

120 0C and was heated for 2 h after which heating was stopped and contents cooled to room temperature overnight. Contents from the Parr reactor were then concentrated to remove excess of solvent and vinyl acetate. The crude obtained was then dissolved in 15 mL of acetone and added batch wise to a refluxing mixture of 13 g of silica gel and 100 mL of hexanes to boil off acetone and precipitate impurities. The slurry obtained was then collected on a glass frit, solids disposed and filtrate reduced on rotovap. The crude obtained was purified by column chromatography using 80:20 (hexanes: diethyl ether) as the eluent system to obtain dark orange colored oil (Rf = 0.35) as the product. Total weight = 1.694 g (%Yield =

15.5%).

For the purposes of NMR assignments, major isomer was isolated. The major isomer crystallizes from hexane solution of mixture of isomers when stored in refrigerator (-20 0C); however, solubilizes in

34 hexane at room temperature. The major isomer was isolated by careful filtration and washing of the crystals at lower temperatures (-20 0C to -10 0C) to obtain the pure isomer as white crystalline material.

1 2 3 For major isomer: H NMR (500 MHz): 2.10 (3H, s, PhCH-OCOCH3), 3.16 (1H, dd, J = 14.7, J = 1.5

Hz, PhCHH), 3.57 (1H, dd, 2J = 14.8, 3J = 4.5 Hz, PhCHH), 5.86 (1H, dd, 2J = 4.6, 3J = 2.0 Hz, PhCH-OAc), 6.93

(1H, d, Jortho = 7.6 Hz, aromatic H meta to iodine), 7.61 (1H, s, aromatic H ortho to iodine and BCB ring),

7.69 (1H, dd, Jortho = 7.7, Jmeta = 1.1 Hz, aromatic H ortho to iodine and meta to BCB ring).

3.4.7 Synthesis of 1-acetoxy-4 and 5-vinylbenzocyclobutene

In a schlenk flask, potassium fluoride (1.1 g, 19 mmol), n-tetrabutyl ammonium chloride (3.5 g, 13 mmol) and toluene (8 mL) were stirred at room temperature to form good suspension. Molecular sieves were then added to the suspension and stirred further for 10 min and degassed using 2 freeze-pump- thaw cycles. The flask with the degassed contents was taken in the dry box where Pd(dba)2 (547 mg, 0.95 mmol) was added, flask sealed with a rubber septum on one neck and removed from the dry box. In the fume hood, a mixture of 1-acetoxy-4-iodoBCB and 1-acetoxy-5-iodoBCB (1.82 g, 6.34 mmol) and vinyl trimethylsilane (3.81 g, 38 mmol) was added through the rubber septum under N2 atmosphere. The

0 contents were degassed by 2 freeze-pump-thaw cycles, backfilled with N2 and heated to 45 C. The conversion of aryl iodide was monitored by 1H NMR spectroscopy which showed complete consumption of the aryl iodide after 39 h. Reaction was stopped, diluted with Et2O, filtered over celite and the filtrate was reduced on rotovap. The crude was purified by multiple column chromatography iterations. First, the crude was purified using 100% CH2Cl2 as eluent to obtain two fractions: (a) a mixture of the desired product and small amount of silylated (non-deprotected) version of the product (Rf = 0.66) and (b) a mixture of the desired product along with some impurity (Rf = 0.53). Each of these fractions were further purified using (a) Hexanes:Et2O (80:20) and (b) Hexanes:Et2O (70:30) as eluent system respectively to obtain the desired product as a mixture of regioisomers in 574 mg quantity (%Yield = 48%) as light yellow oil. Even though 1H NMR shows a mixture of regioisomers, the coupling constants and the splitting pattern are reported only for the major isomer formed (1-acetoxy-5-vinylBCB) wherever possible. In some cases, there was too much overlap to be able to resolve the splitting and calculate the coupling constants

35 correctly. Dr. Bill Storms has performed detailed 2D NMR analysis on regioisomers in order to identify the correct structure of the major and the minor isomer formed.53 1H NMR (300 MHz) of 1-acetoxy-5-

2 3 vinylBCB: 2.11 (3H, s, PhCH-OCOCH3), 3.20 (1H, d, J = 14.3 Hz, PhCHH), 3.63 (1H, dd, J = 14.6, J = 4.7 Hz,

PhCHH), 5.20 (1H, d, J = 10.8 Hz, CHH=CHPh), 5.70 (1H, d, J = 17.6 Hz, CHH=CHPh), 5.89 (1H, m, PhCH-

3 3 OCOCH3), 6.69 (1H, dd, J = 17.6, J = 10.8 Hz, CH2=CHPh), 7.10 (1H, d, J = 7.6 Hz, aromatic H meta to vinyl),

7.35 (2H, m, aromatic Hs ortho to vinyl).

3.4.8 Synthesis of 2,3,5,6-tetrafluoro-4-(2,2,3,3,3-pentafluoropropoxy)-styrene

Scheme 3.4 Synthesis of 2,3,5,6-tetrafluoro-4-(2,2,3,3,3-pentafluoropropoxy)-styrene

NaH (2.23 g, 92.7 mmol) in THF (60 mL) was added to a 250 mL 3-neck round bottom flask equipped with a stir-bar, an air condenser, a gas adapter for N2, an addition funnel and glass stoppers. The resulting suspension was stirred under N2 for 10 min. To this suspension, pentafluoropropanol (9.27 g,

61.8 mmol) was added through addition funnel over 30 min and stirred under N2 at room temperature for

12 h. This suspension was then refluxed for 1 h and then cooled to prepare sodium salt of pentafluoropropanol. Pentafluorostyrene (10.0 g, 51.5 mmol) in THF (40 mL) was added to another 250 mL round bottom flask equipped with a stir-bar, an addition funnel and gas adapters. Sodium salt of pentafluoropropanol in THF was added to this solution using an addition funnel over 1 h upon which, the color of the contents changed from colorless to light yellow and was stirred at room temperature. After

4.5 h, an aliquot of the reaction mixture was taken and its NMR analysis (1H and 19F) indicated complete

36 consumption of the starting compound. The contents were poured over 150 mL of ice/water mixture in a separating funnel that resulted in phase separation overnight. The organic phase was extracted with 15 mL of Et2O (5 mL 3 times each), all the organic phases combined, dried using MgSO4 and concentrated using rotary evaporation. The crude obtained was further purified using vacuum distillation (53-55 0C; 1 mm Hg) to obtain colorless oil in 7.8510 g quantity (%Yield = 47%). 1H NMR (300 MHz): 4.60 (2H, t, 2J =

3 3 12.3 Hz, -OCH2CF2-), 5.69 (1H, d, J = 11.7 Hz, CHHCH-PhF), 6.07 (1H, d, J = 18.2 Hz, CHHCH-PhF), 6.63 (1H, dd, 3J = 18.0, 3J = 11.9 Hz, CHHCH-PhF).

3.5 Synthesis of polymers

3.5.2 Synthesis of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) using free

radical polymerization

In a typical procedure, a solution of benzoyl peroxide (16 mg, 66 μmol), styrene (400 mg, 3.84 mmol) and 1-acetoxy-4 and 5-vinylBCB (474 mg, 2.52 mmol) in benzene (1 mL) was added to a 10 mL schlenk flask. The contents were degassed by 3 freeze-pump-thaw (5-15-5 min) cycles, backfilled with N2, closed to the schlenk line and heated at 70 0C for 21 h. Reaction was stopped by quenching the contents in liquid N2 and exposing to atmosphere. The contents were diluted with 3 mL THF and precipitated in cold methanol (100 mL) to obtain a white precipitate. This precipitate was collected on a glass frit and dried under vacuum (40 0C) overnight to obtain a white powder in 390 mg quantity. %Yield = 45%; GPC:

PS,UV 1 Mn = 22.6 kDa, Ɖ = 3.55; H NMR (300 MHz): 1.39 (2H, br, backbone CH2CH-), 1.78 (1H, br, backbone

CH2CH-), 2.09 (3H, br, CH-OCOCH3), 3.08 (1H, br, PhCHH of BCB), 3.51 (1H, br, PhCHH of BCB), 5.79 (1H, br,

PhCH-OAc), 6.51 and 7.06 (aromatic Hs, br); composition: 65% styrene and 35% 1-acetoxy-4 and 5- vinylBCB.

3.5.3 Synthesis of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) using ARGET

ATRP

In a typical procedure, CuBr2/Me6TREN solution (95 μL, 0.85 μmol/5.1 μmol; (50 mg CuBr2 + 310 mg Me6TREN)/ 25 mL CH3CN) solution) was added to a 15 mL high vacuum line glassware and CH3CN was

37 removed under vacuum. A mixture of ethyl-2-bromoisobutyrate (1.66 mg, 8.5 μmol), styrene (796 g, 7.65 mmol), 1-acetoxy-4 and 5-vinylBCB (160 mg, 0.85 mmol) was then added to CuBr2/Me6TREN and stirred at room temperature for 10 min. Ascorbic acid (0.6 mg, 3.4 μmol) was quickly added to the contents and degassed by 3 freeze-pump-thaw (5-20-5 min) cycles after which, flask was backfilled with N2 and closed

0 to the schlenk line and heated at 90 C for 23 h. Reaction was stopped by quenching the contents in liq. N2 and exposing the catalyst to atmosphere. The crude reaction mixture was diluted with 2 mL THF, passed through basic alumina to remove the catalyst and precipitated in 50 mL MeOH to obtain a white precipitate. This precipitate was collected on a glass frit and dried under vacuum overnight to obtain a

PS,UV 1 white powder in 213 mg quantity. %Yield = 22.3%; GPC: Mn, = 22.1 kDa, Ɖ = 1.28; H NMR (300 MHz):

1.39 (2H, br, backbone CH2CH-), 1.78 (1H, br, backbone CH2CH-), 2.09 (3H, br, CH-OCOCH3), 3.08 (1H, br,

PhCHH of BCB), 3.51 (1H, br, PhCHH of BCB), 5.79 (1H, br, PhCH-OAc), 6.51 and 7.06 (aromatic Hs, br); composition: 90.8% styrene and 9.2% 1-acetoxy-4 and 5-vinylBCB.

3.5.4 Synthesis of poly(methyl methacrylate-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB])

using AGET ATRP

In a typical procedure, copper(0) powder (1.2 mg, 19 μmol) and copper(I) bromide (2.7 mg, 19

μmol) were added to a 25 mL schlenk tube with a stir-bar and kept under N2. A mixture of ethyl-2- bromoisobutyrate (2.1 mg, 11 μmol), PMDETA (3.3 mg, 19 μmol), methyl methacrylate (337 mg, 3.4 mmol) and 1-acetoxy-4- and 1-acetoxy-5-vinylBCB (70 mg, 0.37 mmol) was added to schlenk tube at once under N2. The contents were degassed using 3 freeze-pump-thaw (5-15-5 minute) cycles. After degassing,

0 contents were back-filled with N2, closed to the schlenk line and heated at 70 C for 5 min. Reaction was stopped by quenching the contents in liquid N2 and exposing the catalyst to atmosphere. The crude reaction mixture was diluted with 1 mL THF, passed through basic alumina and precipitated in 30 mL

MeOH to obtain a white precipitate. This precipitate was collected on a glass frit and dried under vacuum

PS,RI 1 overnight to yield a white powder in 70 mg quantity. %Yield = 17%; GPC: Mn, = 11.7 kDa, Ɖ = 2.18; H

NMR (300 MHz): 0.84 (3H, br, CH3C- of PMMA backbone), 2.11 (3H, br, CH-OCOCH3 of BCB ring), 3.02 (1H,

38 br, PhCHH of BCB ring), 3.59 (3H, br, -COO-CH3 of PMMA), 5.85 (1H, br, PhCH-OAc of BCB ring), 6.75-7.25

(aromatic Hs, br); composition: 86% MMA and 14% 1-acetoxy-4 and 5-vinylBCB.

3.5.5 Synthesis of poly(styrene) using ATRP for ATRC purposes

In a typical procedure, copper (II) bromide (11.0 mg, 50 μmol) and copper (I) bromide (142.0 mg,

1 mmol) were added to a dry schlenk flask with a stir-bar and kept under N2. PMDETA (213 mg, 1.2 mmol) was then added to the flask and the mixture was stirred for 10 min. A mixture of styrene (10.4 g, 100 mmol) and ethyl-2-bromoisobutyrate (194 mg, 1 mmol) was added to the flask, the contents degassed by

0 4 cycles of Freeze-Pump-Thaw (5-30-5 min.), backfilled with N2 and heated at 80 C for 3 h. Reaction was stopped by quenching the contents in liq. N2 and exposing the catalyst to atmosphere. An aliquot was taken for conversion calculation and the results are 44% conversion before precipitation. The crude reaction mixture was passed through basic alumina, precipitated in cold methanol, precipitate collected on a glass frit and dried under vacuum overnight to obtain 2.56 g of a white powder. %conversion (1H

PS,RI 1 NMR) = 44%; %Yield = 24.6%; GPC: Mn = 4.42 kDa, Ɖ = 1.08; H NMR (300 MHz): the key resonances here are: 3.50 (2H, br, CH3CH2-OCO- of initiator), 4.50 (1H, br, Ph-CH-Br of chain end); %Chain End

Functionality = 85% (comparison of resonances from initiator and chain end).

3.5.6 Synthesis of poly(styrene) using coupling reaction under atom transfer radical coupling

conditions (ATRP-ATRC sequence)

Copper(0) powder (18.00 mg, 0.272 mmol) and copper(I) bromide (9.76 mg, 0.07 mmol) were added to a schlenk flask with a stir-bar under N2 and sealed with a rubber septum. N2 sparged (30 min)

PS,RI toluene solution of polystyrene (300 mg of polystyrene with Mn = 4.42 kDa, Ɖ = 1.08; %Chain End

Functionality = 85% in 1 mL toluene) and PMDETA (59.00 mg, 0.34 mmol) were added to the schlenk flask using degassed syringe. The contents were degassed by 3 cycles of Freeze-Pump-Thaw (5-30-5 min), back-

0 filled with N2 and heated at 70 C for 6 h. Aliquots were taken at 4 h and 6 h and were analyzed using GPC

1 and H NMR spectroscopy. Reaction was stopped by quenching the contents in liq. N2 and exposing the catalyst to atmosphere. The reaction mixture was passed through basic alumina, precipitated in cold

MeOH and subsequently collected on a glass frit. The product was dried in vacuum oven overnight to

PS,RI 1 obtain a white powder in 126 mg quantity. After 6 h: %Yield = 42%; GPC: Mn = 8.01 kDa, Ɖ = 1.17; H

NMR (300 MHz): the key resonances are: 3.50 (4H, br, CH3CH2-OCO- of initiator).

3.5.7 Synthesis of poly(styrene) using ARGET ATRP

In a typical procedure, CuBr2/Me6TREN solution (215 μL, 2 μmol/12 μmol; (50 mg CuBr2 + 310 mg

Me6TREN)/ 25 mL CH3CN) solution) was added to a 15 mL high vacuum line glassware and CH3CN was removed under vacuum. A mixture of ethyl-2-bromoisobutyrate (3.65 mg, 19 μmol) and styrene (2.0 g, 19 mmol) was then added to CuBr2/Me6TREN and stirred at room temperature for 10 min. Ascorbic acid (1.4 mg, 8 μmol) was quickly added to the contents and the contents were degassed by 4 freeze-pump-thaw

0 (5-25-5 min) cycles, backfilled with N2, closed to the schlenk line and heated at 90 C for 20 h. Reaction was stopped by quenching the contents in liq. N2 and exposing the catalyst to atmosphere. The crude reaction mixture was diluted with 2 mL THF, passed through basic alumina to remove the catalyst and precipitated in 30 mL MeOH to obtain a white precipitate. This was collected on a glass frit and dried

PS,UV under vacuum overnight to obtain a white powder in 742.4 mg quantity. %Yield = 37%; GPC: Mn = 39.8 kDa, Ɖ = 1.27.

3.5.8 Synthesis of poly(styrene-b-[2,3,5,6-tetrafluoro-4-(2,2,3,3,3-pentafluoropropoxy)-styrene])

using ARGET ATRP

CuBr2/Me6TREN solution (230 μL, 2.1 μmol/13 μmol; (50 mg CuBr2 + 310 mg Me6TREN)/ 25 mL

CH3CN) solution) complex was added to a 15 mL high vacuum line glassware and CH3CN was removed

PS,UV under vacuum. A solution of polystyrene macroinitiator (39 mg of polymer with Mn = 19.8 kDa, Ɖ =

1.24; 2 μmol) and 2,3,5,6-tetrafluoro-4-(2,2,3,3,3-pentafluoropropoxy)-styrene (163 mg, 0.5 mmol) in xylenes (1 mL) was added to CuBr2/Me6TREN and stirred at room temperature for 10 min. Ascorbic acid

(1.8 mg, 10.1 μmol) was quickly added to the contents and the contents were degassed by 3 freeze-pump-

0 thaw (5-20-5 min) cycles, backfilled with N2, closed to the schlenk line and heated at 90 C for 20 h.

Reaction was stopped by immersing the flask in liq. N2 and exposing the catalyst to atmosphere. Reaction mixture was diluted with 2 mL THF, passed through alumina and precipitated in MeOH (30 mL). The

40 precipitate was collected on a glass frit and dried under vacuum overnight to obtain a white powder in 85

PS,UV mg quantity. %Yield = 42%; GPC: Mn = 30 kDa, Ɖ = 1.36. Bimodal distribution was observed in GPC. The

1 peak molecular weights observed were: Mp1 = 25777 Da and Mp2 = 48764 Da. H NMR (300 MHz): the key resonances are: 1.40 (2H, br, CH2CH-Ph of backbone), 1.90 (1H, br, CH2CH-Ph of backbone), 4.50 (2H, br, -

CH2CF2CF3 of fluorinated unit), 6.25-7.25 (aromatic Hs, br); Mn,NMR = 46.4 kDa; composition: styrene/F5PTFS = 2.3/1.

3.5.9 Synthesis of poly(styrene-b-[2,3,5,6-tetrafluoro-4-(2,2,3,3,3-pentafluoropropoxy)-styrene]-b-

styrene) using ARGET ATRP

CuBr2/Me6TREN solution (95 μL, 0.86 μmol/5.17 μmol; (50 mg CuBr2 + 310 mg Me6TREN)/ 25 mL

CH3CN) solution) complex was added to 10 mL schlenk tube and CH3CN was removed under vacuum. A solution of poly(styrene-b-[2,3,5,6-tetrafluoro-4-(2,2,3,3,3-pentafluoropropoxy)-styrene]) macroinitiator

PS,UV (40 mg of copolymer with Mn,NMR = 46.4 kDa; Mn = 30 kDa, Ɖ = 1.36, 0.86 μmol) and styrene (52 mg,

0.45 mmol) in xylenes (3 mL) was added to CuBr2/Me6TREN and stirred at room temperature for 10 min.

The xylenes solution of macroinitiator at room temperature was opaque, suggesting poor solubility of the macroinitiator. However, the reaction was carried out in anticipation that the solubility would be higher at reaction temperature (90 0C). Ascorbic acid (0.6 mg, 3.4 μmol) was quickly added to the contents and contents were degassed by 3 freeze-pump-thaw (5-15-5 min) cycles, backfilled with N2, closed to the

0 schlenk line and heated at 90 C for 41 h. Reaction was stopped by immersing the flask in liq. N2 and exposing the catalyst to atmosphere. Reaction mixture was passed through basic alumina and precipitated in MeOH (40 mL) to obtain a very fine white powder. The precipitate was collected on a glass frit and dried under vacuum overnight to obtain copolymer in 10 mg quantity. %Yield = 10.9%; GPC:

PS,UV Mn = 27.3 kDa, Ɖ = 1.33. Bimodal distribution was observed in GPC and the peak molecular weights

1 were: Mp1 = 26218 Da and Mp2 = 41657 Da. H NMR: 1.40 (2H, br, CH2CH-Ph of backbone), 1.90 (1H, br,

CH2CH-Ph of backbone), 4.50 (2H, br, -CH2CF2CF3 of fluorinated unit), 6.25-7.25 (aromatic Hs, br); Mn,NMR =

50.3 kDa; composition: styrene/F5PTFS = 2.78/1.

3.6 Intermolecular crosslinking of polymers

3.6.2 Intermolecular crosslinking of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-

vinylBCB]) using BuLi at room temperature

The procedure mentioned here is of experiment performed in a dry box. Similar experiment when performed in a fume hood by carefully handling BuLi, showed similar result. In a dry box, THF solution of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) (36 mg of copolymer with

PS,RI Mn = 14.7 kDa, Ɖ = 2.57; styrene:BCB = 65:35 molar composition in 0.3 mL THF and [BCB] =0.33 M) was prepared as a clear solution. An approximate excess of BuLi (2.2 M solution in cyclohexane) was added to the copolymer solution at once (2-3 drops were added; actual amount needed was 1.2 μL). On addition of

BuLi, the color of the contents instantaneously changed from colorless to dark orange and turned into a gel, indicating instantaneous crosslinking. After 1 min, excess of BuLi was quenched by adding 1 mL of

MeOH, which turned the color of the contents to light yellow. Outside the dry box, the contents were concentrated on rotovap. The resulting crosslinked polymer partially dissolved in THF and CDCl3 and only

1 PS,RI the soluble part could be analyzed using GPC and H NMR spectroscopy. GPC: Mn = 18.1 kDa; Ɖ = 6.1;

1 H NMR (300 MHz): 2.53 (3H, br, CH3-Ph of o-tolualdehyde units), 6.20-7.20 (aromatic Hs, br), 10.05 (1H,

Ph-CO-H of o-tolualdehyde units).

3.6.3 Intermolecular crosslinking of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-

vinylBCB]) using NaOMe at room temperature

NaOMe (39 mg, 180 μmol, 25% (w/w) solution in methanol) was added to a 25 mL 2-neck round bottom flask with a stir-bar, a glass stopper and a gas adapter and methanol was removed under vacuum to obtain a white powder of NaOMe. The removal of methanol is important in order to maintain aprotic reaction conditions for crosslinking purposes. THF solution of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-

PS,UV [1-acetoxy-5-vinylBCB]) (30 mg of copolymer with Mn = 21.9 kDa, Ɖ = 1.31; styrene:BCB = 90.8:9.2 molar composition in 0.5 mL THF and [BCB] = 50 mM) was prepared as a clear solution. This solution was added at once to NaOMe under N2 at room temperature. Upon addition, the color of the contents instantaneously changed from colorless to light yellow. After 5 min, the reaction mixture was diluted with

0.5 mL THF and quenched by adding it to NH4Cl/MeOH (30 mg/30 mL) solution to obtain a white precipitate. The precipitate was collected on a glass frit and dried under vacuum to obtain a white powder that was difficult to scrap off from the frit. The contents were dissolved in THF, transferred to a vial and

THF removed under vacuum. Product weight = 18.4 mg, [%Yield (w/w) = 61.3%]. The resulting crosslinked polymer was partially soluble in THF and CDCl3 and only the soluble part could be analyzed using GPC and

1 PS,UV 1 H NMR spectroscopy. GPC: Mn = 48.3 kDa; Ɖ = 7.47; H NMR (300 MHz): 1.42 (CH2CH-Ph of backbone), 1.85 (CH2CH-Ph of backbone), 2.55 (3H, br, CH3-Ph of o-tolualdehyde units), 6.20-7.20

(aromatic Hs, br), 10.08 (1H, Ph-CO-H of o-tolualdehyde units).

3.6.4 Intermolecular crosslinking of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-

vinylBCB]) using NaOMe at sub-ambient temperatures

The purpose of this experiment was to perform the crosslinking experiment at sub-ambient temperature. For this purposes, THF solution of copolymer, THF and pipettes were equilibrated at -7 0C overnight by keeping in the refrigerator. In a fume hood, NaOMe (28 mg, 130 μmol, 25% *w/w+ solution in

MeOH) was added to a 25 mL 2-neck round bottom flask with a stir-bar and a glass stopper. MeOH was removed under vacuum to maintain aprotic reaction conditions. NaOMe was then equilibrated at 0 0C for

PS,UV 30 min using an ice bath. The precooled THF solution of copolymer (40 mg of copolymer with Mn =

27.4 kDa, Ɖ = 1.53; styrene:BCB = 96.5:3.5 molar composition in 0.3 mL THF; [BCB] = 43.3 mM) was added to cooled NaOMe quickly under N2 at once using precooled pipettes. The removal of copolymer solution from refrigerator and its addition over NaOMe was done in less than 15 seconds to avoid warming up of a solution above 0 0C. Upon addition, an instantaneous color change from colorless to light yellow was observed. After 5 min, the contents of the reaction mixture were diluted with 0.5 mL THF (equilibrated to

0 -7 C overnight) and quickly added to NH4Cl/MeOH solution to quench the reaction mixture to obtain a white precipitate that was collected on a glass frit and dried under vacuum to obtain a white powder in 18

PS,UV 1 mg quantity (% Yield [w/w] = 45%). GPC: Mn = 31.6 kDa, PDI= 1.97, H NMR (300 MHz): 1.43 (2H, br, backbone CH2CH-), 1.84 (1H, br, backbone CH2CH-), 2.07 (3H, br, CH-OCOCH3), 2.54 (3H, br, CH3-Ph of o-

43 tolualdehyde units), 3.07 (1H, br, PhCHH of BCB), 3.52 (1H, br, PhCHH of BCB), 5.80 (1H, br, PhCH-OAc),

6.25 and 7.25 (aromatic Hs, br), 10.00 (1H, Ph-CO-H of o-tolualdehyde units).

3.6.5 Intermolecular crosslinking of poly(methyl methacrylate-co-[1-acetoxy-4-vinylBCB]-co-[1-

acetoxy-5-vinylBCB]) using NaOMe at room temperature

NaOMe (32.4 mg, 150 μmol, 25% (w/w) solution in methanol) was added to a 25 mL 2-neck round bottom flask with a stir-bar, a glass stopper and a gas adapter. Methanol was removed under vacuum to obtain a white powder of NaOMe. The removal of methanol is important in order to maintain aprotic reaction conditions for crosslinking purposes. THF solution of poly(MMA-co-[1-acetoxy-4-

PS,RI vinylBCB]-co-[1-acetoxy-5-vinylBCB]) (15 mg of copolymer with Mn = 11.7 kDa, Ɖ = 2.18; MMA:BCB =

86:14 molar composition in 0.4 mL THF and [BCB] = 47.5 mM) was prepared as a clear solution. This solution was added at once to NaOMe under N2 at room temperature. Upon addition, the color of the contents gradually changed from colorless to light yellow and turned cloudy. After 5 min, the reaction mixture was diluted with 0.5 mL THF and quenched by adding to NH4Cl/MeOH solution to obtain a white precipitate. The contents obtained were concentrated using rotary evaporation, redissolved using 5 mL

THF and passed through celite plug to remove the salts. The filtrate obtained was concentrated using

PS,RI rotary evaporation to obtain 8 mg of a light yellow oil/powder (%Yield = 53%). GPC: Mn = 33.7 kDa, Ɖ =

1 2.79; H NMR (300 MHz): 0.84 (3H, br, CH3C- of PMMA backbone), 3.59 (3H, br, -COO-CH3 of PMMA),

7.00-7.25 (aromatic Hs, br), 10.24 (1H, Ph-CO-H of o-tolualdehyde units).

3.7 Intramolecular crosslinking of polymers to synthesize single chain polymer nanoparticles

3.7.1 Synthesis of highly fluorinated polymer nanoparticles via intramolecular crosslinking of

poly(pentafluorostyrene-co-[1-ethoxy-4-vinylBCB]-co-[1-ethoxy-5-vinylBCB])

Xylenes (50 mL) was added to a 250 mL 3-neck round bottom flask with a stir-bar, water condensers, a gas adapter, a glass stopper and a rubber septum and preheated to reflux under N2. The

PS,UV copolymer solution in xylenes (9.4 mg of copolymer with Mn = 49.6 kDa, Ɖ = 2.01; PFS:BCB = 91:9 molar composition in 9 mL xylene; [BCB] = 0.49 mM) was added to refluxing xylenes drop wise using a

44 syringe pump at 2 mL/h through the rubber septum. On complete addition, the contents were stirred for additional 30 min and then cooled to room temperature. The xylenes was removed using a trap-to-trap distillation, crude diluted with THF, transferred to a vial and dried till constant weight to obtain a yellow

PS,UV viscous oil in 6.6 mg quantity. %Yield = 70.2%. GPC: Mn = 19.1 kDa, Ɖ = 1.44.

3.7.2 Synthesis of tadpole nanoparticles via intramolecular crosslinking of poly[(styrene-co-4-

vinylBCB)-block-(2,3,5,6- Tetrafluoro-4-(2,2,3,3,3- pentafluoropropoxy)styrene-co-2,3,5,6-

Tetrafluoro-4-(2-(1,2-dihydrocyclobutabenzen-1-yloxy)ethoxy)styrene)] at 150 0C under bad

solvent conditions

o-Dichlorobenzene (o-DCB) (100 mL) was added to a 250 mL 3-neck round bottom flask with a stir-bar, rubber septum, a water condenser, a gas adapter and a glass stopper and preheated to 150 0C

PS,UV under N2. The di-block copolymer solution in mesitylene (30 mg of di-block copolymer with Mn = 56.1 kDa, Ɖ = 1.56; Styrene:VBCB = 49:6 and Fluorostyrene:EtOBCB = 37:9 [mol/mol; 1H NMR] in 35 mL mesitylene) was added to o-DCB at 150 0C dropwise using a syringe pump at 2 mL/h through a rubber septum. On complete addition, the contents were stirred for an additional 30 min and then cooled to room temperature. o-DCB and mesitylene were removed using trap-to-trap distillation, and the crude product was redissolved in 3 mL THF and precipitated in 30 mL MeOH to obtain a white precipitate/suspension. This was stored in refrigerator overnight at 0 0C to obtain a better precipitate.

Upon observing the precipitate at bottom, all of the liquids were carefully decanted using a pipette and the solids were transferred to a vial with a spatula to yield crude in 39 mg quantity. % Yield of crude =

PS,UV 130%. GPC: Mn = 41.1 kDa, Ɖ = 2.18.

3.7.3 Synthesis of amphiphilic nanoparticles via intramolecular crosslinking of tadpole nanoparticles

at 250 0C under poor solvent conditions

Mineral oil (70 mL) was added to a 250 mL 3-neck round bottom flask with a stir-bar and two gas adapters. One of the gas adapters was connected to N2 and another was connected to a 25 mL round bottom flask to collect any THF that would evaporate. Mineral oil was sparged with N2 for 10 min. Tadpole

PS,UV solution in THF (15 mg of tadpole with Mn = 41.1 kDa, Ɖ = 2.18; in 15 ml THF) was prepared and

0 sparged with N2 for 10 min. Mineral oil was heated to 250 C under N2 and THF solution of tadpoles was added dropwise using a syringe pump at 2 mL/h through a rubber septum. On complete addition, the contents had turned into light yellow oil with light brown colored material suspended in it. Contents were transferred to centrifuge tubes (50 mL; polypropylene-based) and centrifuged at 8000 rpm for 30 min to obtain light brown colored material at the bottom of the centrifuge tubes. Mineral oil was decanted carefully, and then 3 mL hexanes was added to dissolve any remaining mineral oil and the liquids were carefully removed using a pipette to obtain 10 mg of crude. % Yield = 67%. The crude/solid obtained was

PS,UV dissolved in THF for 30 min prior to GPC analysis. GPC: Mn = 11.9 kDa, Ɖ = 1.32.

3.7.4 Synthesis of polystyrene nanoparticles via intramolecular crosslinking of poly(styrene-co-[1-

acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) at room temperature using BuLi

The procedure mentioned here is of experiment partially performed in a dry box. Similar experiment when performed in fume hood by carefully handling BuLi, showed similar result. In a dry box,

PS,UV benzene solution of copolymer (50 mg of copolymer with Mn = 22.6 kDa, Ɖ = 3.55; styrene:BCB =

65:35 molar composition in 12 mL benzene; [BCB] = 0.01 M) was prepared and transferred to a plastic syringe. BuLi solution in benzene (0.12 mL BuLi [as a 2.2 M solution in cyclohexane] in 50 mL benzene) was prepared in a dry box and transferred to a 3-neck round bottom flask and the setup was sealed using a glass stopper and a rubber septum. In a fume hood, the copolymer solution was added to the BuLi solution in benzene under N2 using a syringe pump at the rate of 1.0 mL/h at room temperature. On complete addition of BuLi, the color of the contents was colorless and was stirred for additional 30 min.

Excess of BuLi was then quenched by adding 1 mL of MeOH and 1 mL of saturated aqueous NH4Cl solution upon which, formation of some solids (presumably LiOMe) was observed. These solids were filtered and disposed. The filtrate was concentrated using rotary evaporation, redissolved in 3 mL of THF and precipitated in 50 mL of cold MeOH to obtain a white precipitate. This precipitate was collected on a glass frit but, was difficult to scrap off. The entire amount on the frit was dissolved and used for analysis. GPC:

PS,UV 1 Mn = 16.0 kDa, Ɖ = 2.75; H NMR (300 MHz): 2.47 (3H, br, CH3-Ph of o-tolualdehyde units), 6.20-7.20

(aromatic Hs, br), 9.99 (1H, Ph-CO-H of o-tolualdehyde units).

3.7.5 Synthesis of polystyrene nanoparticles via intramolecular crosslinking of poly(styrene-co-[1-

acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) at room temperature using NaOMe

NaOMe (21.2 mg, 98 μmol, 25% (w/w) solution in methanol) was added to a 50 mL 3-neck round bottom flask with a stir-bar, air condensers, gas adapters and a rubber septum. MeOH was removed carefully under vacuum to maintain aprotic reaction conditions. THF (30 mL) was added to NaOMe and

PS,UV stirred at room temperature for 10 min. THF solution of the copolymer (30 mg of copolymer with Mn

= 27.4 kDa, Ɖ = 1.53; styrene:BCB = 96.5:3.5 molar composition in 5 mL THF; [BCB] = 2 mM) was added to

NaOMe using a syringe pump at the rate of 1 mL/h under N2 at room temperature. After complete addition, the contents were stirred for additional 30 min. Reaction was quenched by adding the contents to NH4Cl/MeOH (30 mg/30 mL) solution to obtain a white precipitate. The contents were concentrated using rotary evaporation, redissolved in 5 mL THF, passed through celite plug to remove the salts and the

PS,UV filtrate was concentrated using rotary evaporation for analysis. GPC: Mn = 25.4 kDa, Ɖ = 1.47.

CHAPTER IV

SYNTHESIS AND CHARACTERIZATION OF HIGHLY FLUORINATED

SINGLE CHAIN POLYMER NANOPARTICLES

4.1 Introduction

This chapter pertains to (a) the synthesis of highly fluorinated polymer nanoparticles using pseudo-high dilution continuous addition method and (b) the characterization of these nanoparticles using GPC and TEM.

Functional polymer nanoparticles are gaining much attention as potential candidates for application in various fields such as drug delivery.38 Single chain polymer nanoparticles (SCPNs) are new type of polymeric nanoparticles that are particularly interesting. As the name suggests, these nanoparticles are made from a single polymer chain employing intramolecular crosslinking and provide access to particles in the 5-20 nm size range. Another advantage of this method is that, the functionalities and the properties of the starting linear polymer are carried into the crosslinked SCPNs, providing a useful tool to tune the properties of final SCPNs, as per the requirement. For example, Barner-Kowollik and coworkers synthesized triarylphosphine-containing polystyrene copolymers and converted them into

SCPNs via formation of non-covalent intramolecular crosslinks between triarylphosphine and suitable

Pd(II) complex to prepare Pd(II) containing SCPNs. 84 These Pd(II) containing SCPNs were then utilized as catalysts for Sonogashira coupling. In another example, Loinaz and coworkers prepared thermoresponsive

SCPNs starting from linear copolymer containing thermoresponsive poly(N-isopropylacrylamide) in the backbone.85 The usefulness and versatility of SCPN technology to prepare functional polymer nanoparticles were reviewed extensively by Berda44 and Barner-Kowollik86.

The preparation of SCPNs requires control over the polymer crosslinking reaction to selectively obtain intramolecular crosslinking rather than intermolecular crosslinking. Selective intramolecular

48 crosslinking can be obtained by employing either ultra-high dilution technique or continuous addition technique.30 The former technique is the most widely utilized in the literature and typically uses an ultra- high dilute solution of the linear polymer (around 10-6 M). The preparation of a polymer solution with such a low concentration requires a large amount of solvent and thus, puts restriction on the amount of polymer that can be converted into SCPNs in one single batch. In 2002, Hawker and coworkers developed continuous addition technique which typically uses a relatively concentrated solution of linear polymer

(10-2 – 10-4 M) and hence, requires less amount of solvent. In this technique, the polymer solution was added drop wise continuously under rapid crosslinking reaction conditions using a syringe pump so that, the instantaneous concentration of crosslinker/reactive species would be ultra-dilute and not the concentration of polymer. The technique developed by Hawker has striking similarity to the well- established pseudo-high dilution technique used for the synthesis of macrocycles.87 The successful use of pseudo-high dilution technique requires: (1) the rate of reaction >> the rate of addition and (2) the products formed to be stable under reaction conditions. The former requirement is typically achieved by employing rapid crosslinking reaction conditions and by optimizing parameters such as rate of addition, concentration of crosslinker/reactive species and instantaneous concentration of crosslinker/reactive species. Hawker and coworkers used benzocyclobutene (BCB) groups to form the intramolecular crosslinks, which upon crosslinking form thermally stable C-C bonds that satisfy the second requirement for the use of pseudo-high dilution technique.

Various reports on the synthesis of SCPNs from linear polymer precursors have been published that utilize a wide range of chemistries to form the intramolecular crosslinks. Despite its advantages, very few reports have used pseudo-high dilution technique (or continuous addition technique) for the synthesis of SCPNs.8,17,30 Most of these reports use ultra-high dilution technique, which requires a large amount of solvent. The primary reason for the use of ultra-high dilution technique in these cases is that, the chemistries used are not rapid enough under respective reaction conditions to satisfy an important criterion of the rate of reaction >> the rate of addition (necessary requirement for the success of pseudo- high dilution technique).

The Pugh group recently reported the synthesis of a new monomer/crosslinker, 1-ethoxy-4- and

1-ethoxy-5-vinyl BCB (3.2).18 The copolymer containing this new crosslinker underwent ring-opening followed by crosslinking at much lower temperatures (100-140 0C) than unsubstituted BCB. Dr. James

Baker, a former graduate student in The Pugh group, synthesized highly fluorinated copolymers containing 2,3,4,5,6-pentafluorostyrene (PFS) as the fluorinated comonomer and new crosslinker in the backbone (Scheme 3.1).88 In this project, I investigated the conversion of highly fluorinated linear copolymers into highly fluorinated SCPNs utilizing pseudo-high dilution technique and their characterization using GPC and TEM.

Scheme 3.1 Synthesis of poly([2,3,4,5,6-pentafluorostyrene]-co-[1-ethoxy-4- and 1-ethoxy-5-vinylBCB])88

Fluorinated polymers are known for their interesting properties such as lower surface energy.89

Moreover, fluorinated nanoparticles are useful in other fields such as biomedical.90 Recently, PFS-based polymers have been used as substrates for quantitative post-polymerization modification via nucleophilic substitution of para-F under mild reaction conditions to attach different molecules.91 Highly fluorinated copolymers synthesized by James and their crosslinked SCPNs can potentially be used for these types of applications.

4.2 Parameters responsible for selective intramolecular crosslinking of (3.3) using pseudo-high dilution technique

Scheme 3.2 Synthesis of highly fluorinated SCPNs (3.4) from highly fluorinated copolymer containing

1-ethoxyBCB (3.3)

In order to achieve successful intramolecular crosslinking for a BCB-based crosslinking system using pseudo-high dilution technique, parameters that needed to be optimized were: reaction temperature for rapid crosslinking, concentration of crosslinker in the syringe and the rate of addition. All these parameters combined together should satisfy the rate of reaction >> the rate of addition criterion for selective intramolecular crosslinking. The temperature for ring-opening of BCB and its subsequent reactions can be measured using differential scanning calorimetry (DSC) to determine the temperature corresponding to the maximum exotherm. The Pugh group reported this temperature as 140 0C for 1- ethoxy-4- and 1-ethoxy-5-vinylBCB containing polymers.18 I decided to use 140 0C as the crosslinking reaction temperature since, the rate of crosslinking would be rapid around 140 0C and would likely satisfy the rate of reaction >> the rate of addition criterion.

Figure 4.1 DSC thermogram of polystyrene copolymer containing 1-ethoxyBCB and indicating maximum exotherm around 140 0C.88

Once the reaction temperature for rapid crosslinking was identified, I turned my attention to the remaining two parameters, the concentration of crosslinker and the rate of addition. In order to decide an appropriate range for these parameters, I took some cues from literature30 and some of the conditions developed in our lab for similar experiments.88,92 Based on these, I decided to use concentration of crosslinker in the range of 10-3 to 10-4 M and the rate of addition less than 3 mL/h.

SCPN synthesis is a solution reaction and it is important to choose the correct solvent. For our experiment, an ideal solvent would solubilize highly fluorinated copolymer and would have boiling point not less than 140 0C. James Baker had studied the solubility of highly fluorinated copolymer in few solvents and had found that xylenes solubilizes the copolymer. Xylenes have a boiling point around 140

0C, which is very close to the reaction temperature required for our project. I decided to use xylenes as the solvent for SCPN synthesis.

4.3 Synthesis of Highly Fluorinated Single Chain Polymer Nanoparticles

Dr. James Baker synthesized highly fluorinated copolymers (3.3) with different molecular weights and varying mol% of BCB in the backbone. I converted these linear copolymers into respective highly fluorinated SCPNs using the parameters mentioned above for ‘pseudo-high dilution continuous addition technique’. A representative setup is shown below (Figure 4.2).

Figure 4.2 Representative setup used for synthesis of SCPNs from (3.3) using ‘pseudo-high dilution continuous addition technique’.

In a typical experiment, a copolymer solution with BCB (≈ 10-4 M) was added to the refluxing xylenes (around 50 mL) using a syringe pump under N2. The copolymer solution was added directly from room temperature to the refluxing xylenes since; at warmer temperatures BCBs might undergo premature crosslinking. The results of SCPN synthesis from different copolymer precursors are summarized in Table 4.1.

Table 4.1 Experimental conditions used in the synthesis of SCPNs from different linear precursors and their characterization using GPC.

Entry Composition (% Mole) Linear Rate of Crosslinked Addition Nanoparticle Polymer

PFS VEBCB (mL/h) Mn (kDa) *Ɖ+ M (kDa) *Ɖ+ n

1 91 9 49.6 [2.01] 2.0 19.1 [1.44]

2 95 5 29.5 [1.65] 1.0 9.37 [1.17]

3 95 5 32.8 [1.49] 2.0 8.70 [1.27]

4 95 5 41.3 [2.03] 1.0 11.6 [1.37]

* For all the reactions, [BCB] = 0.5 mM

Hb Hc

Hc Ha Hb

THF THF

Figure 4.3 A representative 1H NMR spectra of linear copolymer (top) and crosslinked nanoparticle

(bottom; zoomed in) [Table 4.1, Entry 1] showing disappearance of the resonances characteristic to the

BCB ring (e.g. methylene protons between 3.0 - 3.5 ppm).

The crosslinking of linear polymers and its subsequent conversion to SCPNs can be analyzed using

NMR spectroscopy and GPC. 1H NMR spectrum in Figure 4.3 showed complete disappearance of methylene proton resonances (3.0 - 3.5 ppm) belonging to the 1-ethoxyBCB unit. The resonances belonging to the benzylic methine protons that resonate around 4.75 ppm are also completely consumed.

This indicates complete consumption of the crosslinking unit because of thermal stimulus and suggests its conversion to the reactive intermediate, o-QDM, responsible for dimerization and crosslinking.

GPC is a very useful tool for the characterization of SCPNs. Table 4.1 reports the GPC analysis that showed reduced apparent molecular weight of SCPNs compared to their linear polymers, indicating reduction in hydrodynamic volume of linear polymer as its shape changes from random coil to crosslinked globule, upon crosslinking. Representative stacked chromatograms are shown in Figure 4.4. There is no evidence of intermolecular crosslinking since there is a clean shift of entire chromatogram.

Table 4.2 Comparison of molecular weights of linear polymers and their SCPN counterparts

Entry Linear Polymer Nanoparticle Linear Nanoparticle %Reduction Compaction polymer composition Mn (kDa) Mn (kDa) Mp Mp in Mp Factor PFS VEBCB (Mp)

1 91 9 49.6 19.1 92287 25770 72 0.28

2 95 5 29.5 9.36 52684 11640 78 0.22

3 95 5 32.8 8.70 52385 12106 77 0.23

4 95 5 41.3 11.6 81534 16310 80 0.20

%reduction in M = 1- and Compaction Factor = p

Figure 4.4 Representative GPC chromatograms of linear copolymers and their corresponding SCPNs.

Top: Table 4.1, Entry 1 and bottom: Table 4.1, Entry 3.

We also wanted to investigate the effect of molecular weight and crosslink density in the linear polymers on the final properties of SCPNs. Hawker and coworkers demonstrated that, the reduction in apparent molecular weight is higher when higher mol% of crosslinker is incorporated in the backbone.30

They also proposed the possibility of being able to control the size of SCPNs (which would be directly proportional to the molecular weight in the case of SCPNs) by controlling the molecular weight of linear polymer and the crosslink density. Table 4.2 compares the peak molecular weights of linear polymers with

56 different molecular weights and different mol% of BCB to their corresponding SCPNs. All the samples showed similar values of %reduction in peak molecular weights and compaction factor. Especially in

Entries 1 and 4, we observed similar values despite some difference in the crosslink density. I believe that in our case, <10 mol% of BCB is not enough to produce a significant difference in the compaction of a polymer chain. Nevertheless, all the NMR spectroscopy and GPC data indicates that successful intramolecular crosslinking has occurred using ‘pseudo-high dilution continuous addition technique’, resulting in the formation of highly fluorinated SCPNs.

4.4 Characterization of Highly Fluorinated SCPNs using TEM

Transmission Electron Microscopy (TEM) is another widely used technique for the characterization of polymer nanoparticles.38 Characterization of materials using TEM requires good contrast between the carbon grid and the material deposited on it. Organic materials typically contain elements with low electron density (such as H, C, O, F, etc.) and do not provide enough contrast with respect to the carbon grid and are difficult to image. Organic materials and polymers in particular are often stained using staining agents such as RuO4 and OsO4 to increase their electron density which in turn,

93 facilitates their imaging using TEM. RuO4 and OsO4 staining agents function as oxidizing agents that oxidize the organic material or polymer and get deposited onto it. Ru-based and Os-based compounds have much higher electron density compared to the carbon grid, thereby providing much needed contrast.94

Figure 4.5 TEM images showing SCPNs prepared from linear copolymer (PFS/VEBCB = 91/9; Entry 1, Table

4.1). Scale bars: 100 nm (left) and 50 nm (right). Size as analyzed using ImageJ = 17 ± 3 nm. SCPNs stained with RuO4 for 15 min.

TEM analysis of our highly fluorinated polymer nanoparticles was first performed without any staining. As expected, the sample did not have enough contrast on its own. I then decided to use OsO4 as the staining agent since it is often recommended for use with the unsaturated systems. However, OsO4 provided very little contrast even after longer staining times and after multiple attempts. I then switched to RuO4 for staining purposes that provided very good contrast for imaging using TEM. This observation of

RuO4 proving to be a better staining agent than OsO4 is in agreement with their respective reactivities since; RuO4 is a stronger oxidizing agent than OsO4. Also, the substrate that I was trying to stain/oxidize was a highly fluorinated polymer with >90 mol% of PFS and <10 mol% of BCB. Fluorinated polymers are known for their resistance towards oxidizing agents. This means, in fluorinated SCNP the most likely oxidizable groups are the BCB units which are too few in numbers. In retrospect, our observation of OsO4 not staining/oxidizing enough of our fluorinated SCNPs agrees well.

Figure 4.6 TEM images showing SCPNs prepared from linear copolymer (PFS/VEBCB = 95/5; Entry 3, Table

4.1). Scale bars: 100 nm (left) and 50 nm (right). Size as analyzed using ImageJ = 17 ± 3 nm (diameter).

SCPNs stained with RuO4 for 15 min.

Representative TEM images obtained for two different samples after staining with RuO4 for 15 min are shown in Figure 4.5 and 4.6. Both the SCPNs in the images show spherical polymer nanoparticles with sizes 17 ± 3 nm (diameter). The sub-20 nm size of these nanoparticles is in agreement with the sizes reported in the literature.44 The spherical shape observed in TEM is in line with the observation by other research groups. Even though both of these images contain SCPNs made from linear polymers with different molecular weights and crosslink density, their sizes are similar. This further substantiates the data in Table 4.2 that <10 mol% BCB in this case is not enough to produce a significant difference in the compaction of a polymer chain. Nevertheless, TEM analysis proves the formation of SCPN by one more characterization method.

4.5 Conclusion

In conclusion, I have reported the development and utilization of pseudo-high dilution technique for the synthesis of highly fluorinated SCPNs. These SCPNs were characterized using NMR spectroscopy,

GPC and TEM. All these techniques support the formation of selective intramolecular crosslinks and TEM analysis showed formation of sub-20 nm spherical polymer nanoparticles.

CHAPTER V

CHARCATERIZATION OF AMPHIPHILIC NANOPARTICLES SYNTHESIZED VIA

STEP-WISE CROSSLINKING OF A SINGLE DI-BLOCK COPOLYMER CHAIN

5.1 Introduction

This chapter discusses different characterization techniques used in the analysis of amphiphilic single chain polymer nanoparticles (SCPNs) synthesized by Dr. Bill Storms (former student in The Pugh group) from di-block copolymer precursor.

In last decade, single chain polymer technology has gained wide attention from polymer community resulting in good number of research reports on SCPNs.44 Linear copolymers consisting of at least 2 monomers in the backbone (uni-block copolymer) have often been used as linear precursors for the preparation of SCPNs. However, utilization of more complex precursors towards intramolecular crosslinking is also reported. For example, Hawker and coworkers reported selective intramolecular crosslinking on a block copolymer with poly(ethylene glycol) (PEG) as one block and polystyrene copolymer containing crosslinker as another block that produced hybrid linear-SCPN type nanostructures.30

In the Pugh group we decided to use this selective crosslinking concept towards the synthesis of a Janus-type crosslinked amphiphilic polymer nanoparticle. Our design included a di-block copolymer with immiscible blocks and a BCB-based crosslinker in each block that would crosslink selectively at distinctly different temperatures (Figure 5.1). Styrene and 2,3,5,6-tetrafluoro-4-(2,2,3,3,3- pentafluoropropoxy)-styrene *TF5FS+ were chosen as the major comonomers (≈ 90 mol%) for each of the blocks since homopolymers of these two monomers are immiscible with each other.88,122 An ethoxy

0 0 substituted BCB (Tcrosslinking = 140 C) and an unsubstituted BCB (Tcrosslinking = 250 C) were chosen as the

BCB-based crosslinkers for each of the blocks. Our idea was that the use of immiscible blocks would help us in obtaining phase-segregated morphology in doubly crosslinked nanoparticle, thereby forming a

Janus-type amphiphilic nanoparticle. Dr. Bill Storms (former student in The Pugh group) developed synthesis of a functional di-block copolymer containing 4 different monomers in the backbone. He performed step-wise thermal crosslinking on a di-block copolymer (150 0C and 250 0C) to prepare doubly crosslinked polymer nanoparticles.53 Solvents for crosslinking reaction chosen in his experiment were good solvents for block undergoing crosslinking. For example, the starting block copolymer was readily soluble in mesitylene at room temperature and was used for first step of crosslinking at 150 0C.

Intermediate tadpole was soluble in THF at room temperature and was used to add tadpole particles to phenyl ether at 250 0C. Tadpole particles were soluble in phenyl ether upon warming.

Scheme 5.1 Schematic representation of di-block copolymer and its conversion to Janus-type amphiphilic polymer nanoparticle via stepwise and selective intramolecular crosslinking in a solvent good for the block undergoing crosslinking.

Bill performed 1H NMR analysis during the synthesis of these nanoparticles. Figure 5.1 shows 1H

NMR spectrum of starting di-block copolymer and crosslinked nanoparticle after crosslinking of both the

62 blocks. The resonances belonging to benzylic methylene protons, 3.0 - 3.5 ppm for ethoxy substituted BCB and 3.0 ppm for unsubstituted BCB, disappeared completely after both the steps of crosslinking, indicating formation of doubly crosslinked polymer nanoparticles.

Figure 5.1 Stacked 1H NMR spectra of linear di-block copolymer (top) and doubly crosslinked amphiphilic polymer nanoparticle (bottom) analyzed by Dr. Bill Storms, indicating disappearance of the resonances belonging to BCBs in both the blocks.

Bill also performed GPC analysis on each of the three morphologies and observed that the hydrodynamic volume of a di-block copolymer chain decreased in a step-wise manner as it went from random coil to tadpole to doubly crosslinked nanoparticle (Figure 5.2). The peak retention time corresponding to the linear di-block copolymer shifted to higher retention times upon crosslinking of the

63 first block, indicating reduction in the peak molecular weight (GPCPS) and change in the shape to form tadpole-like nanostructures. The same tadpole when crosslinked at 250 0C produced nanoparticle that eluted at even higher retention times, indicating further change in the shape. Results from these characterization methods show successful selective and multiple crosslinking of a single polymer chain; however, they do not provide any evidence about the phase-segregated morphology of the final doubly crosslinked nanoparticle. In this project, I characterized these doubly crosslinked nanoparticles by different characterization techniques to evaluate their morphology along with other important features such as size and shape. In particular, I used techniques such as dynamic light scattering (DLS), atomic force microscopy (AFM), transmission electron microscopy (TEM) and diffusion ordered spectroscopy – nuclear magnetic resonance (DOSY-NMR) towards this purpose. Each of these techniques and results will be discussed individually.

GPC overlay of step-wise chain collapse

Elution Time (Mins) 29

Figure 5.2 Stacked chromatograms of different morphologies obtained during the process of step-wise intramolecular crosslinking of a single di-block copolymer chain indicating sequential reduction in hydrodynamic volume and change in the shape (analyzed by Dr. Bill Storms).

In order to promote selective crosslinking of an individual block at each step and minimize encapsulation of the other block, the crosslinking reaction should be performed in a poor solvent for the block undergoing crosslinking. The solubility of individual homopolymers, polystyrene and poly(TF5FS], was evaluated in various solvents to identify a poor solvent for each of the blocks (Table 5.1). Mesitylene dissolved both the homopolymers whereas o-dichlorobenzene (o-DCB) dissolved only polystyrene and was a poor solvent for poly(TF5FS] at room temperature. Block copolymer was dissolved in mesitylene and added dropwise to o-DCB at 150 0C to crosslink the fluorinated block containing ethoxyBCB as the crosslinker (Scheme 5.2; first step)

Table 5.1 Solubility of PS (Mn = 33 kDa; Ɖ = 1.30) and P*TF5FS+ (Mn = 58 kDa; Ɖ = 1.84) at room temperature in different solvents (conc. = 5 mg/mL) and solubility observed over 5 minutes after addition of solvent. +++ = soluble; + = slightly soluble/swells; - = insoluble.

Scheme 5.2 Schematic representation of a di-block copolymer and its conversion to a Janus-type amphiphilic polymer nanoparticle via stepwise and selective intramolecular crosslinking in a solvent poor for the block undergoing crosslinking.

GPC analysis performed after each step of the crosslinking reaction showed reduction in apparent molecular weight and peak molecular weight compared to the precursor (Figure 5.3). This decrease in hydrodynamic volume of the block copolymer as it undergoes stepwise crosslinking reaction supports the formation of intramolecular crosslinks within individual blocks. Results from GPC analysis only show that a single chain of diblock copolymer has been converted to a doubly crosslinked nanoparticle; however, it does not provide any evidence regarding the phase-segregated morphology of these nanoparticles. Namrata Salunke, a graduate student in Dr. Karim’s group, is currently helping with the characterization of morphology of these nanoparticles using AFM analysis.

Figure 5.3 Stacked chromatograms of different morphologies obtained during the process of step-wise intramolecular crosslinking of a single di-block copolymer chain indicating sequential reduction in hydrodynamic volume and change in the shape (performed in a solvent poor for the block undergoing crosslinking).

Multiple and selective crosslinking of a single polymer chain has recently gained attention from other research groups as well. For example, Meijer and coworkers reported controlled crosslinking/folding of an ABA tri-block copolymer chain using orthogonal crosslinking chemistries for each of the blocks.51 The block copolymer used in their case was a methacrylate copolymer all along the backbone with crosslinkable groups pendant to the backbone, which upon crosslinking formed a polymer nanoparticle with uniform surface chemistry. Lutz and coworkers in a separate study reported step-wise intramolecular crosslinking of a sequence controlled polymer to prepare single chain polymer nanoparticles with distinct crosslinked subdomains.52 The copolymer used in their work was a copolymer of styrene and N-substituted maleimide all along the backbone with crosslinkable groups pendant to the backbone. A schematic was shown in Figure 2.6.

An interesting observation from Lutz’s work was that their nanoparticles had two crosslinked subdomains of similar chemical composition separated by a large polystyrene spacer. Also, characterization of these nanoparticles for their morphology was not reported. As opposed to these two examples, our approach uses a di-block copolymer with immiscible blocks, making the copolymer backbone amphiphilic in nature, which in turn makes it a potential candidate for the preparation of Janus- type nanoparticles. At the same time, doubly crosslinked nanoparticles prepared in our group require thorough characterization to study their morphology and prove whether they are phase-segregated or not. I used different characterization techniques to evaluate spatial chemical composition of nanoparticles prepared in our group (crosslinked under good solvent conditions by Dr. Bill Storms) and these will be discussed below.

5.2 Transmission Electron Microscopy (TEM):

Transmission Electron Microscopy (TEM) has become an extremely useful technique for the characterization of nanostructures, especially polymeric nanostructures. TEM has been used to image polymeric nanostructures of different shapes (such as spherical95 and elliptical96) and morphology (such as core-shell, vesicles, micelles and fibers)97. Thin films of block copolymers containing phase-segregating blocks form phase-segregated morphology with nanometer sized features. This morphology is widely studied using TEM since TEM can distinguish between the two phases based on their differential transmission of an electron beam.98 Differential transmission or contrast between two phases in TEM is due to difference in the electron densities of respective phases.93 For our project, we wanted to analyze whether our amphiphilic nanoparticles are phase-segregated or not. Other possible morphologies for our doubly crosslinked nanoparticles are shown in Scheme 5.3. An amphiphilic nanoparticle with two distinctly crosslinked sub-domains would form upon intramolecular crosslinking of each block completely independent to each other and at distinctly different temperatures. However, if the one of the blocks encapsulates the other block during the separate crosslinking steps, then core-shell morphology is likely to form. If both the blocks crosslink in same step in a random manner, then it would likely form randomly crosslinked morphology. We wanted to distinguish between the two phases in our nanoparticle and

68 determine their spatial arrangement with respect to each other. TEM seemed to be a promising technique to study the morphology of our nanoparticles.

Scheme 5.3 Possible morphologies resulting from intramolecular crosslinking of a di-block copolymer precursor.

Characterization of materials using TEM requires good contrast between carbon grid and the material deposited on it. Organic materials typically contain elements with low electron density (such as

H, C, O and F) that do not provide enough contrast with respect to carbon grid and are generally difficult to image. For TEM analysis of our nanoparticles I had to make sure that they not only have enough contrast with respect to the carbon grid but also have contrast between two phases. Our nanoparticles were prepared from a high molecular weight (around 70 kDa based on GPCPS) linear di-block copolymer containing a hydrocarbon block and a fluorocarbon block. I hypothesized that since it is a large molecular weight crosslinked polymer, there would be large number of atoms and hence enough electron density in the nanoparticle on its own as well as enough difference between the two phases for distinguishing purposes. However, my hypothesis proved to be wrong since I could hardly observe any nanostructure due to inadequate contrast between the nanoparticle and carbon grid on which the nanoparticles were deposited. Organic materials and polymers in particular are often stained using staining agents such as

RuO4 and OsO4 to increase their electron density which in turn increases their contrast with respect to carbon grid and facilitates their imaging using TEM. RuO4 and OsO4 staining agents function as oxidizing agents that oxidize the organic material or polymer and get deposited onto it.94 Both Ru-based and Os- based compounds have much higher electron density compared to the carbon grid thereby providing much needed contrast. I then decided to use staining agents to enhance the contrast between our

69 nanoparticles and the carbon grid. I thought that use of staining agents would also help me in obtaining contrast between the two phases present within our nanoparticles. Staining agents would likely oxidize the hydrocarbon phase more than the fluorocarbon phase since fluoropolymers are resistant to oxidizing

89 agents. I had found out that RuO4 was a better staining agent than OsO4 (highly fluorinated SCPNs;

Chapter IV) and decided to evaluate it first.

For TEM analysis, THF solution of nanoparticles was drop-cast on carbon coated grid. Excess of

THF was absorbed by filter paper and samples were dried at ambient temperatures in fume hood overnight. Samples were then vapor-stained using an aqueous solution of RuO4 for less than 15 min. For each sample at least two grids were prepared and at least two sets of images for each grid were used to analyze the size, shape and morphology.

I started my experiments with 20-25 mg/mL THF solution of doubly crosslinked nanoparticles stained for 15 min with RuO4. TEM images for this concentration showed spherical nanoparticles with two size distributions, small sized particles with 20-25 nm (diameter) and other distribution belonging to large sized particles with 50-90 nm (diameter) [Figure 5.4]. Smaller sized distribution is most likely coming from discreet SCPNs whereas larger sized particles are very likely physical aggregates of discreet particles.

Larger sized particles are certainly not because of chemical coupling that could form due to intermolecular crosslinking since GPC results (Figure 5.2) do not show any evidence for intermolecular crosslinking. Most important observation from these images was that we could not observe substantial differential staining across the surface of the nanoparticles and hence could not determine spatial chemical composition. Some of the nanoparticles observed in Figure 5.4 show areas that are slightly lighter as compared to other areas on nanoparticles and may seem as if they are due to differential staining (Figure 5.4 (c) and (e); nanoparticles highlighted with an arrow). However, it is difficult to substantiate the claim of differential staining since, only couple of nanoparticles showed ‘apparent differential staining’. Figure 5.4 (e) shows zoomed in image shown in 5.4 (a) to demonstrate inconsistent

‘apparent differential staining’. Also, this ‘apparent differential staining’ was observed only in larger sized particles and was not observed in smaller sized particles. As mentioned earlier, we were interested in

70 studying the morphology of SCPNs and not their physical aggregates. Thus, two problems that needed attention were: (a) breaking physical aggregates to obtain SCPNs and (b) obtaining differential staining across SCPNs to evaluate whether there is phase-segregated morphology or not.

(a) (b)

100 nm 100 nm

50 nm 50 nm

(e)

100 nm

Figure 5.4 TEM images of samples prepared with THF solution of doubly crosslinked nanoparticles with

20-25 mg/mL concentration and 15 min of staining. Scale bars: (a), (b) and (e) 100 nm; (c) and (d) 50 nm.

In order to break the physical aggregates, I diluted the THF solution of doubly crosslinked nanoparticles in a stepwise manner from 20-25 mg/mL to 5 μg/mL. The dilution helped in breaking the physical aggregates as the number of larger sized particles decreased and the number of smaller sized particles increased upon dilution (based on qualitative observation of TEM images shown in Figure 5.4 through Figure 5.9). However, not all of the physical aggregates were broken in the concentrations attempted since very small amount of larger sized particles still could be seen in lowest concentration samples. Lower concentration samples showed SCPNs distinctly from larger aggregates with enough clarity to determine their morphology. As I started working with lower concentration samples, I kept staining time of 15 min in order to obtain good contrast between nanoparticles and carbon grid. However, in all of the samples that were stained for 15 min, I did not observe any difference in the staining across the nanoparticle (Figures 5.5, 5.6, 5.7). Figure 5.5 (b) and 5.6 (b) clearly show TEM image with spherical, globular polymer nanoparticles with 20 nm size (diameter) and uniform staining across the surface.

(a) (b)

50 nm 100 nm

Figure 5.5 TEM images of samples prepared with THF solution of doubly crosslinked nanoparticles with 15 mg/mL concentration and 15 min of staining. Scale bars: (a) 50 nm and (b) 100 nm.

(a) (b)

100 nm 20 nm

Figure 5.6 TEM images of samples prepared with THF solution of doubly crosslinked nanoparticles with 5-

7 mg/mL concentration and 15 min of staining. Scale bars: (a) 100 nm and (b) 20 nm.

(a) (b)

50 nm 50 nm

Figure 5.7 TEM images of samples prepared with THF solution of doubly crosslinked nanoparticles with 2-

3 mg/mL concentration and 15 min of staining. Scale bars: (a) and (b) 50 nm.

Staining is an oxidation reaction and the extent of this reaction can be controlled by controlling the reaction time just like any other chemical reaction.94 I hypothesized that maybe 15 min is too long of a reaction time and is leading to oxidation of both the phases in our nanoparticles. I further hypothesized that decrease in the reaction time (staining time) might lead to incomplete oxidation reaction for one of the phases and in turn would help in obtaining selective staining. I decreased the reaction time to 10 min and 5 min for two of the samples. Unfortunately, decreasing the reaction/staining time did not help in obtaining contrast within the SCPNs since uniform staining was observed even after multiple attempts

(Figures 5.8 (b), 5.9 (a) and 5.9 (b)). In one of the grids containing sample with 5-7 mg/mL concentration, larger sized particles with non-uniform staining were observed (Figure 5.8 (a)). However, in the same image smaller sized particles with relatively uniform staining were observed. Also, we observed these types of nanoparticles only in one area of one grid. On the same grid in many other areas, I observed smaller sized particles with uniform staining (Figure 5.8 (b)). Non-uniform staining in larger sized particles was not the purpose of my study and hence this unusual and only image observed amongst a set of other consistent images was not analyzed further.

(a) (b)

50 nm 50 nm

Figure 5.8 TEM images of samples prepared with THF solution of doubly crosslinked nanoparticles with 5-

7 mg/mL concentration and 5 min of staining. Scale bars: (a) and (b) 50 nm.

In the lowest concentration sample analyzed by TEM (5 μg/mL), differential staining was not observed across the surface of larger and smaller sized particles (Figure 5.8 (b)).

(a) (b)

500 nm 100 nm

Figure 5.9 TEM images of samples prepared with THF solution of doubly crosslinked nanoparticles with 5

μg/mL concentration and 10 min of staining. Scale bars: (a) 500 nm and (b) 100 nm.

I rationalized non-selective staining in the case of RuO4 to its stronger oxidizing power and

0 decided to evaluate relatively milder oxidizing agent such as OsO4. OsO4 (b.p. = 130 C) has a higher

0 boiling point than RuO4 (b.p. = 40 C) and typically requires longer staining times for vapor staining. I stained lower concentration samples (Entry 7 in Table 5.2) overnight with OsO4. Even after multiple attempts, I could not observe selective staining in the nanoparticles stained using OsO4 and didn’t pursue this staining agent further. Table 5.2 summarizes all the results of TEM analysis performed on our doubly crosslinked SCPNs.

Table 5.2 Summary of TEM analysis results performed on doubly crosslinked SCPNs. d = diameter and r = radius.

Entry Concentration Staining Staining TEM Observation of THF Agent Time solution (minutes)

1 20 – 25 No - Difficult to distinguish between sample and mg/mL staining carbon grid; no contrast

2 20 – 25 RuO 15 2 d = 20-30 nm; r = 10-15 nm 4 mg/mL distributions d = 50-90 nm; r = 25-45 nm

3 15 mg/mL RuO 15 2 d = 20-30 nm; r = 10-15 nm 4 distributions d = 50-80 nm; r = 25-40 nm

4 5 – 7 mg/mL RuO 15 2 d = 20-30 nm; r = 10-15 nm 4 distributions d = 50-80 nm; r = 25-40 nm

5 5 – 7 mg/mL RuO 5 2 d = 20-30 nm; r = 10-15 nm 4 distributions d = 50-80 nm; r = 25-40 nm

6 2 – 3 mg/mL RuO 15 2 d = 20-30 nm; r = 10-15 nm 4 distributions d = 50-80 nm; r = 25-40 nm

7 5 μg/mL RuO 10 2 d = 20-30 nm; r = 10-15 nm 4 distributions d = 50-80 nm; r = 25-40 nm

While trying to rationalize all my observations, I realized that a part of my substrate (specifically the fluorocarbon phase in SCPNs synthesized by Bill in our group) is not completely a fluorocarbon phase

(Scheme 5.1). Each of the repeat units in the fluorocarbon phase has a methylene group attached to an aromatic ring via ether linkage. In the major comonomer structure, this methylene group is part of pentafluoropropoxy tail whereas; in the minor comonomer it is part of BCB unit. Thus, the fluorocarbon phase is not completely resistant to oxidation reaction because of the presence of other hydrocarbon functional groups.94 I believe that the rate of reaction between staining/oxidizing agent and phenyl groups in hydrocarbon phase and methoxy groups in fluorocarbon phase is comparable leading to non-selective staining. Unfortunately, I could not obtain desired contrast between the two phases in our nanoparticles using selective staining.

TEM analysis was useful in obtaining information about size and shape of our nanoparticles but unfortunately not useful for determining morphology of our nanoparticles. Spherical nanoparticles with sub-30 nm size (diameter or width), when discreet, were observed using TEM.

5.3 Atomic Force Microscopy (AFM) analysis of individual nanoparticles:

AFM is also a useful imaging technique for the characterization of polymeric nanostructures. It is often used to determine the size (width and height) and shape of polymer nanoparticles.41,99 Phase imaging, one of the predominant modes of AFM imaging used for polymer characterization, provides contrast between different components of the sample based on differential tip-sample interaction.

Material properties (such as stiffness, adhesion, viscoelasticity, etc.) can affect the tip-sample interaction.

Phase imaging can distinguish various material components in the sample based on differential tip-sample interaction, thus providing much needed contrast. Nanosized feature sizes resulting from phase segregation of block copolymers are often analyzed using phase imaging technique of AFM since, different phases can be distinguished from each other.100

For our project, we wanted to study the spatial chemical composition of our doubly crosslinked nanoparticles to evaluate whether they are phase-segregated or not. Our nanoparticles were synthesized from a di-block copolymer consisting of a hydrocarbon block and a fluorocarbon block. Both of these blocks have very different physical properties and are likely to have different interactions with the AFM

77 tip. I hypothesized that AFM might be able to distinguish between the two phases within our nanoparticle based on differential tip-sample interactions. Previous graduate student from our group, Dr. Bill Storms, had collaborated with Dr. Rebecca Agapov from Dr. Foster’s group for AFM analysis of highly fluorinated

SCPNs (their synthesis is mentioned in Chapter IV of my thesis). They used spin casting to deposit THF solution of SCPNs onto mica substrate. Figure 5.10 (a) is the height image showing presence of discreet

SCPNs with uniform heights around 6 nm across all the features. Higher sized particles that might form because of either chemical crosslinking of multiple chains or physical aggregation of SCPNs were not observed. The aggregation of nanoparticles due to capillary forces was not observed since spin-casting method of deposition avoids such aggregation. They also observed something interesting in the adhesion

AFM image: highly fluorinated SCPNs showed different adhesion with respect to mica substrate (Figure

5.10 (b)). This means that, the AFM tip used had differential tip-material interactions with the two materials present in the sample, fluorinated SCPNs and mica substrate. The color scale in Figure 5.10 (b) shows that, the fluorinated SCPNs demonstrated lower adhesion compared to the mica substrate. I hypothesized that, the adhesion mode in particular would help us distinguish between the two phases of our doubly crosslinked nanoparticle.

(a) (b)

Figure 5.10 AFM images of highly fluorinated SCPNs. (a) height image and (b) adhesion image.

Our nanoparticles were analyzed by AFM in collaboration with Mr. Jacob Scherger (Prof. Foster’s group), who provided kind help in performing AFM analysis. Silicon tips on a nitride cantilever (Bruker:

Scanasyst-Air) with a tip diameter of 4-24 nm (average 10 nm) were used. Typical sample preparation included: drop-casting of THF solution of doubly crosslinked nanoparticles on a freshly cleaved substrate,

78 absorbing excess THF with filter paper and drying the sample overnight in fume hood. Various parameters were changed one-by-one and results will be discussed in the following sections.

We started our AFM studies with a sample made from THF solution of doubly crosslinked nanoparticles [1 mg/mL] on mica substrate deposited using spin-casting method (Figure 5.11). For our sample, we observed individual nanoparticles (width/length around 20-35 nm and height = 1-2 nm) as well as larger aggregates (width/length > 35 nm and height > 2 nm). These larger aggregates are certainly not because of chemical coupling that could form due to intermolecular crosslinking, since GPC results did not show any evidence of intermolecular crosslinking. These larger aggregates are also less likely due to capillary forces since we had used spin-casting method of deposition. As will be shown later in this section, larger aggregates are a result of solution aggregation of individual SCPNs and method of deposition doesn’t really help in avoiding the aggregation. For this reason, we did not pursue spin-casting as a method of deposition further.

(a) (b)

Figure 5.11 AFM images of samples prepared by deposition of THF solution of doubly crosslinked nanoparticles with 1 mg/mL concentration on mica using spin-casting. Scan size: (a) and (b) 1 μm 1 μm.

Our main target was to image individual SCPNs and then try to distinguish between two phases using possible differential tip-sample interaction (e.g. using adhesion mode). In order to obtain mainly individual nanoparticles, we diluted the concentration of THF solution step-by-step and used drop-casting as method of deposition. For a sample on mica substrate with 25 μg/mL concentration in THF, we observed two distributions: individual nanoparticles and larger aggregates (Figure 5.12). Sizes corresponding to the individual nanoparticles are in good agreement with the expected values and those reported in the literature for similar type of nanoparticles. As observed in Figure 5.12 (c), these nanoparticles are loosely crosslinked coils that flatten out when deposited on the surface forming pancake-like morphology as evident from their much larger width/length (20-35 nm) compared to their height (1-2 nm). This observation is also in good agreement with the SCPN literature.51

(a) (b)

0.5 μm 0.5 μm 1 μm 1 μm

(c)

Figure 5.12 AFM images of samples prepared by deposition of THF solution of doubly crosslinked nanoparticles with 25 μg/mL concentration on mica using drop-casting. Scan size: (a) 0.5 μm 0.5 μm, (b)

1 μm 1 μm. (c) represents height linecut for one of the discreet SCPNs (width = 25 nm; height = 1 nm).

Upon further diluting the concentration of nanoparticles to 5 μg/mL and 0.5 μg/mL, we observed some interesting structures in the AFM images (Figures 5.13 and 5.14). Individual nanoparticles and ‘rings’ containing individual nanoparticles were observed. It looked like individual nanoparticles are aggregating in circular fashion to form ‘rings’. These rings are around 200 nm wide and the heights of features vary from 1 nm to 8 nm as observed in 3D height image in Figure 5.14 (a). Some of these features are taller compared to individual SCPNs (1-2 nm). However, the presence of smaller features in the rings suggests that the rings are formed because of the aggregation of discreet SCPNs. We could not rationalize

81 this unusual observation of rings. However, they have striking similarity to the AFM images reported by

Hawker and coworkers for their hybrid SCPNs (Figure 5.14 (b))101. Hawker and coworkers studied hybrid

SCPNs (a poly(butyl acrylate) chain tethered to crosslinked polystyrene globule) using AFM and observed similar ‘rings’; however, they too do not offer any explanation for this unusual observation.

(a) (b)

1 μm 1 μm 1 μm 1 μm

Figure 5.13 AFM images of samples prepared by deposition of THF solution of doubly crosslinked nanoparticles with 5 μg/mL concentration on mica using drop-casting. Scan size: (a) and (b) 1 μm 1 μm.

(a) height image and (b) adhesion image.

(a)

1 μm 1 μm

(b)

1 μm 1 μm 0.25 μm 0.25 μm

Figure 5.14 AFM images of samples prepared by deposition of THF solution of doubly crosslinked nanoparticles with 5 μg/mL concentration on mica using drop-casting. Scan size: (a) 1 μm 1 μm. (a) 3D view height image shown in Figure 5.13 (a). (b) Ring-like structures reported by Hawker and coworkers in

Macromolecules 2005, 38, 2674-2685.

(a)

0.5 μm 0.5 μm

(b)

(c)

0.5 μm 0.5 μm

Figure 5.15 AFM images of samples prepared by deposition of THF solution of doubly crosslinked nanoparticles with (a), (b) 5 μg/mL and (c) 0.5 μg/mL concentration on mica using drop-casting. Scan size:

(a) and (c) 0.5 μm 0.5 μm. (a) height image with linecut on right, (b) adhesion image linecut for larger particle in Figure 5.15 (a), (c) adhesion image with linecut on right.

For the same sample with 5 μg/mL concentration, we also observed presence of discreet SCPNs

(width = 25 nm; height = 2 nm) and larger aggregates (width = 50 nm; height = 9 nm) along with rings in

AFM images (Figure 5.15). We also performed adhesion mode measurements on these samples and observed clear difference between tip-mica interaction and tip-nanoparticle interaction: (i) in Figure 5.13

(b), lower adhesion of rings ( 3-5 nN) compared to mica ( 7-8 nN), (ii) in Figure 5.15 (b), lower adhesion of nanoparticles ( 4-5 nN) compared to mica ( 8 nN) and (iii) in Figure 5.15 (c), lower adhesion of nanoparticles ( 4 nN) compared to mica ( 8 nN). However, we did not observe differential interaction of tip with the two phases present in the nanoparticle since no change in the adhesion across the surface of the particle was detected. (Figure 5.13 (b), 5.15 (b) and 5.15 (c)).

In order to break the physical aggregates further, we diluted THF solution of doubly crosslinked nanoparticles to 0.1 μg/mL and 0.05 μg/mL and deposited them onto mica substrate. AFM images in

Figure 5.16 and 5.17 showed mostly individual nanoparticles with very small amount of larger aggregates, suggesting that the aggregates observed were indeed physical aggregates which could be broken down using excess solvent. For the images shown in Figures 5.16 and 5.17, sizes were calculated in the longer width and shorter width direction and are reported accordingly. For example, average dimensions for image in Figure 5.16 are: 33 nm in longer axis and 23 nm in shorter axis and < 1 nm tall. This demonstrates that, these nanoparticles form pancake-like morphology when deposited on the surface and adopt irregular shape. Adhesion measurements on these samples were not useful for our purposes, since we could not distinguish between the two phases within our nanoparticles. For example, in the adhesion image in 5.17 (b), the nanoparticles have adhesion in the same range ( 8-9 nN) across its entire surface, indicating that two phases were not distinguished.

(a) (b)

1 μm 1 μm 1 μm 1 μm

Figure 5.16 AFM images of samples prepared by deposition of THF solution of doubly crosslinked nanoparticles with 0.1 μg/mL concentration on mica using drop-casting. Scan size: 1 μm 1 μm. (a) height image and (b) adhesion image. Average dimensions calculated are: r₁= 33.7 ± 0.9 nm; and r₂= 23.7 ±

1.2 nm. h= 0.83 ± 0.24 nm; h = height, r₁=long axis, r₂=short axis.

At this stage, we rationalized that, maybe the surface on which these nanoparticles are deposited is playing a role in the orientation of nanoparticles. Our nanoparticles have a hydrocarbon phase and a fluorocarbon phase. It is possible that one of the phases wets mica preferentially over other phase (hydrocarbon phase likely to wet since fluoropolymers have lower surface energies). If there is preferential wetting, one of the phases will ‘sit’ on top of other (fluorocarbon phase likely to be in air) and only the phase on top will get detected by AFM. To evaluate this hypothesis, we decided to deposit our nanoparticles on a different substrate.

(a) (b)

0.5 μm 0.5 μm 0.5 μm 0.5 μm

Figure 5.17 AFM images of samples prepared by deposition of THF solution of doubly crosslinked nanoparticles with 0.05 μg/mL concentration on mica using drop-casting. Scan size: 0.5 μm 0.5 μm. (a) height image and (b) adhesion image. Average dimensions calculated are: r₁=34.8 ± 5 nm and r₂=24 ± 3.8 nm; h=0.68 ± 0.09 nm; h = height, r₁=long axis, r₂=short axis.

We decided to use silicon wafer as our substrate for these experiments and prepared samples with very dilute solutions (concentrations of 0.1 μg/mL and 0.05 μg/mL based on previous experiments) to obtain mostly individual nanoparticles without larger aggregates. The height images in Figure 5.18 (a) and (c) show discreet SCPNs that have adopted irregular, pancake-like morphology (average width 25-40 nm and height 1-3 nm) on the silicon wafer. Unfortunately, even with silicon wafer as substrate, we could not observe any difference in adhesion between the two phases (Figure 5.18 (b) and (d)). For example, adhesion measurements for particles in Figure 5.18 (b) show that the nanoparticles ( 10 nN) have lower adhesion compared to silicon wafer ( 12-13 nN). However, the adhesion remains uniform or non- distinguishable across the surface of the nanoparticle ( 10 nN).

(a) (b)

1 μm 1 μm 1 μm 1 μm

Figure 5.18 AFM images of samples prepared by deposition of THF solution of doubly crosslinked nanoparticles with (a), (b) 0.1 μg/mL and (c), (d) 0.05 μg/mL concentration on silicon wafer using drop- casting. Scan size: 1 μm 1 μm. (a), (c) height image and (b), (d) adhesion image. Average dimensions calculated are: (a) r₁=42.3 ± 5.2 nm and r₂=32.2 ± 3.8 nm; h = 2.5 ± 1.2 nm, (c) r₁ = 37 ± 3.8 nm, r₂ = 26.8 ±

1.7 nm; h=1.3 ± 0.5 nm; h = height, r₁=long axis, r₂=short axis.

Table 5.3 Summary of AFM analysis results performed on doubly crosslinked SCPNs.

Sr. Concentration Substrate Deposition Brief Observations No. (in THF) Method

1 1 mg/ml Mica Spin-casting Larger aggregates + individual nanoparticles

2 25 μg/ml Mica Drop-casting Larger aggregates + individual nanoparticles

3 5 μg/ml Mica Drop-casting Individual nanoparticles + “rings” + aggregates

4 0.5 μg/ml Mica Drop-casting Individual nanoparticles + “rings”

5 0.1 μg/ml Mica Drop-casting Mostly Individual nanoparticles + larger aggregates

6 0.05 μg/ml Mica Drop-casting Mostly individual nanoparticles + larger aggregates

7 0.1 μg/ml Silicon Drop-casting Mostly individual nanoparticles Wafer

8 0.05 μg/ml Silicon Drop-casting Mostly individual nanoparticles Wafer

Table 5.3 summarizes all of the size distribution results obtained with AFM analysis. All of the

AFM images show presence of discreet SCPNs (individual nanoparticles: size: width/length around 20-35 nm and height = 1-2 nm) as globular structures. These discreet SCPNs form larger physical aggregates in solution when the concentration of solution is high and can be broken down upon diluting the solution significantly. Discreet SCPNs are imaged as loosely crosslinked coils that flatten out on substrate forming pancake-like morphology. None of the adhesion images showed distinguishable contrast in the adhesion across the surface of the nanoparticle. This could mean that either there is no difference in adhesion over the surface of the particle or the difference is not large enough to be detected by the current capabilities of the technique.

5.4 Dynamic Light Scattering (DLS):

As mentioned in the previous sections, TEM and AFM did not provide information regarding the phase segregated morphology of our nanoparticles. I had high hopes from these two techniques, since these techniques provide an opportunity to use contrast between two phases of a block copolymer. In

TEM this contrast is a result of differential electron density, whereas in AFM it is due to differential tip- sample interaction. In order to investigate the phase-segregated morphology by another technique, we decided to use DLS. DLS does not provide contrast between two phases as such. However, it can be used

102,103 to determine hydrodynamic radius (Rh) and the shape of nanoparticle utilizing shape factor analysis.

Our hypothesis was that if our nanoparticles are indeed phase-segregated, they are likely to be elongated in shape because of the immiscible nature of blocks. It would mean that they would be more cylindrical/rod-like in shape than spherical. We then decided to use DLS to evaluate the shape of our doubly crosslinked nanoparticles.

DLS studies were performed in collaboration with Dr. Fadi Haso (Prof. Liu’s group), who provided kind help in performing DLS analysis. In a typical experiment, solutions of different concentrations were filtered through 450 nm filter (hydrophilic PTFE), analyzed at different angles using green laser light and data obtained was fit using CONTIN. ΓG(Γ) values obtained are plotted as intensity in Figures 5.19, 5.20 and 5.21). Some of the parameters, either in the sample preparation process or in experimental conditions, were varied one-by-one in order to obtain more information. All the results will be discussed individually in the following sections.

We started DLS experiments using THF solution of our doubly crosslinked nanoparticles with 1 mg/mL concentration. This particular sample was prepared by adding THF at once to nanoparticles.

CONTIN analyzed DLS intensity plot showed presence of two size distributions: smaller sized particle distribution (Rh = 10-20 nm) and larger sized particle distribution (Rh = 40-400 nm) irrespective of the angle of measurement (Figure 5.19). Smaller sized particle distribution is most likely coming from individual

SCPNs since their sizes are in the expected size range for nanoparticles made from a single polymer chain.

Larger sized distribution is very likely coming from physical aggregates of individual nanoparticles, since we had observed similar aggregates in previous two techniques.

Figure 5.19 CONTIN analyzed DLS intensity plot for THF solution of doubly crosslinked nanoparticles with 1 mg/mL concentration. Solution prepared by direct addition of THF to nanoparticles. Data collected at different angles; for some of the angles it was analyzed in duplicates (e.g. 60-2 and 45-2 represent duplicates).

Sizes obtained for individual nanoparticles from DLS are slightly larger than those obtained from

TEM and AFM. We believe that this is due to DLS being a solution-state characterization where solvent can swell the nanoparticles, thereby slightly increasing the sizes compared to dry-state characterization such as TEM and AFM. The data presented in Figure 5.19 is an intensity plot and not the number plot. In light scattering, intensity is directly proportional to (size)6. This means that, for our results shown in Figure

5.19, the number of smaller sized particles is much higher as compared to the number of larger sized particles. As mentioned earlier, we were interested in finding the shape of our SCPNs which required obtaining a single distribution of smaller sized particles. Thus, our first target was to break the larger sized physical aggregates to obtain a single distribution corresponding to smaller sized particles.

In order to break the physical aggregates, I diluted the concentration of nanoparticles to 0.5 mg/mL and 0.125 mg/mL. DLS analysis on these nanoparticles showed two distributions, individual nanoparticles and larger physical aggregates, similar to what was observed in the previous sample (Figure

5.20). We needed to dilute the solution further in order to break down the aggregates. However, when we attempted DLS analysis on solution with concentration less than 0.1 mg/mL, we did not obtain enough signal in DLS. The maximum possible concentration we could use for DLS analysis in order to get good signal to noise ratio was more than 0.1 mg/mL. This concentration was much higher than the concentration required to completely break the aggregates and to obtain a single distribution corresponding to the individual nanoparticles. We took cue from TEM and AFM analysis where we had observed that, individual nanoparticles are obtained when the concentration is less than 0.1 μg/mL. Since obtaining DLS data for single distribution of individual nanoparticles by mere dilution of THF solution was not possible, we had to utilize different strategies to break the physical aggregates.

In order to break physical aggregates we first attempted to change the way we prepared our nanoparticle solution. Previously, we had prepared solution by adding THF directly to nanoparticles. We hypothesized that may be this direct addition is unable to dissolve all the nanoparticles and hence unable to break the physical aggregates. We thought that if we add THF slowly to nanoparticles, it will provide enough time for all the nanoparticles to dissolve and help us in obtaining a single distribution.

Nanoparticle solution was then by adding THF slowly over nanoparticles using a syringe pump (0.5 mL/h).

Interestingly, this solution exhibited fluorescence and this observation remained same even after multiple attempts of preparing the solution in the same way. When green laser light travelled through this solution, it emitted yellow light instead of scattering the green light. This behavior was surprising and we could not find any explanation for this observation. Unfortunately, changing the sample preparation technique did not help us. We did not pursue slower addition of THF method for sample preparation further and continued with direct addition of THF for all the remaining experiments.

Figure 5.20 CONTIN analyzed DLS intensity plot for THF solution of doubly crosslinked nanoparticles with

0.5 mg/mL (top) and 0.125 mg/mL (bottom) concentration. Solutions prepared by direct addition of THF to nanoparticles. Data collected at different angles; for some of the angles it was analyzed in duplicates

(e.g. 45-2 represents duplicate).

For next experiment, we hypothesized that it might be possible to break the aggregates at warmer temperatures since the physical forces holding individual nanoparticles together might be broken when heated. We analyzed 1 mg/mL THF solution of nanoparticles at 45 0C and observed two distributions with similar size ranges as observed before. Unfortunately, slightly warmer temperatures did not produce the much needed single distribution.

In all our experiments so far, we had observed two size distributions, one belonging to <20 nm in size (discreet SCPNs) and other belonging to >40 nm in size (physical aggregates of SCPNs). We thought that if we filter our solution through a 20 nm filter, we should be able to remove the larger sized distribution and obtain single distribution belonging to SCPNs. We tried this easy approach by filtering 1 mg/mL THF solution through a 20 nm filter (Whatman Anotop 25; Alumina-based membrane with polypropylene housing). Surprisingly, this filtered solution exhibited fluorescence and emitted yellow light upon interaction with the green laser light. We had observed similar behavior before in the case of sample prepared via slow dissolution process. We could not explain this observation too; however, it might be possible that our nanoparticles might be interacting with the filter material in some way leading to this unusual behavior.

After some failed attempts in breaking the physical aggregates, we thought that may be THF is not as good of a solvent for either of the blocks in our nanoparticles as we think it is. If THF is not a good solvent for either of the blocks, our nanoparticles might self-assemble into larger aggregates in a similar way amphiphilic di-block copolymers self-assemble to form micelles and/or other structures in selective solvents.104 Our nanoparticles have a polystyrene block and a fluorostyrene block. THF is known to be a good solvent for polystyrene but, the same cannot be said true for fluorostyrene.105. α,α,α- trifluorotoluene [TFT] was chosen as the good solvent for fluorostyrene block solvent, since we had found that it dissolves fluorostyrene as well as the block copolymer well.53 We prepared nanoparticle solutions in various compositions of (THF + TFT) mixture and evaluate whether any of the solvent mixtures breaks the physical aggregates. We prepared 4 different compositions as follows (all vol./vol. basis): (a) THF/TFT

= 75/25, (b) THF/TFT = 60/40, (c) THF/TFT = 50/50 and (d) THF/TFT = 25/75.

Computation of correct hydrodynamic radius (Rh) using DLS requires using correct values of refractive index and viscosity of the solvent.102,103 We could not find refractive index and viscosity values of different compositions of (THF + TFT) mixture in the literature and hence these values were measured in-house. Refractive indices were measured using Abbe Refractometer (Bausch and Lomb) at 23 0C.

Dynamic viscosity values were computed using efflux times measured using Ubbelohde viscometer

(Canon Instruments; size 50; viscometer constant = 0.004 cSt/sec) at 23 0C.

Table 5.4 Measured physical properties of different compositions of (THF + TFT) mixture

Composition Refractive Index Dynamic Viscosity (vol/vol) (Measured using Abbey (calculated using Ubbelohde Refractometer) Viscometer) [cP]

THF/TFT (75/25) 1.4090 0.591

THF/TFT (60/40) 1.4103 0.599

THF/TFT (50/50) 1.4110 0.609

THF/TFT (25/75) 1.4125 0.631

TFT 1.4143 0.619

DLS analysis was performed on 1 mg/mL and 0.1 mg/mL solution of nanoparticles in all of the compositions of (THF + TFT) mixture prepared. Surprisingly, even these solutions exhibited bimodal distribution of nanoparticle sizes in DLS (Figure 5.21). Smaller sized distribution in all these compositions was in the similar size range (Rh < 20 nm). However, larger sized distribution changed depending upon the solvent, since solutions in THF/TFT (75/25), THF/TFT (25/75) and TFT showed narrower distribution of sizes as compared to pure THF as solvent. This substantiated our previous observation and rationalization that discreet SCPNs are undergoing self-assembly in solution and this self-assembly can be controlled by utilizing different solvents. Unfortunately, even the mixture of solvents did not help us in obtaining a single distribution of discreet SCPNs.

Figure 5.21 CONTIN analyzed DLS intensity plot for solutions of doubly crosslinked nanoparticles prepared in different compositions of (THF + TFT) mixture with 1.0 mg/mL concentration. Solutions prepared by direct addition of solvent to nanoparticles. Data collected at different angles.

Table 5.5 summarizes all the data obtained from DLS analysis. In summary, all DLS experiments showed presence of two distributions of nanoparticle sizes. All the results not only showed the presence of discreet SCPNs but also showed the presence of larger physical aggregates formed due to self-assembly of fraction of discreet SCPNs. All the attempts to break the physical aggregates were unsuccessful. Since all the results showed two distributions, we could not use this data to calculate Rh(0) (Rh extrapolated to zero angle) and hence could not determine the shape of our nanoparticles using shape factor analysis

(Rg/Rh(0)).

Table 5.5 Summary of results obtained from DLS analysis

Solution Conc. Sr. Solvent Method of Temp filtered 0 analyzed DLS observation No. used sample prep through ( C) (mg/mL)

1 Adding THF at 2 distributions; 450 nm 1 THF once to 25 0.5 6-20 nm and

nanoparticle 40-400 nm 0.125

Adding THF at 2 distributions; 450 nm 2 THF once to 45 1 6-20 nm and

nanoparticle 40-400 nm

Added THF Solution exhibited slowly using fluorescence; upon syringe pump 3 THF 450 nm 25 1 shining green laser to solution emitted yellow nanoparticle light instead of scattering (0.5 mL/h)

Solution exhibited Adding THF at fluorescence; upon 4 THF once to 20 nm 25 1 shining green laser nanoparticle solution emitted yellow light instead of scattering

Adding 1 2 distributions; THF/TFT solvent at 5 450 nm 25 6-12 nm and (75/25) once to 0.1 30-160 nm nanoparticle

Adding 1 2 distributions; THF/TFT solvent at 6 450 nm 25 6-15 nm and (60/40) once to 30-300 nm nanoparticle 0.1

Adding 1 2 distributions; THF/TFT solvent at 7 450 nm 25 10-20 nm and (25/75) once to 30-100 nm nanoparticle 0.1

Adding 1 2 distributions; solvent at 8 TFT 450 nm 25 10-25 nm and once to 50-120 nm nanoparticle 0.1

5.5 Diffusion-Ordered Spectroscopy Nuclear Magnetic Resonance (DOSY-NMR):

Our efforts to determine whether our doubly crosslinked nanoparticles are phase- segregated/Janus in nature or not did not succeed using TEM, AFM and DLS techniques. We thought if our nanoparticles are not phase-segregated they are very likely to adopt core-shell type morphology where one of the phases would be encapsulated by other. During literature search I came across a report that utilized DOSY-NMR technique to determine the morphology of polymer nanoparticles. Darcos and coworkers were able to demonstrate the formation of micelles/core-shell morphology using DOSY-NMR spectroscopy.106 I decided to use this technique with the hope of finding more information about the morphology of our nanoparticles.

DOSY-NMR is a 2D NMR technique that measures the signal due to self-diffusion of molecules/species in solution. This leads to one dimension being the diffusion coefficient (D) of the species in solution and other dimension being the chemical shift of the nucleus.107 The diffusion coefficient (D) of a species (molecule, nanoparticle, aggregate, etc.) depends on its size, mass, charge, solvent, temperature, etc. Thus, if there are more than one species with different diffusion coefficients in the solution, they can be resolved on the diffusion coefficient dimension. The corresponding cross-peaks of each of these species in 2D NMR can provide information regarding the functional groups present in that species. Because of these unique advantages, DOSY has been used to characterize mixtures and aggregates. Recently, there is growing utilization of this technique in the polymer community for various purposes such as: (a) determination of the molecular weight distribution of PEO,108 (b) characterization of drug-loaded polymer nanoparticles,109 (c) characterization of SCPNs and comparison with respect to their linear precursors.85

Darcos and coworkers’106 use of DOSY for the characterization of micelles caught my attention. In their work, an amphiphilic tri-block copolymer PLA-b-PEG-b-PLA was characterized using DOSY NMR spectroscopy in CDCl3 which is a good solvent for both blocks. Only one cross-peak belonging to the tri- block was observed by DOSY, demonstrating the absence of any other impurities such as macroinitiator

98 and homopolymer. Corresponding chemical shifts of this cross-peak showed 1H NMR resonances belonging to both blocks, PLA and PEG. Upon adding this amphiphilic block copolymer to D2O, which is a selective solvent for the hydrophilic block, micelles formed. When these micelles were analyzed using

DOSY, only one spot corresponding to the PEG block was detected and no cross-peak corresponding to the hydrophobic PLA part was observed (Figure 5.22). They attributed this observation to the formation of micelles in D2O where PEG block would encapsulate PLA blocks. They rationalized that encapsulated PLA blocks will have limited molecular mobility and hence, will be muted in the DOSY spectrum. They extended this technique for the determination of critical micellar concentration (CMC) and found that the numbers match well compared to other well established techniques. Their results show that if a polymer nanoparticle contains two phases, and if one of the phases is encapsulated by the other, then it is possible to identify the phase that is encapsulated. My hypothesis was that if I analyze our nanoparticles using

DOSY NMR spectroscopy, I might be able to identify whether our nanoparticles are of core-shell morphology or not.

Figure 5.22 DOSY-NMR spectra reported by Darcos and coworkers for the determination of polymer nanoparticle morphology. Linear tri-block copolymer (left) and micelles (right) show two different diffusion coefficients corresponding to two distinct morphologies and sizes.

In order to analyze our nanoparticles using DOSY NMR spectroscopy, I chose d8-THF as the deuterated solvent since in all other techniques I had used THF to dissolve our nanoparticles. First, I performed DLS analysis on doubly crosslinked nanoparticles in d8-THF (1 mg/mL) to obtain the diffusion coefficient values. I observed two distributions, one belonging to discreet SCPNs and another belonging to larger physical aggregates, similar to the one observed when non-deuterated THF was used (Figure 5.23).

For each distribution, the average diffusion coefficient values were calculated using the Stokes-Einstein equation. Since we observed two distributions in DLS that differ by approximately one order of magnitude in size, and hence in diffusion coefficient values, I was also expecting to observe two distributions in the

DOSY spectrum.

Figure 5.23 CONTIN analyzed DLS intensity plot for d8-THF solution of doubly crosslinked nanoparticles with 1 mg/mL concentration. Solutions prepared by direct addition of THF to nanoparticles. Data collected at 900. Average diffusion coefficient values were calculated using the Stokes-Einstein equation where, T =

303.15 K (30 0C), η = 0.48 * 10-3 kg/(m2. s).

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DOSY-NMR analysis was performed with the kind help of Dr. Jessi Baughman who ran the NMR experiment and its subsequent processing and analysis. DOSY-NMR analysis on 1 mg/mL of our doubly crosslinked nanoparticles in d8-THF is shown in Figure 5.24. Surprisingly, only one distribution was

-10 detected in the spectrum. The diffusion coefficient value obtained from DOSY NMR (DDOSY-NMR = 5.2 10 m2/sec) for this cross-peak was closer to the value calculated from DLS data for the smaller sized

-11 2 distribution (Peak 1 in Figure 5.23 and DPeak1 = 2.6 10 m /sec). We thus concluded that the only spot seen in DOSY NMR is representing the smaller sized particle distribution. Even though we observed two size distributions in DLS, we believe that the concentration of larger sized particles in solution is too small to be detected by DOSY NMR spectroscopy and hence, the cross-peaks corresponding to the second distribution are not observed. DLS data shown in Figure 5.23 is an intensity plot and not number distribution plot. In DLS analysis, intensity signal has a very strong dependence on the size since it varies with (size)6. Thus, the number of smaller sized particles >> the number of larger sized particles in our case, and hence the concentration of large sized particles is too small to be detected by DOSY NMR spectroscopy.

We further analyzed the only cross-peak obtained in DOSY NMR spectroscopy and analyzed the

1H NMR resonances on the chemical shift dimension. Resonances belonging to the methylene oxy protons in the fluorocarbon phase as well as the aromatic protons in the hydrocarbon phase were detected. In other words, for the smaller sized distribution, both phases were detected in DOSY NMR spectroscopy.

We first thought that detection of resonances belonging to both phases suggests that our nanoparticles do not have core-shell morphology. However, we also realized that for DOSY-NMR analysis we had used

THF as solvent, which is a good solvent for both blocks (styrene and fluorostyrene) in our nanoparticles since nanoparticles as well as homopolymers of individual blocks are readily soluble in THF. This will lead to swelling of both blocks by THF. Darcos and coworkers observed only one phase in their micelles because they had used a solvent (D2O) that was bad for one of blocks (PLA) in their system. The PLA blocks thus formed the “core” part of the micelle, and therefore had much lower molecular mobility compared to the hydrophilic “shell” of PEG. Hence, PLA resonances were not observed by DOSY-NMR

101 spectroscopy. Unlike Darcos and coworkers’ example, we had used good solvent for both blocks of our nanoparticles, which very likely swelled both the blocks instead of selectively solubilizing one block.

Swelling of both phases will help in their easy detection in DOSY-NMR since neither will be a solid core with lower molecular mobility. DOSY-NMR results in THF-d8 only show that resonances for both phases are observed in NMR for the discreet nanoparticles. These results do not allow us to differentiate between the core-shell and Janus-type morphology.

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Figure 5.24 DOSY-NMR spectrum of doubly crosslinked nanoparticles in d8-THF (1 mg/mL) acquired at 30

0C. An average value corresponding to diffusion coefficient is reported.

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5.6 Atomic Force Microscopy (AFM) analysis of nanoparticles and polystyrene homopolymer blend films:

With all of the above techniques not providing conclusive evidence on the morphology of our

doubly crosslinked nanoparticles, I decided to study blend of doubly crosslinked nanoparticles and

polystyrene (PS) homopolymer by AFM (Bruker AXS) analysis. This work was performed in

collaboration with Ms. Namrata Salunke (Prof. Karim’s group), who provided kind help in performing

AFM analysis and contact angle measurements. Silicon tips on a nitride cantilever with a tip radius of

≤ 10 nm and spring constant of 7.8 N/m were used. Typical sample preparation included: preparing a

blend solution of doubly crosslinked nanoparticles and homopolymer in THF and flow coating this

solution onto a silicon wafer cleaned using UV ozone exposure.

TEM, AFM (of individual nanoparticles), DLS and DOSY-NMR techniques were unable to

differentiate between the two phases of our doubly crosslinked nanoparticles. In order to help with

the differentiation between two phases, we blended our nanoparticles in polystyrene homopolymer.

We hypothesized that when such a doubly crosslinked nanoparticle-homopolymer blend film was

cast, the PS part would be miscible with the PS homopolymer while the fluorinated part would be

immiscible. In such a case, the fluorinated lobe, owing to its low surface energy, would phase

separate to the film surface, but the PS lobe would remain anchored to the film.

In order to prepare films, 3% doubly crosslinked nanoparticles and 2% PS homopolymer (w/w)

were dissolved in THF. The molecular weight of the polystyrene part (as measured by GPC) of our

doubly crosslinked nanoparticles was around 30 kDa. Taking this into consideration, PS homopolymer

with two different molecular weights were investigated, Mn = 3 kDa and Mn = 37 kDa, to evaluate the

suitable PS homopolymer for uniform blending. Films of these blends were prepared by flow coating

on silicon wafers with a film thickness of around 30 nm. (the film thickness was controlled by

controlling the flow coating velocity.) These films were annealed above the Tg of the homopolymer in

order to obtain an equilibrium morphology. Upon annealing the films for 4 hours at 110 0C under

vacuum, films with the 3 kDa PS homopolymer as matrix showed early stage dewetting (Figure 5.25,

left). Under the same annealing conditions, films with the higher molecular weight PS homopolymer

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(37 kDa) as matrix produced a uniform film (Figure 5.25, right). The surface morphology of these uniform films was investigated using AFM and their contact angles were measured using tensiometer.

100 μm 100 μm Figure: 5.25 Optical microscopy images of flow coated films cast from THF solution of blend of doubly crosslinked nanoparticles and PS homopolymer. Films were annealed for 4 h at 110 0C under vacuum.

(Left) annealed film with 3 kDa PS homopolymer showing early stage dewetting. (Right) annealed film with 37 kDa PS homopolymer showing uniform film.

AFM analysis of these films showed the presence of discreet nanostructures at the air-film interface. These nanostructures were 20-30 nm in width and 1-2 nm in height and are comparable in size to the features we observed in the AFM analysis of individual nanoparticles (section 5.3 of this thesis). Phase imaging110 showed a clear difference between the nanostructures on the surface of the film and the matrix material indicating that these nanostructures have different tip-sample interactions compared to the PS matrix (Figure 5.26). Fluorinated polymers have lower surface energies than their hydrocarbon counterparts, and in an equilibrium state, the low surface energy fluorinated part is expected to phase separate to the film-air interface. A representative schematic depicting the arrangement of doubly crosslinked nanoparticles at the air-film interface is shown in

Figure 5.27. In the image shown in Figure 5.26, a large number of these discreet nanostructures are observed with a relatively narrow distribution of sizes (Figure 5.28).

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(a) (b)

(c)

(d)

Figure: 5.26 AFM images of annealed films cast from THF solution of blend of doubly crosslinked nanoparticles and PS homopolymer [Mn = 37 kDa] (a) height/topography image showing the presence of a number of discreet nanostructures, (b) phase image showing contrast between the matrix material and the nanostructures at the air-film interface, contrast arises from varying phase signal from the nanoparticle and the background matrix, (c) and (d) linecut from height image showing features with width ≈ 20-30 nm and height ≈ 1-2 nm.

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PFS

PSPS = 37– 3k kDa

Figure 5.27 Representative schematic showing the arrangement of nanoparticles at the air-film interface wherein the fluorinated part has phase separated to the surface while PS part is miscible with the film.

Figure 5.28 Analysis of all the nanostructures observed at the air-film interface in this AFM image

(same image shown in Figure 5.25 (a); with all the particles selected) showing their narrow height

distribution.

To further confirm that fluorinated parts of the nanostructures are at the air-film interface, we

measured the contact angles of different liquids on these films. Liquids that were bad solvents for

both, polystyrene and polyfluorostyrene parts, were chosen. Water, ethylene glycol and

diiodomethane were chosen since they met the criteria. If the fluorinated part is at the air-film

interface, the contact angle of liquids will be higher compared to the film of neat PS. Contact angles

107 were measured on the films containing double crosslinked nanoparticles and compared to neat PS films. Both water and ethylene glycol showed significantly higher contact angles for nanoparticle- containing films compared to neat PS films substantiating our conclusion that the nanostructures at the air-film interface are fluorinated. This analysis helps in determining the spatial chemical composition of our doubly crosslinked nanoparticles proving that our nanoparticles are amphiphilic in nature. This in turn suggests that our nanoparticles contain two separate lobes, each of which were formed separately during their respective chain collapse stages (Scheme 5.1).

Figure 5.29 Contact angles for different liquids measured on neat PS film (left) and film containing nanoparticle/homopolymer blend (right).

We then calculated surface energies of these films of the nanoparticle/PS homopolymer blend based on the contact angle data. Using diiodomethane and water as test liquids, Fowkes theory111 was utilized to calculate the surface energy. This theory relies on polar and dispersive components

108

(with one test liquid consisting of only a dispersive component) of surface energies of the test liquids

to obtain substrate surface energy. Compared to the PS film, which showed a surface energy of 45.47

mJ/m2, films with nanoparticles gave surface energy values of 44.82 mJ/m2. Lower surface energy

value obtained is due to the presence of the fluorinated lobe at the air-film interface.

The preparation of thin films containing a blend of our doubly crosslinked nanoparticles and PS

homopolymer helped in selectively separating the styrene and the fluorostyrene part of the

nanoparticles in the film. The presence of the fluorinated part is clearly observed in the height and

phase AFM images. Contact angle measurements further substantiated the presence of the

fluorinated part at the air-film interface, thereby suggesting that both blocks in our nanoparticles

retain their immiscibility and the nanoparticles have Janus-type morphology.

5.7 Conclusions

In conclusion, four different techniques were used for the characterization of doubly crosslinked nanoparticles in order to evaluate their size, shape and morphology. All of the techniques support the formation of discreet SCPNs since their presence was clearly detected in all the analyses. TEM and AFM demonstrate that these SCPNs are loosely crosslinked coils that flatten out when deposited on the surface, adopting a pancake-like shape, which when viewed from above looks like a spherical shape. In all of the techniques, two size distributions were detected: smaller sized distribution (individual SCPNs; approximately < 20 nm size) and larger sized distribution (physical aggregates of SCPNs formed due to self-assembly; approximately > 40 nm in size). TEM and AFM on individual nanoparticles were unable to provide information whether our nanoparticles had phase-segregated morphology or not due to insufficient contrast between the two phases. Different parameters were varied to try to obtain detectable contrast between the two phases, but with no success. DLS analysis could not provide information regarding the shape of our nanoparticles due to the bimodal size distribution in solutions.

Multiple attempts to break the physical aggregates utilizing different strategies failed. DOSY-NMR spectroscopy analysis showed that resonances belonging to both phases were observed in DOSY-NMR analysis of discreet nanoparticles. Since DOSY-NMR spectroscopy was performed in a good solvent for

109 both blocks (THF-d8), it does not provide any information regarding the morphology, whether core-shell or phase-segregated. Preparation of films of nanoparticle and homopolymer blend helped in selectively anchoring one phase (PS) onto the film and pushing the other phase (fluorostyrene) to the surface of the film owing to its lower surface energy. Height and phase imaging in AFM analysis clearly detected the distinct presence of discreet nanostructures of chemical composition different than that of the matrix at the air-film interface. Water contact angle studies further confirmed that nanostructures at the air-film interface are fluorinated since they showed higher contact angles compared to neat PS films. These studies confirmed that our doubly crosslinked nanoparticles were prepared via stepwise and selective crosslinking of di-block copolymer and they still retain immiscibility between its two blocks and possess

Janus-type morphology.

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CHAPTER VI

DEVELOPMENT OF A NEW ROOM TEMPERATURE POLYMER CROSSLINKING SYSTEM

BASED ON 1-FUNCTIONALIZED BENZOCYCLOBUTENE

6.1 Introduction

The contents in this chapter pertain to (a) the need for development of a new low temperature crosslinker based on BCB and (b) the development of a new 2-component room temperature crosslinking system based on 1-functionalized BCB.

As previously described, BCBs have attracted attention from polymer community because of their utility as thermal crosslinkers. BCBs undergo thermal ring-opening isomerization to form highly reactive intermediate, o-quinodimethane (o-QDM), which in the absence of a dienophile either dimerizes or oligomerizes with itself to form stable C-C bonds and crosslinked material. Pure thermal crosslinking, no need of metal catalyst and absence of any byproducts are some of the advantages of this chemistry.28

However, for unsubstituted BCBs the temperature required for ring-opening followed by crosslinking is in excess of 200 0C, which limits its use in temperature-sensitive applications.34 The ring-opening isomerization temperature of BCBs can be reduced by functionalizing the 4-membered ring.2,3,14,16,17,18,19

Recently, 1-functionalized BCBs based molecules have been developed and used as low temperature crosslinkers for polymer crosslinking purposes by Pugh18, Harth17 and Wilson112. These groups report 1- functionalized BCB based polymers that undergo crosslinking in the range of 100-150 0C, although their approaches to incorporate BCB-molecules in the polymer backbone are different.

For our project, we needed a polymerizable 1-functionalized BCB that crosslinks at a sufficiently lower temperature compared to unsubstituted BCB. Our idea was to prepare high molecular weight (50 kDa – 100 kDa) di- and tri-block copolymers where the blocks will be immiscible with each other and also contain BCBs within each block that crosslink at distinctly different temperatures. As explained in a

111 previous chapter, we chose styrene and highly fluorinated styrene-containing blocks that are immiscible with each other. We wanted then to crosslink each of these blocks selectively and in a stepwise manner to prepare Janus amphiphilic polymer nanoparticles using BCB chemistry (as outlined in Figure 6.1).

Scheme 6.1 Schematic representation of the proposed di-block copolymer and its conversion to Janus amphiphilic polymer nanoparticle via stepwise and selective intramolecular crosslinking.

Dr. James Baker and Dr. Williams Storms-Miller, previous graduate students in the Pugh group, worked towards developing the synthesis of diblock copolymers containing immiscible blocks and two different BCBs within each block. They mainly worked with 1-ethoxy-4- and 1-ethoxy- 5-vinyl BCB as the second BCB monomer that upon incorporation in the polymer backbone would crosslink at a lower temperature. In this case, the crosslinking temperature of the 1-ethoxyBCB unit (100-150 0C) was sufficiently different from unsubstituted BCB (220-250 0C) to selectively crosslink each of the blocks without affecting the BCB units in the other block. However, they faced a number of challenges while establishing this diblock copolymer synthesis as well as working with 1-ethoxyBCB in general.

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6.2 Problems with 1-ethoxy-4 and 5-vinylBCB

The Pugh group developed the synthesis of a new monomer/crosslinker, 1-ethoxy-4- and 1- ethoxy-5-vinylBCB, which can be incorporated in the polymer backbone via vinyl group polymerization.4

One of the previous theses from the Pugh group demonstrated the polymerizability of this new monomer via radical polymerization.18,88 Though attractive as a low temperature crosslinker, an ethyl ether substituent on the 4-membered ring makes this molecule thermally unstable. Dr. James Baker and William

Storms-Miller53, former students in the Pugh group, observed that this monomer had limited shelf life at room temperature and would undergo premature crosslinking within a month at room temperature. In some cases, premature crosslinking was also observed while it was being stored in a refrigerator.

Utilization of this monomer as a low temperature crosslinker warranted its use soon after it was synthesized.

Scheme 6.2: New monomer (1-ethoxy-4- and 1-ethoxy-5-vinylBCB) synthesized by the Pugh group and maximum temperature that can be used for its (co)polymerization without premature ring-opening.

James studied the thermal stability of this monomer using NMR-Temperature studies. He heated a 10 mol% solution of 1-ethoxyBCB in hexafluorobenzene at different temperatures and followed the consumption of 1-ethoxyBCB using resonances corresponding to the 4-membered ring by 1H NMR spectroscopy. A 10 mol% solution of BCB was used to mimic the possible polymerization conditions since copolymers containing 10 mol% BCB in the backbone were planned for the synthesis of crosslinked single chain polymer nanoparticle. No BCB consumption was observed in 48 hours at 50 0C, whereas 4% of 1- ethoxyBCB was consumed upon heating the solution at 60 0C for 48 hours. The consumption increased

113 upon increasing the temperature: 33% at 70 0C and 76% at 80 0C in 48 hours. This thermal instability of 1- ethoxyBCB above 60 0C restricted the temperature that can be used for the polymerization of styrenic derivative of 1-ethoxyBCB (1-ethoxy-4-and 1-ethoxy-5-vinylBCB). Ideally 50 0C would be a good temperature for polymerization since no premature crosslinking of BCB will take place. However, the rate of polymerization of styrenic monomers (using one of the controlled radical polymerization techniques such as ATRP) would be too slow at that temperature. ATRP-based polymerizations of styrenic monomers are often conducted above 80 0C to prepare high molecular weight polymers. James and Bill chose 60 0C as the polymerization temperature since it offered a better compromise between polymerization rate and the minimum possible premature crosslinking. However, even at 60 0C, it required longer reaction times

(>90 hours) to prepare styrene and fluorostyrene-based high molecular weight copolymers. Limitations of thermal instability and lower rates of polymerization convinced me to look for an alternative monomer/crosslinker to 1-ethoxy-4- and 1-ethoxy-5-vinylBCB.

6.3 Screening of potential 1-functionalized BCB-based molecules for room temperature crosslinking

The criteria for an alternate crosslinker to 1-ethoxyBCB-based crosslinker were that the desired crosslinker: a) would be thermally stable enough to handle and have long enough shelf life, b) would allow its incorporation into a polymer backbone under typical radical polymerization conditions to prepare high molecular polymers, c) would have a sufficiently distinct crosslinking temperature compared to unsubstituted BCB to help in the preparation of Janus polymer nanoparticles as outlined in Scheme 6.1.

I began my literature search for a different substituent on the 4-membered ring that would help satisfy all of the criteria mentioned above. Choy and coworkers14 reported that the acetate group on 1- acetoxyBCB can be deprotected using a nucleophile such as n-butyl lithium to form 1-alkoxideBCB, which in turn undergoes ring-opening isomerization to form the corresponding o-QDM at temperatures as low as -78 0C to 25 0C (Scheme 6.3). This lower temperature requirement to undergo ring-opening isomerization was attributed to the powerful electron-donating ability of the naked alkoxide anion.

Formation of alkoxide o-QDM was proved by trapping it with different dienophiles and characterizing the

114 resulting cycloadducts. The formation of highly reactive o-QDM at such low temperatures caught my attention.

Scheme 6.3: Schematic representation of deprotection of 1-acetoxyBCB using nucleophiles and subsequent reactions as reported by Choy and coworkers.14

BCBs in general undergo ring-opening isomerization to form the corresponding o-QDM species, which are the key intermediates towards the formation of crosslinked materials. The polymer community has been aware of unsubstituted BCB (o-QDM formation temperature 200 0C – 250 0C)30 and recent development in 1-functionalized BCB-based crosslinkers (o-QDM formation temperature 100 0C – 150

0C).17,18,110 However, the polymer community was unaware of formation of alkoxide o-QDM at ambient and sub-ambient temperatures. The literature reports on small molecules showed the possibility of crosslinking of polymers at room temperature via formation of alkoxide o-QDM at room temperature.

Room temperature crosslinking would be sufficiently different compared to unsubstituted BCB

(crosslinking in the range of 220 0C – 250 0C) and would satisfy one of the criteria outlined above for the synthesis of Janus polymer nanoparticles. I decided to explore the 1-alkoxideBCB molecule further.

6.3.1 Thermal stability of 1-acetoxyBCB and 1-hydroxyBCB

1-AlkoxideBCB can be formed by either treating 1-hydroxyBCB with a suitable base113 or by deprotection of 1-acetoxyBCB using a suitable nucleophile. In order to choose a better candidate between the two, I performed small molecule NMR-temperature studies.

1-HydroxyBCB undergoes thermal decomposition mainly via rearrangement to o-tolualdehyde.114

In order to determine the maximum possible temperature that can be used to safely handle 1- hydroxyBCB, I prepared a solution of 1-hydroxyBCB (50 mg, 0.42 mmol) with 1,1,1,2-tetrachloroethane

(35 mg, 0.21 mmol) as an internal standard in 3 mL of DMSO-d6. This solution was then equally split into 3

115

NMR tubes and heated at different temperatures for different amounts of time and the consumption of 1- hydroxyBCB was monitored using 1H NMR spectroscopy. The NMR spectra and tabulated results are reported below. As shown by the decrease in the methylene resonances corresponding to 1-hydroxyBCB and the appearance of an aldehydic proton at 10.22 ppm, 60% of 1-hydroxyBCB underwent thermal decomposition at 60 0C in 24 hours mainly by forming o-tolualdehyde. Thermal decomposition increased to 85% at 70 0C in 24 hours, clearly demonstrating the thermal sensitivity of the 4-membered ring in this molecule. This 1-hydroxy substituent didn’t offer the much needed thermal stability for our project and hence was not pursued further.

C2H2Cl4

Hb Ar Hc H c Hc

DMSO

Hf C2H2Cl4 He

Hf DMSO He Ar Hc Hc

Figure 6.1: Stacked 1H NMR spectra of 1-hydroxyBCB solution (with 1,1,1,2-tetrachloroethane as internal standard in 1 mL DMSO-d6) heated to 60 0C and NMR collected at different time intervals: (a) 0 h (top) and (b) 24 h (bottom).

116

1 Table 6.1: Comparison of integration of methylene protons (Hc) in H NMR of 1-hydroxyBCB solution (with

50 mol% 1,1,1,2-tetrachloroethane as internal standard in 1 mL DMSO-d6) with respect to the integration of internal standard protons (methylene protons) at different temperatures and 24 h time intervals.

1-AcetoxyBCB was then evaluated for its thermal stability. Endo and coworkers16 have reported

DSC analysis of 1-acetoxyBCB that showed the possible thermal stability of this molecule around 90 0C before it starts decomposing. They proposed that this molecule undergoes thermal decomposition via elimination of acetic acid, forming benzocyclobutadiene, which undergoes further dimerization to form benzobiphenylene. However, they didn’t provide any molecular characterization (such as NMR spectroscopy) of the resulting molecule to support this mechanism. In order to determine the possible temperature range for use of 1-acetoxyBCB, I heated a 10 mol% solution of 1-acetoxyBCB in bromopentafluorobenzene with 5 mol% 1,1,1,2-tetrachloroethane as an internal standard at different temperatures. The concentration of BCB can affect the rate at which it is consumed since higher the concentration, the higher is the probability for the ring-opened isomer to undergo dimerization. The 10 mol% solution in fluorinated solvent was used to mimic the possible polymerization conditions for the preparation of poly(pentafluorostyrene-co-1-acetoxy-4,5-vinylBCB) with 90:10 as the comonomer feed ratio. Any solvent with a boiling point higher than 100 0C can be used for this NMR-Temperature study.

Aliquots were taken at different time intervals (0 hours, 24 hours and 48 hours) and analyzed by 1H NMR spectroscopy. The loss of the benzylic methine proton in 1-acetoxyBCB (labeled as Hb in Figure 6.2) was

117 monitored by comparing the integration of the benzylic methine proton to the integration of internal standard. The stacked 1H NMR spectra of aliquots taken at different times at 100 0C are shown in Figure

6.2. No change in the integration of methine proton was observed and no new resonance was observed, indicating that the 4-membered ring with an acetate group is stable for at least 48 hours at 100 0C.

H b Ha Ha Hc C2H2Cl4

Hc Hc Hb

Ha C2H2Cl4 Hc H Hb c

Ha C2H2Cl4 Hc Hc Hb

Figure 6.2: Stacked 1H NMR spectra of 10 mol% solution of 1-acetoxyBCB in bromopentafluorobenzene with 5 mole% 1,1,1,2-tetrachloroethane as internal standard heated to 100 0C at different time intervals:

0 h (top), 24 h (center) and 48 h (bottom).

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1 Table 6.2: Comparison of integration of methine proton (Hb) in H NMR of 1-acetoxyBCB solution (10 mol% solution in bromopentafluorobenzene with 5 mol% 1,1,1,2-tetrachloroethane as internal standard) with respect to the integration of internal standard protons (methylene protons) at different temperatures and various time intervals.

These results showed that 1-acetoxyBCB is thermally stable. It also showed that its styrenic derivative, 1-acetoxy-4- and 1-acetoxy- 5-vinylBCB, once synthesized may potentially be subjected to higher temperatures under radical polymerization conditions. This would provide faster rates of polymerization and higher molecular weight polymers in shorter amounts of time.

6.3.2 Deprotection of 1-acetoxyBCB using a nucleophile and characterization of its products

Choy and coworkers14 deprotected the acetate group on 1-acetoxyBCB using carbanions as nucleophiles (such as n-butyl lithium, methylmagnesium bromide) at -78 0C to 25 0C to produce 1- alkoxideBCB, which in turn formed alkoxide o-QDM. Formation of alkoxide o-QDM was confirmed by trapping it with different dienophiles and characterizing the resulting cycloadducts (Scheme 6.3). I was particularly interested in the dimerization of alkoxide o-QDM since, in my project, I wanted the two o-

QDM units on the same polymer chain to dimerize together and from crosslinks. In order to mimic the possible polymer crosslinking mechanism, I decided to perform the deprotection of 1-acetoxyBCB in the

119 absence of a dienophile at room temperature. NaOMe as the nucleophile was chosen over BuLi for this model experiment since it is easier and safer to handle.

Scheme 6.4: Reaction showing unusual behavior associated with alkoxide o-QDM species

When an o-QDM forms starting from unsubstituted BCB, it either dimerizes to form dibenzocyclootadiene type structures or oligomerizes to form polyphenylene type structures. Therefore, I expected that alkoxide o-QDM would form a dimerized structure similar to dibenzocyclootadiene but with two additional hydroxyl groups on the benzylic positions. On addition of a THF solution of 1-acetoxyBCB to

NaOMe at room temperature, the color of the contents instantaneously changed from colorless to orange to light yellow, suggesting that rapid deprotection was immediately followed by dimerization. The organic content from the crude reaction mixture was isolated only by liquid-liquid extraction and was not purified further. However, analysis of this crude mixture did not show any resonances that would belong to dibenzocyclootadiene type structures. 1H NMR analysis showed complete deprotection of the acetate group indicating, that alkoxideBCB was indeed formed; however, no evidence of the formation of dibenzocyclootadiene structures was seen. This was surprising and unexpected to me. It turned out that alkoxide o-QDM is a special case of o-QDMs. These alkoxide o-QDMs have a resonance stabilized structure in the form of o-formylbenzhydryl anion. It is this o-formylbenzhydryl anion that undergoes dimerization with itself to form lactol type structures instead of alkoxide o-QDM.113 This behavior is in stark contrast to

120 other 1-substituted o-QDMs, which form upon thermal ring-opening isomerization of 1-functionalized

BCBs.

Hd Ha

Aromatic Hd

Hc THF THF Hb

Figure 6.3: 1H NMR spectrum of crude reaction mixture showing the presence of dimerized lactol as the major reaction product obtained after deprotection of 1-acetoxyBCB by NaOMe in the absence of a dienophile.

Figure 6.3 shows 1H NMR spectrum of crude reaction mixture obtained after reaction of NaOMe with 1-acetoxyBCB in THF in the absence of a dienophile. 1H NMR resonances corresponding to the dimerized lactol, 3-(2-methylphenyl)-1-isochromanol, are reported in the literature83 and are clearly seen in the spectrum shown above. The key resonances to look for are: (a) methine protons on the 6- membered ring at 6.14 ppm (s; Hd) and 5.45 ppm (dd; Hb), (b) Hc methylene protons on the 6-membered ring between 2.80-3.10 ppm and (c) benzylic methyl protons at 2.38 ppm (s). Lactol was formed as the major product along with some other side-products; however, no attempt was made to purify the crude

121 reaction mixture since the lactol is already reported in the literature. Molecular characterization of the dimerized structure gave me an idea about the structure of the crosslinked unit that would form upon treating polymer containing 1-acetoxyBCB with a nucleophile.

1-AcetoxyBCB was thus chosen as the desired BCB-based molecule for my project since it satisfied all of the criteria outlined earlier. 1-AcetoxyBCB is thermally stable under standard radical polymerization conditions and can be deprotected to form the dimerized structure at room temperature.

I wanted to develop a polymerizable 1-acetoxyBCB-based molecule on the same lines as of other

1-functionalized vinylBCBs developed in our lab. 1-acetoxy-4- and 1-acetoxy-5-vinylBCB was then designed as the monomer/crosslinker that can be incorporated into the polymer backbone through the vinyl group, and the acetate group on it can be deprotected using a nucleophile at room temperature to form the desired crosslinks.

6.4 Synthesis of a new monomer/crosslinker: 1-acetoxy-4- and 1-acetoxy-5-vinylBCB

In order to prepare the new monomer/crosslinker, I utilized a synthetic route developed in our lab18 for the synthesis of 1-functionalized vinylBCBs. I first regioselectively halogenated anthranilic acid to obtain 5-iodo-2-aminobenzoic acid, which on subsequent diazotization produced the corresponding diazonium carboxylate salt. This salt was handled carefully and heated to form a benzyne, which was trapped in situ via [2+2] cycloaddition with vinyl acetate to obtain a mixture of regioisomers of 1-acetoxy-

4- and 1-acetoxy-5-iodoBCB (Scheme 6.5).

The mixture of aryl iodide obtained in this case was separated to obtain pure isomer by crystallization in order to facilitate assignment of its 1H NMR resonances. The isolated isomer was identified as the major isomer formed in the reaction with 1,5 arrangement as shown in Figure 6.4. Dr. Bill

Storms-Miller performed extensive 2D NMR analysis of the isomers formed in the cycloaddition reaction,53 and those results were useful in identifying the correct structure of the isolated pure isomer.

122

The key resonances of the major isomer to look for are: (a) the characteristic resonances of methine proton (5.86 ppm, dd) and methylene protons (3.16 ppm, d and 3.57 ppm, dd) of the 4- membered ring, (b) the methyl protons of the ester (2.10 ppm, s), (c) the aromatic proton ortho to iodine and the 4-membered ring (7.60 ppm, s), and (d) the remaining two aromatic protons as doublets (6.93 ppm and 7.68 ppm).

Scheme 6.5: Synthesis of new monomer/crosslinker, 1-acetoxy-4- and 1-acetoxy-5-vinylBCB (6.6).92

123

cdcl3_01 2.10

H f Ha Hb

H e Hc Hd

Hf H H

He Hd Hb c c

7.26

0.00

7.60

6.94

6.92

7.68

7.69

5.86

3.18

5.87

3.56

5.86 3.55

3.14

3.59 3.58

0.93 0.88 0.97 1.00 1.08 1.05 3.05

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

Figure 6.4: 1H NMR spectrum of the major isomer, 1-acetoxy-5-iodoBCB.

The polymerizable styrenic derivate of this compound was prepared by introducing a vinyl group onto the aromatic ring employing transition metal-catalyzed cross-coupling reactions. Here, the aryl iodide has a nucleophile-sensitive group on it since in our model compound studies we observed that

NaOMe completely deprotected the acetate group instantaneously at room temperature (Scheme 6.4).

This nucleophile-sensitivity restricted the choice of the cross-coupling reaction that could be used. Heck couplings that employ silane-based reagents and quaternary ammonium salts - also known as ‘Jeffery

Conditions’ - proved to be promising.115 The vinyl source used in these couplings was vinyl trimethylsilane, which is the least nucleophilic amongst other vinyl donors used in other couplings (e.g. Kumada, Suzuki, etc.), hence providing broader substrate scope. Other factors such as, milder reaction conditions (25 0C –

50 0C) and cost, toxicity and ready availability of vinyl silanes were favorable.116 These cross-couplings produced yields in the range of 40-50% to produce an isomeric mixture of the desired monomer. The aryl

124 iodide was used as a mixture of regioisomers and hence the vinylated derivative was also obtained as a mixture of regioisomers.

Figure 6.5 shows the 1H NMR spectrum of the mixture of regioisomers for the new monomer.

The structures of the major and minor isomers are shown. 2D NMR studies performed by Dr. Bill Storms were useful in assigning the resonances in the figure.53 The key resonances here are: (a) methine proton of the vinyl group resonating at 6.70 ppm (dd) and (b) two methylene protons of the vinyl group resonating at around 5.70 ppm (d) and 5.20 ppm (d). The coupling constants corresponding to the major isomer were reported in the experimental section. Resonances characteristic of the 4-membered ring resonate in a similar region as that of aryl iodide, indicating that the cross-coupling reaction used here selectively reacts with the iodine group on the aromatic ring.

125

H Hg g’ Hf’ Hf He’ He Hd Hd’ Ha H He H e’ b H Hb’ c’ Hc

Ha Ha’ Ha’

HeHdHd’ He’ HbHb’ H H H H g g’ H Hc’ c c’ Hf Hf’ c

Ha H Hg’ g H Hf f’ He’ He Hd Hd’ H Ha’ He H e’ b H Hb’ c’ Hc

Ha Ha’

HeHd He He’ Hd’ HbHb’ Hg’ Hg Hf Hf’

Figure 6.5: 1H NMR spectrum of a mixture of 1-acetoxy-4-vinylBCB and 1-acetoxy-5-vinylBCB. Entire spectrum shown on top and expanded regions on bottom.

126

6.5 Synthesis of copolymers containing 1-acetoxyBCB

Once the new monomer (6.6) was available, its polymerizability was evaluated. Polymerization of vinyl monomers can be carried out by either ionic or radical polymerization.117 Within ionic polymerization, anionic polymerization using a carbanion as an initiator for polymerization of styrenic monomers was ruled out because of the highly nucleophilic nature of initiator. Cationic polymerization could be employed since it is compatible with the new BCB monomer reported here, but it often requires the use of stringent reaction conditions that are not often accessible. Radical polymerization on the other hand offers broader functional group tolerance, accessible reaction conditions that can be used with readily available resources.37 Hence, it was pursued to make copolymers containing the new monomer/crosslinker, 1-acetoxy-4- and 1-acetoxy-5-vinylBCB (6.6). As a proof of concept, the copolymerization of the new monomer was attempted using free radical polymerization. A degassed mixture of benzoyl peroxide as initiator and a 60:40 (mol/mol) of feed of styrene and (6.6) was heated at

0 70 C to obtain copolymers with Mn = 15-20 kDa and broader molecular weight distributions analyzed using GPC. Two examples of free radical polymerization are summarized in Table 6.3. 1H NMR analysis showed 65:35 (styrene: 1-acetoxy-4- and 1-acetoxy-5-vinylBCB) (mol/mol) as the composition of the copolymer, indicating successful incorporation of the new monomer into the polymer backbone.

Table 6.3: Table of different copolymers of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5- vinylBCB]) prepared using Free Radical Copolymerization and their characterization

Sr. Molar Feed Ratio Reaction Yield Mn (Ɖ) Composition No. Time 1 (St:(6.6):BPO) (hours) (%) GPC ( H NMR) (St:VAcOxyBCB)

1 60:40:1 14 60.5 mg 14.6 kDa 65:35

(33) (Ɖ = 2.57)

2 58:38:1 21 390 mg 22.9 kDa 65:35

(45) (Ɖ = 3.50)

127

Conventional free radical polymerization method does not allow good control over the polymerization. For our project, we were interested in synthesizing block copolymers in which one of the blocks would contain 1-acetoxy-4- and 1-acetoxy-5-vinylBCB. Synthesizing block copolymers using radical polymerization requires the use of controlled radical polymerization techniques, which offer control over the molecular weight distribution and retention of the chain-end functionalities that are essential for chain extension of the first block.37 I decided to prepare copolymers containing 1-acetoxy-4- and 1- acetoxy-5-vinylBCB using controlled radical polymerization. Atom transfer radical polymerization (ATRP),55 reversible addition fragmentation chain transfer (RAFT)118 and nitroxide-mediated radical polymerization

(NMP)119 are some of the commonly used controlled radical polymerization techniques. I used One of such techniques, ARGET ATRP,77 to copolymerize styrene and (6.6) to obtain copolymers.

Scheme 6.6: Synthesis of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) using ARGET

ATRP.

A series of styrenic (co)polymers with different molecular weights and different compositions was synthesized. These copolymers have slightly broader molecular weight distributions, especially at high molecular weights and when using a higher mol% of BCB in the feed ratio (Entries 2 and 3 in Table

6.4). This is may be due to the possibility of chain transfer events occurring during polymerization at the benzylic protons on (6.6). One of my group members, Isamu Ono, has studied similar compounds and the results indicate possible chain transfer events on the 4-membered ring containing an acetate group.120 1H

NMR analysis of these new copolymers indicated successful incorporation of the new monomer into the polymer backbone (Figure 6.6). In the 1H NMR spectrum, the resonances characteristic of the 4- membered ring are broad: methine proton (5.79 ppm), methylene protons (3.08 ppm and 3.52 ppm) and

128 methyl ester protons (2.06 ppm), indicating that the new monomer is incorporated into the polymer backbone and the pendant BCB ring is intact. GPC analysis of one of the copolymers is presented in

Figure 6.7.

H d H PS_VAcOBCB copolymer_aa222a_A_final g Hf Hg

Hf 7.04

He He He

Hd Hb Hd Hd Hc Hg

He Ha

1.42

6.57 6.47

Hf 1.82 Ha

7.23 H

b Hc Hc 2.06

2.19

5.79

3.08 0.00 3.52

142.46 1.00 0.94 0.89 88.37

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

Figure 6.6: 1H NMR spectrum of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) (Entry

2, Table 6.4). For this example, the molar composition based on the aromatic protons: St/(6.6) = 96.5/3.5.

129

Table 6.4: Table of different copolymers of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5- vinylBCB]) prepared using ARGET ATRP (for all entries molar ratios are, EBib:CuBr2:Me6TREN:Ascorbic Acid

= 1:0.1:0.6:0.4; 90 0C) and their characterization

Entry Molar Feed Ratio Reaction Yield Mn (Ɖ) Composition Time 1 (hours) (%) GPC ( H NMR)

(St:(6.6):EBib) (St:(6.6))

1 665:35:1 11.5 65 mg 8.3 kDa 95:5

(7) (1.29)

2 931:49:1 25 543 mg 31.8 kDa 96:4

(35) (1.42)

3 900:100:1 23 213 mg 22.8 kDa 91:9

(22) (1.29)

Figure 6.7: An example GPC chromatogram (RI detector) of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1- acetoxy-5-vinylBCB]) (Entry 3 in Table 6.4).

130

Radical polymerization also offers the advantage of broader functional group tolerance, and hence can be used to incorporate different monomers in the backbone. In order to expand the comonomer scope, I decided to use methyl methacrylate as another comonomer for polymerization with the new monomer (6.6). Copolymerization of MMA and styrene-based monomers is a tricky system and control over the polymerization in this case depends on the feed ratio of two monomers.117 I was interested in making a MMA copolymer with 10-20 mol% of BCB monomer. There are only a few reports on MMA and styrene copolymerization with 10-20 mol% of styrene in the feed ratio.121 I chose AGET-

ATRP as the controlled radical polymerization technique because it demonstrated relatively good control over MMA and styrene copolymerization. Copolymers with different molecular weight and mol% of BCB were synthesized during the process of optimization of this synthesis and the results are summarized in

Table 6.5.

I observed very broad polydispersities in this copolymer synthesis. I believe that this is not only due to the very different reactivities of MMA and styrene in radical polymerization, but also due to chain transfer events with 1-acetoxyBCB unit.120 I hypothesized that it might be possible to reduce the polydispersities in this synthesis by stopping the reaction at lower conversion. As shown in Table 6.5, I reduced the polymerization time progressively to as low as 5 min (Entry 5 in Table 6.5). The polydispersities narrowed upon shortening the reaction time due to lower conversions obtained during polymerization.

Scheme 6.7: Synthesis and characterization of poly(MMA-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5- vinylBCB]) copolymers using AGET-ATRP.

131

Hi Hh

Hh Hg H f Hf

H e He

Hi Hd Hb

Ha Hg

Hc Hd He Hb

Figure 6.8: An example 1H NMR spectrum of poly(methyl methacrylate-co-[1-acetoxy-4-vinylBCB]-co-[1- acetoxy-5-vinylBCB]) (Entry 5 in Table 6.5). For this example, molar composition based on methine proton of BCB and methoxy protons of MMA is: MMA/(6.6) = 86/14.

1H NMR analysis confirms successful incorporation of new monomer (6.6) into the backbone

(Figure 6.8). The characteristic resonances corresponding to the 4-membered ring are broad: methine proton (5.86 ppm), methylene protons (3.00-3.25 ppm) and methyl ester protons (2.11 ppm). This indicates that the new monomer is incorporated into the backbone and the pendant BCB ring is intact.

Even though the polydispersities are broader (Figure 6.9; GPC) than the ones typically obtained from controlled radical polymerization, the copolymers synthesized are good enough to show the crosslinking of methacrylate-based copolymers at room temperature.

132

Table 6.5: Table of different copolymers of poly(methyl methacrylate-co-[1-acetoxy-4-vinylBCB]-co-[1- acetoxy-5-vinylBCB]) prepared using AGET ATRP at 70 0C and their characterization.

Entry Molar Feed Ratio Reaction Yield Mn (Ɖ) Composition Time 1 (MMA:(6.6):EBib CuBr:PMDETA:Cu(0)) (min) (%) GPC ( H NMR)

(MMA:(6.6))

1 200:20:0.3 1:1.2:1 45 140 mg 31.7 kDa 82:18

(22) (3.37)

2 185:20:0.3 1:1.1:1 45 104 mg 23.9 kDa 79:21

(18) (3.44)

3 180:20:0.5 1:1:1 15 35 mg 8.8 kDa 80:20

(9) (2.76)

4 180:20:0.6 1:1.1:1 15 47 mg 11.7 kDa 75:25

(12) (2.69)

5 180:20:0.6 1:1:1 5 70 mg 11.7 kDa 86:14

(17) (2.18)

133

Figure 6.9: An example GPC chromatogram (RI detector) of poly(methyl methacrylate-co-[1-acetoxy-4- vinylBCB]-co-[1-acetoxy-5-vinylBCB]) (Entry 5 in Table 6.5).

6.6 Intermolecular crosslinking of polymers containing 1-acetoxyBCB at room temperature

New copolymers containing the 1-acetoxyBCB unit were then evaluated for their ability to undergo crosslinking reaction in the presence of a nucleophile at room temperature. First, we attempted to crosslink acetoxyBCB containing copolymers under concentrate conditions using n-butyl lithium (BuLi) as the nucleophile at room temperature (Scheme 6.8).

Scheme 6.8: Crosslinking of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) using BuLi at room temperature.

134

BuLi was added at once over THF solution of copolymer ([BCB] = 0.33 M]) which resulted in an instantaneous color change from colorless to dark orange and the contents turned into a gel, indicating instantaneous crosslinking at room temperature. Excess BuLi and alkoxides formed in the reaction were quenched after 1 minute by NH4Cl/MeOH solution. Analysis of the soluble fraction of the resulting product by GPC showed formation of higher molecular weight species, indicating intermolecular crosslinking (Figure 6.10).

Figure 6.10: GPC chromatogram of starting copolymer and the soluble extract from crosslinked polymer showing intermolecular crosslinking using BuLi as nucleophile.

135

He He He

Hd Inter_PS_BuLi_aa195b_THF_soluble Hd

Hd Hj 1.43

7.26 3.74

Hd 1.24

2.27

6.98 0.00

He Hk 5.00

Hj 7.02

1.15

0.07

2.53

6.52

6.45

1.82

10.00 10.11

1.00 3.85 3.06 2.11

10 9 8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)

Figure 6.11: 1H NMR spectrum of crosslinked polymer obtained using BuLi as nucleophile. No BCB resonances are observed at 5.80 ppm. New resonances belonging to o-tolualdehyde units are seen at around 10 ppm.

1H NMR analysis of the soluble fraction of the crosslinked polymer indicated complete disappearance of the resonances belonging to methine and methylene protons of 1-acetoxyBCB indicating complete deprotection of the acetate group (Figure 6.11). Resonances belonging to o-toluladehyde units were also observed in the 1H NMR of crosslinked material, for example, aldehydic proton resonance around 10.00 ppm and benzylic proton resonances around 2.50 ppm. I believe that these units are formed due to incomplete dimerization of o-formylbenzhydryl anion units formed along the backbone as a result of formation of alkoxideBCB. The results proved my hypothesis that alkoxideBCB containing polymers can be rapidly crosslinked at room temperature. This is a significant improvement in the field of BCB- containing polymer crosslinking since typical temperatures required for crosslinking were always higher

136

(>200 0C in the case of unsubstituted BCB and >140 0C in case of ether substituted BCB, which is a recent development). Our new crosslinker reduces the temperature required for crosslinking by approximately one order of magnitude, although by chemical activation of BCBs. Nucleophile activation of 1-acetoxyBCB- containing polymer to obtain crosslinked polymer can also be looked at as a 2-component crosslinking/curing system similar to the classical 2-component epoxy-amine122 system, but with much faster rates of crosslinking at room temperature.

Successful room temperature polymer crosslinking using BuLi encouraged me to check the feasibility of utilizing other milder nucleophiles such as NaOMe. Upon adding a THF solution of polystyrene copolymer ([BCB] = 25 - 50 mM) all at once over NaOMe at room temperature, an instantaneous color change from colorless to light yellow was observed (Scheme 6.9). Upon quenching the reaction after 5 minutes using NH4Cl/MeOH, a white precipitate was observed that was isolated. The resulting material was partially soluble in THF and its analysis using GPC indeed showed intermolecular crosslinking as seen by formation of polymer with higher molecular weight and multi-modal molecular weight distribution (Figure 6.13).

Scheme 6.9: Crosslinking of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) using

NaOMe at room temperature.

137

Hg Hg Hg Hf Hf Hf

He He He

Hd Inter_PS_NaOMe_aa236b Hd Hg

Hd Hj 7.08

H 1.42 Hd k

6.58 7.25

Hf 1.85

THF

0.00 0.07

Hj 3.74

2.55

9.96 10.13

1.00 63.16 1.80 41.37

10 9 8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)

Figure 6.12: 1H NMR spectrum of the partially crosslinked polystyrene using NaOMe as nucleophile at room temperature. No BCB resonances are observed at 5.80 ppm. New resonances belonging to o- tolualdehyde units are seen at around 10 ppm.

138

Mn = 48.3 kDa Ɖ = 7.47

Mn = 21.9 kDa 1.2 Ɖ = 1.31

0.8

Normalized Linear UV Signal 0.6 Cross-linked 0.4

0.2

0 23 28 33 38 (minutes)

Figure 6.13: GPC chromatogram of starting copolymer and soluble fraction of crosslinked polymer obtained using NaOMe as nucleophile showing intermolecular crosslinking (Entry 3 in Table 6.6).

The 1H NMR spectrum in Figure 6.12 indicated complete disappearance of the resonances belonging to 1-acetoxyBCB, indicating complete deprotection of the acetate group. Resonances belonging to o-tolualdehyde units were also observed in 1H NMR of crosslinked material, for example, aldehydic proton resonance at 10 ppm and benzylic proton resonances at 2.5 ppm) indicating formation of reactive precursors responsible for dimerization and crosslinking of polymer.

Crosslinking of 1-acetoxyBCB containing copolymers using NaOMe as nucleophile is a very useful development since it is easier and safer to use NaOMe as nucleophile than a carbanion such as BuLi. This development clearly increases the accessibility of this system. Also, successful use of milder nucleophiles widens the scope of the comonomers that can be used in the backbone without having to worry about reaction of nucleophile with them. For example, a MMA copolymer containing 1-acetoxyBCB unit will not be selectively crosslinked using BuLi since BuLi can very easily undergo reaction with the ester units of

MMA, thereby consuming BuLi. Use of NaOMe in such case guarantees the availability of nucleophilic species required for deprotection of 1-acetoxyBCB and in turn for crosslinking of polymers. A series of

139 copolymers with different molecular weights and different mol% of BCB were crosslinked using NaOMe.

The results are tabulated in Table 6.6. In all cases, the molecular weight increased and the molecular weight distribution broadened compared to starting linear polymers.

Table 6.6: Results of crosslinking 1-acetoxyBCB-containing polystyrene copolymers using NaOMe.

*BCB+ ≈ 25 - 50 mM for all entries.

Sr. No. Linear Copolymer Temp. Intermolecularly (mol/mol) from 1H NMR (0C) crosslinked

Composition Mn (Ɖ) Mn (Ɖ) 1 95/5 (St/BCB) 8.06 kDa (1.19) RT Rerunning GPC 2 96.5/3.5 (St/BCB) 27.4 kDa (1.53) RT 40.9 kDa (4.01) 3 91/9 (St/BCB) 21.9 kDa (1.31) RT 48.3 kDa (7.47)

As mentioned earlier, this NaOMe-mediated rapid crosslinking can also be applied to polymers other than polystyrenes, such as PMMA. A methacrylate copolymer that was synthesized in-house

(Scheme 6.7) was crosslinked under concentrated conditions in THF using NaOMe as a nucleophile at room temperature. Upon addition of NaOMe, the color of the contents changed slowly from colorless to light yellow. This observation was in contrast to the observation in the case of polystyrene copolymers where the color change to light yellow was very rapid. The reaction was quenched after 5 min and was analyzed using GPC and NMR spectroscopy. Crosslinking reaction in this case occurred as evident from the increase in the molecular weight and broadening of the molecular weight distribution (Figure 6.14). The

1H NMR spectra of the products indicated disappearance of resonances belonging to 1-acetoxyBCB, indicating deprotection of ester group (Figure 6.15). It was difficult to dissolve crosslinked PMMA in CDCl3 and 1H NMR had poor signal-to-noise ratio. However, no BCB resonances were detected confirming deprotection of the ester group on the 4-membered ring. Also, very small resonances belonging to o- tolualdehyde units were observed, suggesting formation of the reactive species responsible for dimerization and crosslinking. Successful crosslinking of methacrylate copolymers widens the range of functional polymers that can be crosslinked using this unique crosslinker and a nucleophile.

140

Scheme 6.10: Crosslinking of poly(methyl methacrylate-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5- vinylBCB]) using NaOMe at room temperature.

1.2 Mn = 33.7 kDa Mn = 11.7 kDa Ɖ = 2.79 Ɖ = 2.18

0.8 Normalized Before crosslinking RI Signal 0.6 After crosslinking

0.4

0.2

0 23 28 33 38 43 (minutes)

Figure 6.14: GPC chromatogram (RI detector) of starting copolymer and soluble fraction of the crosslinked polymer obtained using NaOMe as nucleophile indicating intermolecular crosslinking.

141

H i Hj

Hh Aromatic

Figure 6.15: 1H NMR spectrum of the partially crosslinked methacrylate copolymer using NaOMe as nucleophile at room temperature. No BCB resonances are observed.

Rapid crosslinking of styrenic copolymers using NaOMe as the nucleophile at room temperature encouraged me to attempt crosslinking at sub-ambient temperatures. My hypothesis was that upon decreasing the temperature, the rate of deprotection followed by crosslinking would decrease, but it would still be possible to perform crosslinking in a few minutes at sub-ambient temperatures considering that rapid crosslinking was observed at room temperature.

Scheme 6.11: Crosslinking of poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-[1-acetoxy-5-vinylBCB]) using

NaOMe at sub-ambient temperature.

142

Carefully performing the crosslinking of polystyrene copolymers at 0 0C yielded intermolecularly crosslinked polymer, albeit with lower extent of crosslinking as evident by partial consumption of 1- acetoxyBCB and appearance of smaller intensity resonances corresponding to o-tolualdehyde units seen by the 1H NMR spectrum in Figure 6.17 and by a smaller increase in molecular weight seen by GPC (Figure

6.16).

1.2 Mn = 31.6 kDa Ɖ = 1.97

Mn = 27.4 kDa 0.8 Ɖ = 1.53

Normalized Before crosslinking UV Signal 0.6 After crosslinking

0.4

0.2

0 25 30 35 40 (minutes)

Figure 6.16: GPC chromatogram (UV detector) of starting copolymer and partially crosslinked polymer showing intermolecular crosslinking at 0 0C using NaOMe as nucleophile.

This experiment indicated that the crosslinking reaction rate and hence the extent of crosslinking can be controlled by varying the temperature, which may be a useful feature for applications such as coatings and adhesives. Also to the best of our knowledge, this is the first example of polymer crosslinking using a nucleophile at sub-ambient temperatures.

143

Hg Hg Hg Hf Hf Hf

He He He Hd Hg He Hd

Zero degree C_Inter_PS_NaOMe_aa241a_A Hd

6.57 1.43 7.08 Hd Hj

Hk 6.48 H g Hf Hg Hf

He He He 1.84 Hd 7.24 H 0.00 Hd b Hd Hc

0.07 2.07

Hb Hc Hc Hk

5.80

3.52 3.07 Hj 2.54

240.75 1.00 0.86 0.24 0.46 145.57

10 9 8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)

Hg Hg Hg Hf Hf Hf

He He He

Hd Hd Hd Hj Zero degree C_Inter_PS_NaOMe_aa241a_A

7.26 7.08 7.05 6.58 6.50

Hb 5.82

11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 Chemical Shift (ppm)

Figure 6.17: 1H NMR spectrum of the partially crosslinked polystyrene (expanded region at bottom) using

NaOMe as nucleophile at 0 0C. Resonances belonging to o-tolualdehyde indicate partial deprotection of 1- acetoxyBCB units in the backbone.

144

6.7 Intramolecular crosslinking of polymers containing 1-acetoxyBCB at room temperature

Rapid room temperature crosslinking of 1-alkoxideBCB containing polymers was further utilized to make single chain polymer nanoparticles (SCPNs). SCPNs are increasingly gaining attention as nanostructures in the sub-20 nm size range for potential applications in various fields.123 As the name suggests, SCPNs are nanoparticles made out of a single polymer chain via selective intramolecular crosslinking by forming (non)covalent bonds between the units of the same chain. In the last decade, a wide range of chemistries have been employed towards the synthesis of these nanoparticles, including radical, click, photo and thermal.44 Even though varying chemistries were used, most of the reports utilize

‘ultra-high dilution method’ in order to achieve selective intramolecular crosslinking over intermolecular crosslinking. This method typically requires a large excess of solvent to keep the concentration of polymer low enough for selective intramolecular crosslinking [typical concentration of polymer is around 10-6 M].

The other method called as ‘pseudo-high dilution continuous addition method’ utilizes much less solvent and relies on rapid crosslinking reaction conditions [typical concentration of polymer is around 10-2 – 10-4

M; rate of reaction >> rate of addition] to achieve selective intramolecular crosslinking. The latter method that uses atleast two orders of magnitude concentrated solution has clear advantage since it uses much less solvent. Despite this advantage, only a few reports8,17,30 utilize pseudo-high dilution method. This is mainly because not all the chemistries employed for the synthesis of SCPNs are ‘rapid’ enough, a key requirement for the use of pseudo-high dilution method. As mentioned earlier, our new monomer/crosslinker gave access to BCB-containing polymers that could be crosslinked rapidly at room temperature. I decided to utilize this unique chemistry to make SCPNs using pseudo-high dilution method at room temperature.

For the purposes of synthesizing SCPNs, I used BuLi as the nucleophile. Achieving selective intramolecular crosslinking over intermolecular crosslinking requires optimization of various parameters involved in the pseudo-high dilution continuous addition method. These parameters were first optimized using BuLi as the nucleophile, including concentration of BCB, mode of addition and rate of addition. The results of experiments and the parameters that were varied during the optimization process are

145 summarized in Table 6.7. BCB (10 mM) and addition rate of 1 mL/h provided selective intramolecular crosslinking. Only qualitative observations are mentioned in the table to investigate the parameters and their effect on the crosslinking process. A detailed characterization and discussion will then be presented for optimization for a successful intramolecular experiment.

Table 6.7: Table showing different parameters and their effect on the crosslinking process as observed during the optimization of the pseudo-high dilution continuous addition method.

Entry Concentration of Mode of addition Rate of Observation from GPC BCB in solvent addition analysis

1 0.33 M in THF BuLi over copolymer At once Intra and intermolecular solution addition of BuLi crosslinking

2 0.003 M in benzene BuLi over copolymer At once Intra and intermolecular solution addition of BuLi crosslinking

3 0.1 M in benzene Copolymer solution 1.0 mL/h Intra and intermolecular over BuLi crosslinking

4 0.01 M in benzene Copolymer solution 1.0 mL/h Intramolecular over BuLi crosslinking

In an optimized and successful intramolecular crosslinking experiment with BuLi as the nucleophile, a concentrated solution of polystyrene copolymer in benzene ([BCB] = 10 mM) was added continuously to a solution of BuLi in a large amount of benzene (0.12 mL BuLi in 50 mL benzene) using a syringe pump (rate of addition = 1 mL/h). GPC analysis of the crosslinked polymer nanoparticle indicated reduced apparent molecular weight compared to the linear polymer precursor and did not show any higher molecular weight species, indicating selective intramolecular crosslinking (Figure 6.18). However, better proof of selective intramolecular crosslinking would be light-scattering GPC (LS-GPC) analysis in which the absolute molecular weight is measured. When a polymer chain undergoes selective

146 intramolecular crosslinking, there should not be any change in the absolute molecular weight since it is merely undergoing a change in the chemical bonds, and the shape changes from random coil in solution to crosslinked globule in solution. This change in shape is detected by a change in elution times between linear copolymer and crosslinked copolymer in GPC and is reflected in a reduced apparent molecular weight as seen in Figure 6.18.

Figure 6.18: GPC chromatograms of linear and crosslinked polymer showing intramolecular crosslinking

In order to further substantiate the claim of intramolecular crosslinking, I performed LS-GPC analysis on this optimized and successful intramolecular crosslinking experiment. First I determined the refractive index increment of the starting linear copolymer (with St/BCB = 65/35 on mol/mol basis), which is 0.1738 mL/g (Figure 6.19).

147

Determine dn/dc from RI

data fit

-4 5.0x10

-4 4.0x10

-4 3.0x10

-4 2.0x10

differential indexrefractive -4 1.0x10 dn/dc: 0.1738 (±5.1463%) mL/g

0.0

0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 Concentration (g/mL) Fit R²=0.9921

Figure 6.19: Plot to determine the dn/dc value of linear copolymer (composition: St/VAcOxyBCB = 65/35).

Stacked LS-GPC chromatograms of linear and crosslinked polymer are shown in Figure 6.20. The absolute molecular weight measured is similar for both the shapes. Here, dn/dc value of the crosslinked material was assumed to be the same as that of the linear precursor, which was used to calculate the absolute molecular weight of the crosslinked material. The elution volume clearly changes due to the reduction in the hydrodynamic volume as linear copolymer goes from random coil to crosslinked globule.

This indicates that there is no change in the absolute molecular weight, but only a change in the shape, substantiating selective intramolecular crosslinking.

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Molar Mass vs. time

aa202a aa194b_expt2

8 1.0x10 LS 7 Crosslinked 1.0x10 Mn = 72.4 kDa 6 Ɖ = 1.63 1.0x10 Linear 5 1.0x10 Mn = 70.2 kDa Ɖ = 2.88 4 1.0x10

1000.0

100.0 Molar Molar Mass (g/mol) 10.0

1.0

0.1

0.0

20.0 25.0 30.0 35.0 time (min)

Figure 6.20: Stacked chromatograms of linear copolymer (blue) and crosslinked nanoparticles (red) measured using LS-GPC.

The 1H NMR spectrum of crosslinked polymer shows complete disappearance of the characteristic resonances belonging to 1-acetoxyBCB, and resonances belonging to o-tolualdehyde are observed (Figure 6.21). This indicates that intramolecular crosslinking occurred through 1-acetoxyBCB units. Even though SCPNs have been made previously at room temperature, relatively slower chemistries were employed, making it necessary to use the ultra-high dilution method. In contrast, our method uses a concentrated solution to obtain SCPNs at room temperature.

149

Hg Hg Hg Hf Hf Hf

He He He

Intra_PS_BuLi_aa202a_500 Hd

Hd 0.00 Hd Hj 1.50 Hk

Hd 6.98

He Hk

2.47

6.38

6.50

2.31

9.93 10.05

1.00 4.45 1.64

10 9 8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)

Figure 6.21: 1H NMR spectrum of partially crosslinked polymer nanoparticle showing complete absence of resonances belonging to 1-acetoxyBCB (5.80 ppm) and appearance of resonances belonging to o- tolualdehyde (10.00 ppm).

The synthesis of SCPNs was also attempted using NaOMe as the nucleophile. In this case, THF was used as solvent and NaOMe was added to a large amount of THF ( 50 mL) so that polystyrene copolymer solution could be added to a mixture of NaOMe and THF. However, NaOMe did not dissolve in

THF and always remained as a suspension. The use of toluene as solvent also did not help in dissolution of

NaOMe. For intramolecular crosslinking experiment, NaOMe was not as effective as BuLi. All of the results are summarized in Table 6.8.

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Table 6.8: Comparison of the intramolecular crosslinking performed using NaOMe and BuLi as the nucleophile

Entry Linear Copolymer Linear Nucleophile Crosslinked 1H NMR composition crosslinked Mn [kDa] (equiv. w.r.t. BCB) Mn [kDa] material (1H NMR)(St:BCB) (Ɖ) (Ɖ)

1 65:35 22.8 BuLi (2) 16.0 All BCB consumed; (3.51) (2.75) aldehyde observed

2 65:35 22.8 NaOMe (2.8) 12.8 50% BCB consumed; (3.51) (25% [w/w] solution (2.06) of NaOMe in MeOH 25% aldehyde was used) observed

3 65:35 22.8 NaOMe (3) 20.6 25% BCB consumed; (3.51) (added extra MeOH (3.01) to solubilize very small amount NaOMe) of aldehyde observed

4 91:9 21.9 NaOMe (8) 21.2 BCB resonances clearly seen; (1.31) (aprotic system) (1.30) very small aldehyde observed.

5 96:4 27.4 NaOMe (10) 25.4 BCB resonances clearly seen; (1.53) (aprotic system) (1.47) no aldehyde observed.

The solubility of the nucleophile is important for complete deprotection of acetate group. Higher mol% of BCB was important to achieve partial deprotection and hence partial crosslinking (Entries 2 and 3 in Table 6.8). When a linear copolymer with lower mol% BCB was used, hardly any deprotection of BCB and hence little crosslinking was observed (Entries 4 and 5 in Table 6.8). The use of excess NaOMe in the

151 case of low mol% BCB in the starting copolymer did not improve the results, indicating the importance of solubility of NaOMe (Entries 4 and 5 in Table 6.8). Even though intramolecular crosslinking showed some success using NaOMe, the results are more promising with BuLi (Entry 1 in Table 6.8).

6.8 Conclusion

In conclusion, I have reported the synthesis of a new monomer/crosslinker based on 1- acetoxyBCB. Once incorporated into the polymer backbone, this crosslinker can be activated by a nucleophile to obtain crosslinked material via rapid crosslinking at room temperature. An application of this new 2-pack curing system is demonstrated by the synthesis of single chain polymer nanoparticles at room temperature utilizing a pseudo-high dilution continuous addition method. I have also shown an example of crosslinking at 0 0C. I believe that the broader comonomer scope available with this new crosslinker, coupled with lower temperature, will enable its application in more fields.

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CHAPTER VII

SYNTHESIS OF TRI-BLOCK COPOLYMERS CONTAINING HIGHLY IMMISCIBLE BLOCKS

7.1 Introduction

This chapter discusses use of tri-block copolymer precursors for the synthesis of single chain polymer nanoparticles (SCPNs) of different morphologies. Specifically, this chapter discusses: (a) our design of tri-block copolymers and proposed morphologies of SCPNs likely to form as a result of intramolecular crosslinking, (b) preliminary results showing successful synthesis of tri-block copolymers.

The field of SCPNs has witnessed great interest from polymer community. Most of the research reports have focused on synthesis and application of SCPNs prepared from simple precursors such as, linear uni-block copolymer containing at least 2 monomers and crosslinkers.44 The Pugh group has synthesized SCPNs from more complex precursors such as di-block copolymer containing immiscible blocks and orthogonal crosslinking chemistry for each block.53 Use of multi-block copolymer precursors has recently gained attention from other research groups as well. For example, Meijer and coworkers prepared SCPNs with uniform surface chemistry via controlled crosslinking/folding of ABA tri-block copolymer chain using orthogonal crosslinking chemistries for each of the blocks.51 Lutz and coworkers in a separate study reported step-wise intramolecular crosslinking of an ABA tri-block copolymer to prepare

SCPNs with distinct crosslinked subdomains and uniform surface chemistry.52 The Pugh group’s approach involving a di-block copolymer with amphiphilic backbone presents a route to prepare SCPNs with amphiphilic surface chemistry. This approach is in stark contrast to the work by Meijer and Lutz where

SCPNs with uniform surface chemistry were reported. We wanted to further increase the complexity in the linear polymer precursor used for SCPN synthesis to afford nanoparticles with different and unique morphologies. We decided to explore an amphiphilic ABA tri-block copolymer as our precursor.

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7.2 Proposed design of ABA tri-block copolymer precursors and possible morphologies of their corresponding SCPNs

Our design included a symmetric ABA tri-block copolymer with poly(styrene) and poly(2,3,5,6- tetrafluoro-4-(2,2,3,3,3-pentafluoropropoxy)-styrene) [poly(TF5FS)] as immiscible blocks and BCBs with two distinct crosslinking temperatures in each of these blocks (Figure 7.1). Our idea was that, the use of immiscible blocks would help in preparing Janus/amphiphilic type of nanoparticles.

Scheme 7.1 Representative design of tri-block copolymer precursor in The Pugh group. Each of the immiscible blocks contains BCB that would crosslink at distinctly different temperature compared to BCB in other block.

A general design of symmetric ABA tri-block copolymer is discussed below along with possible morphologies of SCPNs resulting from its intramolecular crosslinking (Figure 7.2). (M-1) in Figure 7.1 depicts representative symmetric ABA tri-block copolymer containing immiscible blocks and BCBs with distinct crosslinking temperatures represented by colored squares with R1 and R2 labels. Depending on the number of hydrocarbon/fluorocarbon blocks, there are two different possibilities for (M-1). There can either be two fluorocarbon blocks as end blocks or two hydrocarbon blocks as end blocks. For the purposes of predicting the possible morphology, it was assumed that the crosslinking reaction was performed in a solvent that was bad for the crosslinking block and good for non-crosslinking block. This way, the block that is undergoing crosslinking would likely remain in collapsed form and the non- crosslinking block would likely remain in chain-extended conformation in solution.

If the central block contains a BCB that crosslinks at a lower temperature, it is likely to form morphology (M-2) upon selective intramolecular crosslinking using pseudo-high dilution technique.

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Advantages and unique features of pseudo-high dilution technique for the synthesis of SCPNs are explained in detail in Chapter IV. Further crosslinking of (M-2) at higher temperature would likely have 3 different possibilities. If both the end blocks on a single chain of (M-2) crosslink separately with respect to each other, then it would likely form (M-5). Since our block copolymer contains immiscible blocks and the crosslinking reaction is assumed to have been performed in bad solvent for the crosslinking block, it is possible that the central block in (M-2) keeps the end blocks away from each other forming morphology depicted in (M-5). However, if the end blocks on a single polymer chain of (M-2) crosslink with each other, then we would likely form (M-6) where the size of one of the lobes would be approximately twice compared to the lobe formed due to crosslinking of central block. Even though our design contains immiscible blocks, possibility of both the blocks mixing (miscible) with each other, upon crosslinking, cannot be ruled out. In such a case, formation of core-shell type morphology (M-7) is expected since end blocks would encapsulate the central block during the second step of crosslinking.

Figure 7.1 Proposed morphologies likely to form upon intramolecular crosslinking of tri-block copolymer precursor.

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Another possibility for starting tri-block copolymer is the case where end blocks contain lower temperature BCB crosslinker. Crosslinking of such a precursor would likely have two paths to follow: (a) formation of (M-3) morphology where two end blocks on a single polymer chain have reacted with each other and/or (b) formation of (M-4) where two end blocks have crosslinked in an intra-block fashion forming distinctly crosslinked sub-domains. Subsequent higher temperature crosslinking of these morphologies would likely result in the formation of (M-8) and (M-9).

All the morphologies presented in Figure 7.2 are proposed morphologies. Whether these morphologies form or not, is best evaluated by actual synthesis and characterization of each of the morphologies. Synthesis of SCPNs from complex precursors is often a very time consuming process and requires extensive optimization at each and every step. Computer simulations can provide an early insight into this crosslinking process and predict the morphologies that are likely to form when a tri-block copolymer chain is subjected to multiple and selective crosslinking. We were fortunate to receive help from Dr. Gary Leuty (Prof. Tsige’s group) who performed computer simulations (molecular dynamics; bead-spring model) on our proposed tri-block copolymer crosslinking. Preliminary results of the work performed by Gary showed that it is possible to obtain the morphologies proposed in Figure 7.2. Only some of the snapshots from his work are presented here. The details are not reported here since it is primarily Gary’s work.

Figure 7.2 Snapshots from simulations predicting that, when a tri-block copolymer chain (M-1) [left] is subjected to intramolecular crosslinking in a solvent bad for the central block, it is likely to form (M-2).

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Figure 7.3 Snapshots from simulations predicting that, when a tri-block copolymer chain (M-1) [left] is subjected to intramolecular crosslinking in a solvent bad for the end blocks, it is likely to form (M-4)

[right].

Figure 7.4 Snapshots from simulations predicting that, when a SCPN (M-4) [left] is subjected to intramolecular crosslinking in a solvent that is bad for end blocks to start with and then slowly becoming bad for the central block, it is likely to form (M-9) [right].

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Figure 7.5 Snapshots from simulations predicting that, when a SCPN (M-2) [left] is subjected to intramolecular crosslinking in a solvent that is bad for the central block to start with and then slowly becoming bad for the end blocks, it is likely to form (M-5) [right].

Synthesis of SCPNs with different morphologies depicted in Figure 7.1 would require synthesis of symmetric tri-block copolymer containing immiscible blocks as the first step. I decided to first establish the conditions for symmetric ABA tri-block copolymer.

7.3 Preliminary results on the synthesis of ABA tri-block copolymer

The symmetric ABA tri-block copolymer containing immiscible blocks has two design possibilities.

It can either have two styrene end blocks or two fluorostyrene end blocks. I decided to explore the synthesis of one of the possible designs: poly(styrene-b-[2,3,5,6-tetrafluoro-4-(2,2,3,3,3- pentafluoropropoxy)-styrene]-b-styrene) [poly(S-b-TF5FS-b-S)].

The monomers that I was working with are all compatible with radical polymerization and I decided to use this method for polymerization purposes. Block copolymer synthesis using radical polymerization is typically carried out using chain extension technique where, the first block is treated as macroinitiator and is then chain extended with the second monomer.37 During my literature search, I came across atom transfer radical polymerization-atom transfer radical coupling (ATRP-ATRC) technique

158 that seemed to be a suitable technique for the synthesis of symmetric tri-block copolymer.63 I decided to evaluate this technique first.

7.3.1 ATRP-ATRC approach

For poly(styrene), ATRP-ATRC approach typically involves: (a) preparation and isolation of poly(styrene) with very high chain end functionality under ATRP conditions, (b) use of polystyrene as macroinitiator to generate polystyryl radical using Cu(I)/ligand catalyst complex and (c) reacting polystyryl radical under monomer free conditions to favor termination reaction. Polystyryl radicals undergo preferential coupling rather than disproportionation during termination reaction; thereby forming polystyrene with double the molecular weight compared to the starting polystyrene.59 ATRP-ATRC approach provides an easy access to symmetric polymers because of this coupling reaction. Synthesis of symmetric ABA tri-block copolymer for our project using ATRP-ATRC approach would involve synthesis depicted in Scheme 7.2. I first decided to attempt this approach on poly(styrene) alone to establish the technique and conditions.

Scheme 7.2 Schematic depicting synthesis of symmetric ABA tri-block copolymer using ATRP-ATRC approach.

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Scheme 7.3 Synthesis of poly(styrene) using ATRP-ATRC approach.

Poly(styrene) with high chain end functionality was prepared using ATRP (Scheme 7.1) conditions developed by Matyjaszewski and coworkers.59 1H NMR spectrum of poly(styrene) was used to calculate % chain end functionality (%CEF) which is ratio of integration of resonances belonging to benzylic methine proton at the chain end to the resonances of initiator end (Figure 7.8). In my case, %CEF was 85% suggesting that 85% of the chains had halogen at their chain end. This poly(styrene) was then reacted under ATRC conditions to prepare poly(styrene) with approximately double the molecular weight (Scheme

7.3). The success of ATRC reaction was confirmed by GPC and 1H NMR spectroscopy analysis. 1H NMR spectrum showed complete disappearance of benzylic methine resonance that was present in poly(styrene) precursor at 4.5 ppm, suggesting that all the polymer chains bearing halogen at the chain end were activated (Figure 7.6, bottom). Molecular weight of resulting polymer was 8 kDa as analyzed by

GPC (Figure 7.7). The efficiency of this coupling reaction can be calculated using GPC data by calculating the ratio of molecular weight of poly(styrene) after coupling to molecular weight of precursor poly(styrene). Coupling efficiency (%CE) in my case was 87%. In any ATRC reaction, there will always be some chains that would: (a) not form the polystyryl radical because there is no halogen at the chain end,

(b) undergo disproportionation reaction forming poly(styrene) with the same molecular weight as that of precursor poly(styrene) and (c) form the polystyryl radical but would not undergo coupling because of the low instantaneous concentration of radical.59 All these factors are reflected in % CE and this efficiency is likely to decrease as the molecular weight of starting polymer is increased.

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Figure 7.6 Stacked 1H NMR spectra of poly(styrene) synthesized using ATRP (top) and ATRP-ATRC (bottom) approach. Inset shows zoomed in region corresponding to polymer chain ends.

161

GPCPS,RI: GPCPS,RI:

Mn = 8.01 kDa; Ɖ = 1.17 Mn = 4.42 kDa; Ɖ = 1.08

Figure 7.7 GPC traces of poly(styrene) synthesized using ATRP (left) and ATRP-ATRC (right) approach.

For our project, we were interested in synthesizing higher molecular weight tri-block copolymers

(around 25 kDa for each block and 75 kDa for entire tri-block). The use of ATRP-ATRC approach for this case would involve radical coupling of two polymers of around 35-40 kDa (Scheme 7.2). This is much higher molecular weight than what I had attempted for ATRP-ATRC model reaction. Also, in the literature the maximum molecular weight of polystyrene used for ATRC purposes was found to be < 10 kDa. I imagined that it would be much more difficult for me to achieve the higher coupling efficiencies in the case of higher molecular weight precursors. Considering this limitation, I decided to explore chain extension approach which is a widely used method for higher molecular weight block copolymer synthesis.

7.3.2 Chain extension approach

Block copolymers are typically prepared via chain extension approach that involves preparing an end block first followed by its reinitiation to add the second block.37 Dr. James Baker and Dr. Bill Storms, previous graduate students in our group investigated synthesis of di-block copolymer poly(S-b-TF5FS) using chain extension approach.53,88 Both James and Bill attempted this synthesis to establish the conditions for the synthesis of di-block copolymer containing immiscible blocks and two different BCBs.

At that time, our group was working with 1-ethoxy-4- and 1-ethoxy-5-vinylBCB as one of the BCB-based monomer/crosslinker. This particular BCB is very temperature sensitive and requires the use of lower

162 temperatures (60 0C) to avoid premature crosslinking. At such lower temperatures, Bill and James found that: (a) synthesis of each of the blocks is very slow because of the low rates of polymerization at 60 0C ,

(b) reinitiation of the first block is very difficult at lower temperatures leading to very few chains undergoing chain extension and (c) solubility of the components of both the blocks is very poor due to their inherent immiscible nature and lower temperatures for polymerization; making it necessary to use large amounts of solvent which in turn slows down the polymerization further. Hvilsted and coworkers have reported synthesis of poly(S-b-TF5FS) using chain extension approach.124 However, they performed all the polymerizations and chain extensions at 110 0C since they were not restricted with respect to the reaction temperature. James and Bill could not use reaction conditions reported by Hvilsted primarily because of the reaction temperature limitations.

As explained in Chapter VI of my thesis, I developed an alternative to thermally sensitive 1- ethoxy-4 and 5-vinyl BCB. A BCB-based molecule, 1-acetoxy-4 and 5-vinylBCB, was developed as a thermal stable alternative that is thermally stable for at least 48 hours at 100 0C. This was an important improvement since, I could perform my polymerizations at much higher temperatures and not remain restricted to 60 0C. I decided to evaluate the synthesis of tri-block copolymer poly(S-b-TF5FS-b-S) using chain extension approach at higher temperatures.

In the first step, I polymerized styrene using ARGET ATRP conditions at 90 0C to prepare poly(styrene) with 19.8 kDa molecular weight. For the next step of chain extension, I decided to use xylenes as the solvent since it dissolved the mixture of polystyrene macroinitiator and fluorinated styrene

(2,3,5,6-tetrafluoro-4-(2,2,3,3,3-pentafluoropropoxy)-styrene) well [here, 4:1 (xylene: macroinitiator and monomer) (wt./wt.)]. Successful chain extension using ARGET ATRP in this case was confirmed by 1H NMR spectroscopy and GPC analysis (Scheme 7.4). Resonances belonging to methoxy protons in the fluorostyrene block were clearly detected at 4.5 ppm indicating successful incorporation of TF5FS (Figure

7.9, top). The composition of the di-block copolymer was styrene:TF5FS = 2.3:1 (mol/mol) based on 1H

NMR spectroscopy. Molecular weight based on NMR spectroscopy was also estimated for the di-block

163 copolymer as Mn,NMR = 46.4 kDa. GPC analysis showed a bimodal distribution for the di-block copolymer sample indicating that only some chains reinitiated and underwent successful chain extension (Figure 7.8).

Scheme 7.4 Synthesis of poly(styrene-b-[2,3,5,6-tetrafluoro-4-(2,2,3,3,3-pentafluoropropoxy)-styrene]-b- styrene) using ARGET ATRP and chain extension approach.

M = 27.3 kDa; M = 19.8 kDa; n n Mn = 30.0 kDa; Ɖ = 1.24 Ɖ = 1.36 Ɖ = 1.33

Figure 7.8 GPCPS,UV traces of poly(styrene) [left], poly(S-b-TF5FS) [center] and poly(S-b-TF5FS-b-S) [right] synthesized using ARGET ATRP and chain extension technique.

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Figure 7.9 Stacked 1H NMR spectra of poly(S-b-TF5FS) [top] and poly(S-b-TF5FS-b-S) [bottom] prepared using ARGET ATRP and chain extension technique.

In order to demonstrate the possibility of tri-block copolymer synthesis, I used the di-block copolymer synthesized above as the macroinitiator and styrene as monomer for third block. I found that, the mixture of macroinitiator and styrene was not completely soluble even after adding a large excess of xylenes as solvent [here, 28:1 (xylene: macroinitiator and monomer) (wt./wt.)]. The solution was opaque at room temperature. I thought that reaction temperature (90 0C) might help in increasing the solubility of the contents and decided to perform the polymerization with opaque mixture. Upon stopping the chain extension reaction, I noticed that the contents still looked opaque. Successful chain extension using

ARGET ATRP at 90 0C in this case was confirmed by 1H NMR spectroscopy analysis where increase in the integration belonging to the styrene resonances compared to the fluorostyrene resonances (Figure 7.9,

165 bottom). The composition of tri-block copolymer was styrene:TF5FS = 2.8:1 (mol/mol) based on 1H NMR spectroscopy. Molecular weight based on NMR was also estimated for the tri-block copolymer as Mn,NMR =

50.8 kDa indicating that only few units of styrene were added in the third block. GPC analysis of this polymer looked like a bimodal distribution and expected the tri-modal distribution was not observed. I believe that the concentration of tri-block copolymer is too low to be seen as an individual distribution.

Also, 1H NMR spectroscopy showed that only few units of styrene were added, indicating that the molecular weight of tri-block is not significantly different than the di-block macroinitiator. Thus, it is likely that the tri-block copolymer distribution is hidden within the higher molecular weight distribution observed in GPC. An interesting observation from GPC analysis was that, Mn of tri-block copolymer (Mn =

27.3 kDa; Ɖ = 1.33) was slightly lower than Mn of di-block copolymer macroinitiator (Mn = 30.0 kDa; Ɖ =

1.36). This was confusing and surprising. I believe that this apparent decrease in molecular weights calculated using poly(styrene) calibration is probably due to interaction of our block copolymer with the column material. Our block copolymer has styrene and highly fluorinated styrene in the backbone and the phenyl rings in these blocks may interact with polystyrene beads in GPC columns via interactions such as

π-π stacking, which in turn may affect their elution times.125 For these experiments, NMR spectroscopy analysis clearly showed successful formation of the tri-block copolymer. I did not attempt to fractionate these polymers to obtain a single distribution belonging to tri-block copolymers. However, these preliminary results show that higher molecular weight tri-block copolymers containing styrene and fluorostyrene-based immiscible blocks can be synthesized using chain extension approach. The major problem with the synthesis of this block copolymer is the inherent immiscibility between the two blocks.

This leads to inconsistent results between different batches and very often no chain extension. Utilizing a different monomer that would be not as immiscible as TF5FS yet, impart amphiphilicity to the block copolymer will be helpful. The preparation of symmetric ABA tri-block copolymer with narrow molecular weight distribution would require optimization of various parameters in the synthesis as well as purification step and would suit well as a long term project.

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7.4 Conclusion

In conclusion, symmetric ABA tri-block copolymer containing immiscible blocks is proposed as a precursor for multiple and selective intramolecular crosslinking. Different morphologies likely to form during this crosslinking process are also proposed. Preliminary computer simulation experiments

(performed by Dr. Gary Leuty from Prof. Tsige’s group) show that the proposed SCPN morphologies are likely to form during this crosslinking process. Two different approaches to prepare new ABA tri-block copolymer are also presented. Proof-of-concept experiments demonstrate successful synthesis of ABA tri- block copolymer using chain extension approach.

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CHAPTER VIII

SUMMARY AND FUTURE DIRECTIONS

My research involved synthesis and characterization of single chain polymer nanoparticles

(SCPNs) prepared using benzocyclobutene (BCB) chemistry and intramolecular crosslinking. Each of the four sub-projects is summarized individually below.

Highly fluorinated SCPNs were successfully synthesized from highly fluorinated copolymer containing pentafluorostyrene and 1-ethoxy-4- and 1-ethoxy-5-vinyl BCB (copolymer was synthesized by

Dr. James Baker) using pseudo-high dilution technique. These SCPNs were characterized using 1H NMR spectroscopy, GPC and TEM. Selective intramolecular crosslinking was observed in GPC where reduction in hydrodynamic volume of polymer was observed compared to its linear precursor. Analysis of these nanoparticles using TEM required the use of staining agent in order to obtain better contrast. RuO4 is a very strong oxidizing/staining agent and stained these nanoparticles better than OsO4. TEM images showed formation of sub-20 nm spherical nanoparticles.

In another project, I characterized new type of amphiphilic polymer nanoparticles using 4 different techniques: TEM, AFM, DLS and DOSY-NMR. These new amphiphilic polymer nanoparticles were synthesized by Dr. Bill Storms using step-wise intramolecular crosslinking of a di-block copolymer chain containing immiscible blocks. Solvents used for crosslinking reaction were good solvents for the block undergoing crosslinking. Since we had used immiscible blocks in order to prepare these nanoparticles, we were expecting final SCPNs to have phase-segregated (or Janus) morphology. My work in this project involved evaluating whether these SCPNs are phase-segregated or not and obtain information about their other features such as size and shape. Some of this work was done in collaboration with other research groups: Mr. Jacob Scherger (Dr. Foster’s group; AFM analysis of individual nanoparticles) and Ms. Namrata

Salunke (Dr. Karim’s group; AFM analysis of nanoparticle/homopolymer blend). TEM and AFM showed

168 presence of discreet SCPNs along with their physical aggregates at concentrations above 50 ng/mL. These aggregates were broken down upon significantly diluting the THF solution of nanoparticles (≤ 50 ng/mL).

Figure 8.1 TEM images (top) and AFM images (bottom; height and adhesion) showing discreet SCPNs as observed by individual techniques.

169

Discreet SCPNs were observed as loosely crosslinked coils that flattened out when deposited on the surface forming pancake-like morphology. These pancake-like nanostructures had much larger diameter/width (≈ 20-30 nm) compared to their height (≈ 1-2 nm) in TEM and AFM images (Figure 8.1).

Unfortunately, TEM and AFM analysis of individual nanoparticles did not distinguish between the two phases within discreet SCPNs. TEM and AFM were unable to provide information whether our nanoparticles had phase-segregated morphology or not due to either insufficient between the two phases or poor resolution capabilities of these techniques. Different parameters were varied in hope to obtain detectable contrast between two phases, but with no success.

DLS analysis was performed with the help of Dr. Fadi Haso (Dr. Liu’s group) to analyze the shape of our SCPNs in solution. However, DLS could not provide information regarding the shape of our nanoparticles due to the bimodal size distribution in solution. The two distributions were: (a) smaller sized distribution with Rh between 10-20 nm most likely representing discreet SCPNs and (b) larger sized distribution with Rh > 40 nm most likely representing physical aggregates of SCPNs. Multiple attempts to break the physical aggregates utilizing different strategies failed. DOSY-NMR spectroscopy was used with the help of Dr. Jessi Baughman to evaluate whether our SCPNs have core-shell morphology or not. In this

2D NMR spectroscopy technique, both the phases (hydrocarbon and fluorocarbon) were detected on the chemical shift dimension for discreet SCPNs. This analysis was performed in THF-d8, which is a good solvent for both blocks of our nanoparticles and it likely swelled both blocks leading to their easy detection in DOSY-NMR spectroscopy. DOSY-NMR analysis did not allow us to differentiate between the core-shell and Janus-type morphology.

Films of our doubly crosslinked nanoparticles and polystyrene (PS) homopolymer blend were analyzed by AFM to evaluate whether PS matrix will help the polystyrene part of nanoparticles anchor within the matrix and immiscible fluorinated part will phase separate at air-film interface. AFM analysis clearly detected the presence of discreet nanostructures of chemical composition different than that of the matrix at the air-film interface (Figure 8.2).

170

Figure: 8.2 AFM images of annealed films cast from THF solution of blend of doubly crosslinked nanoparticles and PS homopolymer [Mn = 37 kDa] (a) height/topography image showing the presence of a number of discreet nanostructures, (b) phase image showing contrast between the matrix material and the nanostructures at the air-film interface, contrast arises from varying phase signal from the nanoparticle and the background matrix, (c) and (d) linecut from height image showing features with width ≈ 20-30 nm and height ≈ 1-2 nm.

171

Water contact angle studies further confirmed that nanostructures at air-film interface are fluorinated since they showed higher contact angles compared to neat PS films. These studies confirmed that our doubly crosslinked nanoparticles possess Janus-type morphology.

In order to selectively crosslink each block of the di-block copolymer chain without encapsulation of the other block, intramolecular crosslinking reaction was performed in poor solvents for the block undergoing crosslinking. Thin films of doubly crosslinked nanoparticles crosslinked under poor solvent conditions and PS homopolymer blend are being analyzed by AFM with the kind help from Namrata

Salunke (Dr. Karim’s group).

I also successfully developed a new 2-component room temperature crosslinking system based on BCB. Development of this system involved developing synthesis of new monomer 1-acetoxy-4- and 1- acetoxy-5-vinylBCB (VAcOxyBCB) using multi-step organic synthesis. The polymerizability of this new monomer was demonstrated by its copolymerization with styrene and MMA using ATRP. The copolymers obtained were then crosslinked using a nucleophile at room temperature and the crosslinking reaction was found to be very rapid affording intermolecular crosslinking within minutes (Scheme 8.1). The crosslinking reaction was characterized using GPC and 1H NMR spectroscopy. Two different nucleophiles,

BuLi and NaOMe, were evaluated for crosslinking purposes and both were found to be successful. Two different types of crosslinking were attempted: intermolecular and intramolecular. Parameters for both of these mechanisms were successfully optimized. The success of intramolecular crosslinking was confirmed by 1H NMR spectroscopy, GPC and LS-GPC analysis.

172

Scheme 8.1: Schematic showing structure of new copolymer poly(styrene-co-[1-acetoxy-4-vinylBCB]-co-

[1-acetoxy-5-vinylBCB]) and its subsequent crosslinking using NaOMe at room temperature. Structure of the crosslinking unit is also shown.

The crosslink unit that would form upon dimerization of each of the BCB unit was also obtained by performing and analyzing the model small molecule reaction. Surprisingly, 1-acetoxyBCB in the presence of a nucleophile dimerizes in a completely different mechanism as compared to the thermal activation and dimerization of other 1-substituted BCBs. The structure of the crosslinking unit is shown in

Scheme 8.1. An example of intermolecular crosslinking at sub-ambient temperatures (0 0C) was also demonstrated. This new 2-component room temperature crosslinking is an important development in the field of BCB-based polymer crosslinkers since, it reduces the temperatures required for crosslinking by one order of magnitude as compared to unsubstituted BCBs (typical crosslinking temperature 250 0C) and by at least 100 0C as compared to ethoxy substituted BCBs (typical crosslinking temperature 100-150 0C).

This system also provides new chemistry for polymer community for rapid room temperature crosslinking purposes.

The use of VAcOxyBCB for selective crosslinking of an amphiphilic block copolymer to prepare crosslinked amphiphilic/Janus-type nanoparticles can be envisaged by the following systems (Scheme

8.2). These proposed systems replace TF5FS as the comonomer for the block providing amphiphilicity with respect to the styrenic block. Few potential comonomers proposed include styrene with a pendant hydrophilic oxyethylene chain and trimethylsilyl styrene derivatives. These proposed comonomers are very likely to facilitate chain extension of the first block to prepare block copolymer since they are not as

173 immiscible with the styrenic block as TF5FS monomer is. Synthesis of block copolymer via chain extension of polystyrene by TF5FS is difficult since this fluorinated monomer is highly immiscible with polystyrene.

Once available, this block copolymer can be crosslinked at room temperature using BuLi to crosslink

VAcOxyBCB containing block to prepare tadpole nanoparticles which can be crosslinked further at 250 0C to crosslink BCB containing block. Cyclohexane and mineral oil can be used as poor solvents for the block undergoing crosslinking in respective steps whereas THF can be used as a good solvent.

Scheme 8.2 Proposed di-block copolymer containing VAcOxyBCB crosslinker and suitable styrenic comonomers. Subsequent crosslinking reaction conditions are proposed in the following step.

During the course of development of VAcOxyBCB crosslinking chemistry, I came across literature that suggested 1-hydroxyBCB unit as a potential new candidate for thermal crosslinking of polymers between 60 0C and 100 0C. Liwen Xing, a (3+2) Master’s degree student in our group, is developing a new thermal crosslinking system based on 1-hydroxyBCB. She is also studying UV-light triggered cycloaddition

174 of o-tolualdehyde which is formed as a result of thermal rearrangement of 1-hydroxyBCB. The preliminary results from her research work are promising and are likely to provide two new crosslinking systems for polymer community.

In another project, I evaluated the possibility of symmetric ABA tri-block copolymer precursors for multiple and selective intramolecular crosslinking. Some of the possible nanoparticle morphologies resulting from this process are proposed. We were fortunate to receive help from Dr. Gary Leuty (Dr.

Tsige’s group) who performed computer simulations that predicted likely formation of proposed morphologies during the crosslinking process. Preliminary results demonstrating successful synthesis of

ABA tri-block copolymer containing immiscible blocks using chain extension approach are also reported.

However, further optimization of synthesis and purification is needed in order to establish ABA tri-block copolymer synthesis.

175

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