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Non-covalent affinity materials for protein structuredetermination via single particle reconstructionanalysis cryo-electron microscopyMinji HaPurdue University

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Recommended CitationHa, Minji, "Non-covalent affinity materials for protein structure determination via single particle reconstruction analysis cryo-electronmicroscopy" (2015). Open Access Theses. 510.https://docs.lib.purdue.edu/open_access_theses/510

PURDUE UNIVERSITY GRADUATE SCHOOL

Thesis/Dissertation Acceptance

To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification/Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material.

Minji Ha

NON-COVALENT AFFINITY MATERIALS FOR PROTEIN STRUCTUREDETERMINATION VIA SINGLE PARTICLE RECONSTRUCTION ANALYSISCRYO-ELECTRON MICROSCOPY

Master of Science

David H. Thompson

Chengde Mao

Mingji Dai

David H. Thompson

R. E. Wild 04/17/2015

i

NON-COVALENT AFFINITY MATERIALS FOR PROTEIN STRUCTURE

DETERMINATION VIA SINGLE PARTICLE RECONSTRUCTION ANALYSIS

CRYO-ELECTRON MICROSCOPY

A Thesis

Submitted to the Faculty

of

Purdue University

by

Minji Ha

In Partial Fulfillment of the

Requirements of the Degree

of

Master of Science

May 2015

Purdue University

West Lafayette, Indiana

ii

This work is dedicated to my parents, Gookhyun Ha and Hyekyeong Lee.

iii

ACKNOWLEDGEMENTS

I would like to appreciate many people who have encouraged and helped me

throughout my graduate study. I especially express my gratitude to my research advisor

Professor David H. Thompson who gave me a great opportunity to work on the intriguing

project and served as an incredible mentor for my graduate life.

Also, I would like to extend my sincere appreciation to many others. First, I

would like to recognize my committee members, Professor Chengde Mao and Professor

Mingji Dai for their guidance. I also gratefully thank my lab members, Yawo Mondjinou,

Vivek Badwaik, Christopher Benjamin, Brad Loren, Ross Verheul, Kyle Wright, Young

Lee, Chris Collins, Scott Bolton, Dr. Seokhee Hyun, Terry Villarreal, Zinia Jaman, and

Shayak Sammadar. Their tremendous support and kindness made me feel at home. I

strongly believe that they became my lifelong friends and collaborators.

Last but not least, I would like to thank my parents, Gookhyun Ha and

Hyegyeong Lee and my grandmothers for their endless trust and love. I always respect

and love you all.

iv

TABLE OF CONTENTS

Page

LIST OF TABLES ............................................................................................................. vi

LIST OF FIGURES .......................................................................................................... vii

LIST OF SCHEMES........................................................................................................ viii

LIST OF ABBREVIATIONS ............................................................................................ ix

ABSTRACT ....................................................................................................................... xi

CHAPTER 1. INTRODUCTION ........................................................................................1

1.1 Challenges in the Protein Structure Determination.................................................1 1.2 Development of Polyrotaxane for Protein Structure Determination.......................4

CHAPTER 2. LYSINE-NTA MODIFIED α-CD POLYROTAXANE SCAFFOLD SYNTHESIS FOR PROTEIN STRUCTURE DETERMINATION USING SINGLE PARTICLE CRYO-EM ANALYSIS. ................................................................................7

2.1 Introduction ..............................................................................................................7 2.2 Results and Discussion ............................................................................................9 2.2.1 Synthesis of the First Generation Polyrotaxane and Post Modification of

α-Cyclodextrin with Lys-NTA .................................................................................9 2.2.2 Synthesis of the Second Generation Polyrotaxane and Post Modification

of α-Cyclodextrin with EBE-NTA .........................................................................11 2.3 Conclusion and Future Plans .................................................................................17 2.4 Experimental Details ..............................................................................................18

CHAPTER 3. THE FORMATION OF THE GOLD NANOPARTICLE CROSS-LINKED POLYROTAXANE NETWORK ......................................................................33

3.1 Introduction ............................................................................................................33 3.2 Results and Discussion ..........................................................................................35 3.3 Conclusion and Future Plans .................................................................................40

v

Page

3.4 Experimental Details ..............................................................................................41

LIST OF REFERENCES ...................................................................................................47

APPENDICES

Appendix A. NMR Spectra Order of Chapter 2 ...............................................................54 Appendix B. NMR Spectra Order of Chapter 3 .................................................................79

VITA ..................................................................................................................................82

vi

LIST OF TABLES

Table Page

Table 2.1 Characterization of NTA modified 1st generation polyrotaxanes ......................10

Table 2.2 Characterization of unmodified 2nd generation polyrotaxanes ..........................14

Table 2.3 Characterization of EBE modified 2nd generation polyrotaxanes ......................15

Table 2.4 Characterization of N3-α-CD threaded polyrotaxane end capped with 2,4,6-trinitrobenzene .................................................................................................... 15

vii

LIST OF FIGURES

Figure Page

Figure 1.1 The proposed schematic diagram of adsorption of His-tagged proteins onto the Lys-NTA modified α-cyclodextrin threaded polyrotaxane scaffolds ..............5

Figure 2.1 Analysis of binding efficiency of polyrotaxane with GFP by ITC ..................11

Figure 3.1 The schematic illustration of gold nanoparticle cross-linked polyrotaxane network .................................................................................................. 34

Figure 3.2 The interfacial reaction setup for gold nanoparticle cross-linked polyrotaxane network .................................................................................................. 34

Figure 3.3 UV-Vis data for gold nanoparticle layers of AuNP cross-linked PRTx network ....................................................................................................................... 36

Figure 3.4 TEM images of cross-linked gold nanoparticles .............................................. 38

Figure 3.5 TEM images of AuNP from the interfacial network setup under different conditions ...................................................................................................... 39

viii

LIST OF SCHEMES

Scheme Page

Scheme 2.1 Synthesis of α-CD polyrotaxane modified with NTA ....................................10

Scheme 2.2 Synthesis of poly(ethyleneglycol)-bis-alkynyl amide ....................................12

Scheme 2.3 Synthesis of 5-azido fluorescein ................................................................... 12

Scheme 2.4 Synthesis of NTA modified polyrotaxanes by post modification with fluorescein end capped with α-CD polyrotaxane ......................................................... 13

Scheme 2.5 Synthesis of nitriloacetic acid-isocyanate ...................................................... 16

Scheme 2.6 Synthesis of NTA modified polyrotaxanes by post modification with fluorescein end capped with NH2-α-CD polyrotaxane ................................................ 16

Scheme 2.7 Synthesis of BCN-EBE-NTA modified polyrotaxane by copper free click reaction ........................................................................................................................ 17

ix

LIST OF ABBREVIATIONS

PRTx

α-CD

MsCl

p-TsCl

DCM

THF

NTA

DMSO

EDTA

TEA

DMF

EDC

PEG

DIEA

EBE

FLUOR

Cryo-EM

AuNP

ACN

EtOAc

His tag

Polyrotaxane

Alpha cyclodextrin

Methanesulfonyl chloride

p-Toluenesulfonyl chloride

Dichloromethane

Tetrahydrofuran

Nitrilotriacetic acid

Dimethyl sulfoxide

Ethylenediaminetetraacetic acid

Trietheylamine

N’, N’-Dimethylformamide

Ethyl-3-(3-dimethylaminopropyl)carbodiimide

Poly(ethyleneglycol)

N,N-Diisopropylethylamine

2, 2’-(Ethylendioxy)bis(ethylamine)

Fluorosceien

Cryogenic electron microscopy

Gold nanoparticle

Acetonitrile

Ethyl acetate

Histidine tag

x

TLC

DSC

AFM

SEM

GPC

Hex

EDC

SPA

MW

TNB

Rb flask

TMS

DEAD

OtBu3

GFP

TBAF

CDI

TNBS

BME

BCN

Thin layer chromatography

N,N'-Disuccinimidyl carbonate

Atomic force microscopy

Scanning electron microscope

Gel permeation chromatography

Hexane

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

Single particle analysis

Molecular weight

2, 4, 6-trinitrobenzene

Round bottom flask

Trimethylsilyl

Diethyl azodicarboxylate

Tri butyl ester

Green fluorescent protein

Tetra-n-butylammonium fluoride

Carbonyldiimidazole

2,4,6-Trinitrobenzenesulfonic acid

β-mercaptoethanol

bicyclo[6.1.0]non-4-yn-9-ylmethyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate

xi

ABSTRACT

Ha, Minji. M.S., Purdue University, May 2015. Non-covalent Affinity Materials for Protein Structure Determination via Single Particle Reconstruction Analysis Cryo-Electron Microscopy. Major Professor: David H. Thompson.

Single particle analysis (SPA) by cryogenic electron microscopy (Cryo-EM) is

one of the powerful tools for determining biological macromolecule structure,

particularly when combined with computational methods or X-ray crystallography.

However, the SPA is challenged by sample processing efficiency. In this work,

polyrotaxane (PRTx) was designed and synthesized such that laterally and rotationally

mobile lysine nitrolicacetic acid (Lys-NTA) modified α-cyclodextrins (α-CD) were

threaded onto a polyethylene glycol (PEG) core. Polyrotaxanes threaded with amine

modified α-CD were also designed and synthesized for stoichiometric control of His-

tagged protein capture by PRTx. For higher efficiency of single particle analysis along

a single strand of the polyrotaxane, gold nanoparticles were used to cross-link the

polyrotaxanes into a network using an interfacial approach. The gold nanoparticle

distribution in TEM images and the UV-Vis spectroscopic changes suggested that a

polyrotaxane network was formed. In addition, to better control the ligands on the

network and to make the reaction on the grid facile, α-CD was functionalized with

xii

mono-6-azido moieties prior to network formation. NMR evidence suggested that mono-

6-azido CD (N3-α-CD) was successfully threaded onto 10K PEG to form a polyrotaxane.

This polyrotaxane was combined with Lys-NTA via a bio-orthogonal copper-free click

reaction with N3-α-CD to produce a polymer network for specific binding with his-tag T7

phage.

1

CHAPTER 1. INTRODUCTION

1.1 Challenges in the Protein Structure Determination

The study of protein structure is an important tool for studying the shape,

function, and intermolecular interactions of protein targets.1 X-ray crystallography

(XRD), nuclear magnetic resonance (NMR), and cryo-electron microscopy (Cryo-EM)

are the most commonly used methods for protein structure determination. These

techniques are often applied in combination to produce a high resolution structure.

X-ray crystallography has long been used to determine bio-macromolecule

structure since it often provides high resolution structures (i.e <1.5 Å) when adequate

qualities of the target are available to support extensive crystallization trials.

Unfortunately, some types of proteins such as heterogeneous samples, membrane

proteins, and protein complexes are not readily amenable to crystallization.2,3

NMR of site specifically labeled proteins is another way to study the protein

structure. This approach also can provide good resolution (2-5 Å), enabling the structural

study of non-crystallizable proteins. Moreover, it is possible to see the relative disorder

of proteins by comparing the NMR peaks in specific regions. One drawback of this

method is that it requires large amount of proteins (>1 mg) and highly concentrated

2

sample to provide high quality of data. Also, mixtures and high molecular weight

proteins are difficult to analyze by NMR.2,3 Alternatively, cryo-EM is an increasingly

popular and powerful method for protein structure determination where a thin layer of the

sample is vitrified at the cryogenic temperature. The sample is then transferred to a cryo-

stage in the electron microscope for imaging, where the low temperature of the sample

reduces or slows down the radiation damage caused by electrons emitted from an electron

gun, and native macromolecular structure can be retained.4 Cryo-EM SPA for 3D image

reconstruction has some distinct merits compared to X-ray crystallography and NMR.5

First, only ng to μg of protein is needed for sample preparation and staining and

embedding in the physiological media is not necessary. As for samples, species with

various types of structures such as helical, 2D crystallines, multi-protein complexes, or

large single particles can be used as a sample, including objects that are 30-200 nm in

size. Time-resolved studies also can be readily performed with cryo-EM enabling

visualization of the transient species in biochemical reactions via a flash freezing of the

sample at various time points during the transformation. Phase information of the

structure can be obtained as the diffracted electron beam is refocused on to the real space

image on the image plane. In addition, modestly impure samples or non-homogeneous

samples can be used because the particles of interest can be manually selected for

reconstruction.5 Also, a variety of materials including affinity lipids6-11, facial

amphiphiles12,13, conformationally restricted amphiphiles14,15, lysolipid guided cubic

phase formation16, and polymer nucleants17 have been used for assisting protein

crystallization for XRD and single particle analysis in Cryo-EM.

3

Meanwhile, the resolution of cryo-EM is highly dependent on the sampling rate,

where more than 30,000 images of the targeting proteins are necessary to obtain the

images with the resolution of 10 Å or better. Furthermore, relatively low concentrations

of the samples used in cryo-EM (μg to mg/mL) make the data collection more labor

intensive and time consuming for elucidation of targeting proteins present in few copies

in high magnification.18

To overcome these challenges in sample analysis and data collection of cryo-EM,

various efforts have been made. For example, automated data collection methods such as

Legnion have been developed,19-22 and a number of methods to concentrate the samples at

the material interfaces have been developed. Kornberg reported capture of targeting

proteins on the lipid monolayer surfaces with antibody and antigen for cryo-EM, thereby

increasing concentration and achieving higher specific binding of the samples.23 In the

meantime, Thompson et al. developed the lipid monolayers for 2D crystallization of the

His-tagged MoCA proteins in water-air interface, and their structure was elucidated with

TEM with 9.5 Å resolution.24 Walz also adapted this strategy of the lipid monolayer for

the purification of the His-tagged transferrin-transferrin receptor (Tf-TfR) complexes

with Ni2+-NTA modified lipid.25 In addition, the Rice group studied the ultrastructure of

the tagged Hepatitis C virus (HCV) with α-HCV antibody modified EM grid and Ni2+-

NTA lipid modified affinity grid.26 However, the main drawback of this lipid monolayer

approach is that only two-dimensional crystals can be used for the imaging, which can be

mediated by tilt series of the images to produce three-dimensional structure. However,

obtaining the high resolution images or structures by tilt series is difficult due to the

technical limits on the tilting in the microscope (maximum tilting angle of ~60°). To

4

overcome this limitation of the tilt series, the helical crystallization method using lipid

nano-tubules was developed, which enabled obtaining the protein images in all

orientation the TEM. However, this approach requires consistent nanotube diameters to

obtain the images with the high resolution.27 DNA template protein arrays developed by

Turberfield induced more dense, but non-overlapping arrays of protein molecules for

SPA, but lysate conditions are incompatible for the protein structure analysis as it can

destroy the DNA lattice.28 Therefore, further refinements of single particle analysis of the

cryo-EM are clearly needed, including the ability to faithfully recognize different

orientations of the proteins for three dimensional reconstructions and efficient sample

preparation methods to achieve higher concentrations for SPA analysis.

1.2 Development of Polyrotaxane for Protein Structure Determination

To address various challenges for proteins structure determinations by SPA cryo-

EM mentioned above, a new family of organic materials, called polyrotaxanes, were

designed to capture the proteins for SPA via cryo-EM. Polyrotaxanes are necklace-like

molecular structures in which one or more macrocyclic molecules (e.g. α-cyclodextrin, α-

CD) is/are threaded onto a linear polymer (e.g. polyethylene glycol, PEG or poly(lactic-

co-glycolic acid) (PLGA)). To prevent the dethreading of the macrocyclic molecules,

both ends of the polymer termini are locked in place with bulky end cap molecules.29-36

Polyrotaxanes have previously been used for drug delivery due to its multi-functionalities

coming from the unique structure properties.31,35,37,38 Threading of α-CD to a linear

polymer axle with end caps enables sliding and rotation of the macrocyclic molecules

al

p

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an

ar

so

F

w

re

p

long the thre

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Figure 1.1 ThLy

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ecognize and

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eaded polym

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r SPA Cryo

more, the af

rotaxane scaf

on.

proteins ontffolds.

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ted Lys-NTA

the design o

ure and stru

5

units

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of the

o-EM

ffinity

ffold,

to the

xanes

A can

of the

ucture

6

analysis because the sliding α-CD along the PEG can capture the particles in various

orientations. Also, the rod-like structure of this polymer can minimize the information

loss from overlapping copies of bound protein, which improves the efficiency on the data

collection as the captured proteins are aligned and concentrated rather than dispersed

randomly on the EM image.

7

CHAPTER 2. LYSINE-NTA MODIFIED α-CD POLYROTAXANE SCAFFOLD SYNTHESIS FOR PROTEIN STRUCTURE DETERMINATION USING SINGLE

PARTICLE CRYO-EM ANALYSIS

2.1 Introduction

Novel materials for efficient protein structure determination by single particle

construction via Cryo-EM have been developed. Polyrotaxanes threaded with α-

cyclodextrin along different lengths of poly(ethylene glycol) can help to overcome

current challenges of SPA by cryo-EM. The sliding ability of α-cyclodextrin enables

captured proteins to possess lateral and rotational freedom, which is significant as it

allows capturing the targeting ligand proteins in multiple orientations.37 Also, one copy of

the polyrotaxane can capture more than one targeting protein, which improves the

efficiency of the data processing and sample preparation.

To capture His-tagged proteins, one of the OH groups on the α-CD was

conjugated with Lys-NTA. To minimize the constrained mobility of the captured proteins

caused by the crowding of the bulky protein molecules, a short spacer was added between

the Lys-NTA and α-CD. (Scheme 2.4)

Using conventional methods, the ligand was conjugated onto the polyrotaxane

scaffold by modifying OH groups of the α-CD after completion of the threading and

endcapping reactions.37 This approach provides no consistent control over the number of

modification ligands since this can be controlled only by changing the stoichiometry of

8

the coupling reagents. Therefore, this leads to difficulties in controlling of the

number and distribution of ligands conjugated to the individual CD monomers as well as

the polyrotaxane as a whole. In an attempt to obviate this limitation, we sought to modify

α-CD with azide (N3) before threading onto the PEG core to produce a polyrotaxane with

a known number of 6-azido-α-CD units so that subsequent click reactions with Lys-NTA

would generate an affinity polyrotaxane with well-controlled protein binding

properties.37,38,40 (Scheme 2.7) In this study, the N3 α-CD threaded polyrotaxane was

prepared and characterized by NMR. Also, similar to the azido CD, threaded amine CD

was modified with isocyanate NTA to capture his-tagged proteins. (Scheme 2.5, Scheme

2.6)

This project is still in the preliminary stage, but the synthesized polyrotaxanes

look promising as demonstrated with its efficient binding constant.

9

2.2 Results and Discussion

2.2.1 Synthesis of the First Generation Polyrotaxane and Post Modification of α-Cyclodextrin with Lys-NTA

In this study, the polyrotaxane was synthesized to improve the protein structure

determination by SPA cryo-EM. The first generation polyrotaxane was an NTA-modified

polyrotaxane end-capped with TNB. (Scheme 2.1) Unfortunately, the deprotection of

NTA after threading and insufficient size of the endcapping molecules induced the

dethreading of the α-CD. Therefore, four different kinds of polyrotaxane were

synthesized with different molecular weights of the PEG (10 kDa and 35 kDa) and

different protecting groups of the NTA (trimethylsilyl and t-butyl ester). The

characterization of this polyrotaxane is shown in Table 2.1. Among this family of

polyrotaxanes, Compound 6b was chosen for protein binding analysis using isothermal

calorimetry (ITC) as the number of α-CDs before and after dethreading remained most

consistent. (Figure 2.1) When polyrotaxane (500 μM) was added to GFP (35 μM), the

binding constant (KD) was 1.19 μM, and the enthalpy of reaction, which is the amount of

heat released upon ligand binding to the polyrotaxane, was 140 cal/mol. The

stoichiometry of the reaction was 0.797, which was close to the theoretical value

expected of 1. When GFP (357 μM) was added to the polyrotaxane (35 μM), the

observed KD was 1.43 μM, the reaction enthalpy was -2.602 cal/mol, and the calculated

stoichiometry was 0.934. This ITC data is consistent with a 1:1 binding ratio of protein

and α-CD on PRTx, which also corresponds to our assumption that the NTA was

modified on the α-CD with 1:1 ratio. Therefore, ITC analysis shows that the architecture

10

of polyrotaxane could capture protein targets in stoichiometric fashion for subsequent

structural analysis.

Scheme 2.1 Synthesis of α-CD polyrotaxane modified with NTA; A: Modification of

PRTx with TMS protected NTA; B: Modification of PRTx with OtBu3 protected NTA.

Table 2.1 Characterization of NTA modified 1st generation polyrotaxanes; Compound 6a and 6b were synthesized with 10 kDa PEG, and Compounds 7a and 7b were synthesized with 35 kDa PEG. ; A: Protected by trimethylsilyl (TMS), B: Protected by tri t-butyl ester

(OtBu3).

Entry

Before deprotection After deprotection

# of CDs threaded

NTA/CD MW

(kg/mol)

# of CDs

threaded NTA/CD

MW

(kg/mol)

6a 6 3.55 16.73 9 2.56 31.68

6b 28 0.95 17.75 3 1.38 15.26

7a 39 2.12 77.33 35 2 108.5

7b 24 2.12 67.95 20 1.02 65.72

Fμ

d

(S

th

T

d

fl

Jo

te

(S

Figure 2.1 AμM polyrotax

2.2.2 Syn

In ord

ethreading,

Scheme 2.4)

he OH group

The polyrota

ifferent mol

luorescein v

ones reagen

erminal alky

Scheme 2.3)

Analysis of bxane 6b, Syr

500

nthesis of the

der to optim

a new reac

) However, t

ps of α-cyclo

xane was p

ecular weigh

ia Cu2+ cata

nt followed

ne end group

) was used

inding efficiringe: 357 μM0 μM polyro

e Second Geα-Cycl

mize the stoi

ction schem

the synthesis

odextrin thre

repared by

hts (3.4 kDa

alyzed click

by CDI m

ps. (Scheme

for end-ca

iency of polyM GFP, KD:otaxane 6b, K

eneration Pollodextrin wi

ichiometry o

me for NTA

s was not co

eaded on the

threading α

a, 10 kDa, an

reaction. Bo

mediated cou

e 2.2) 5-Azid

apping the p

yrotaxane w: 1.43± 0.13KD: 1.19±0.0

lyrotaxane ath EBE-NTA

of protein li

A-modified p

ompleted unt

e polyrotaxan

α-CDs onto

nd 20 kDa) a

oth ends of

upling of p

dofluorescein

pseudorotax

with GFP by IμM; B: Cell04 μM.

and Post MoA

igands and

polyrotaxan

til the modif

ne with a sp

the poly(eth

and end-capp

the PEG we

ropargyl am

n provided b

ane to prev

ITC; A: Cell: 35 μM, Sy

dification of

to prevent α

ne was desig

fication of o

acer was for

hylene glyco

ping with 5-

ere oxidized

mine to gen

by Kyle J. W

vent dethrea

11

l: 35 yringe:

f

α-CD

gned.

one of

rmed.

ol) of

azido

d with

nerate

Wright

ading.

12

Elucidation of the end group of the polymer was challenging due to the low mass of end

group compared to the mass of the polymer. However, after the click reaction of the end

group of the polyrotaxane, the presence of the fluorescein was apparent as the color of the

polyrotaxane was bright yellowish orange m and the NMR peaks at 7 ppm to 9 ppm

confirmed the presence of fluorescein.

Scheme 2.2 Synthesis of poly(ethyleneglycol)-bis-alkynyl amide.

Scheme 2.3 Synthesis of 5-azido fluorescein (provided by Kyle J. Wright).

The synthesized polyrotaxanes were characterized by NMR and GPC to

determine the threading efficiency and the molecular weight of these intermediates. The

calculated results are summarized in Table 2.3. As one α-CD can include two ethoxy

units within the CD cavity, the threading efficiencies of the polymers ranged from 43% to

almost 60% depending on the length of the polymer. These threading efficiencies were

reasonable considering rotational and lateral freedom of the α-CD and the captured

proteins. The molecular weight of the polyrotaxanes acquired from GPC agreed with the

one from NMR, and the polydispersity index was close to 1:1. To reduce the hindrance

13

among the captured targeting proteins, 2, 2’-(ethylendioxy)bis(ethylamine) (EBE) was

conjugated between α-CD and NTA through CDI mediated coupling. This short spacer

could provide the bound proteins with more room to rotate and enable more random

orientations, especially for larger proteins. The polyrotaxanes after EBE modification

were also characterized by NMR and GPC. (Table 2.4)

For better stoichiometric control of the conjugated ligand and simpler post

modification of the polyrotaxane, the α-cyclodextrin was modified before threading.

Scheme 2.4 Synthesis of NTA modified polyrotaxanes by post modification with fluorescein end capped with α-CD polyrotaxane.

The polyrotaxane was synthesized with the N3-α-CD formed by end capping with

TNP and characterized with NMR. (Table 2.5) The number of N3-α-CD and threading

efficiency was lower than that of α-CD threaded polyrotaxanes. However, for higher

14

stability of the product, bulkier end capping groups are needed. For enhanced threading

efficiency, optimized conditions should be developed.

The protocol to modify the α-CD with azide for the N3 CD threaded PRTx above

was performed with the Apple reagent and NaN3. However, the percent yield of the

product was approximately 10% after the purification. To improve the yield and make the

reaction condition milder, different synthesis protocol where α-CD was tosylated

followed by azidation in water. The yield increased about two-folds with this protocol,

but purification of tosyl α-CD was difficult due to high solubility of the α-CD in water

during recrystallization. In addition, it was hard to separate the product from α-CD via

column chromatography due to similar polarities. The highest purity of synthesized tosyl

α-CD was obtained after 4th recrystallization, which was 62%. This compound was

azidated, but it was found that the compound was hydrolyzed back into α-CD in water.

Therefore, the optimized protocol for synthesis of efficient azido α-CD and amine α-CD

should be pursued in the future study.

Table 2.2 Characterization of unmodified 2nd generation polyrotaxanes.

Entry MW of PEG

(kg/mol) # of CDs Threaded

Threading efficiency (%)

MW (kg/mol) PDI

(Mw/Mn) NMR GPC

4a 3.4 20 51.9 23.6 28.7 1.039

4b 10 68 59.3 76.3 44.7 1.169

4c 20 98 43.2 116.2 103.4 1.110

15

Table 2.3 Characterization of EBE modified 2nd generation polyrotaxanes.

Entry MW of

PEG

(kg/mol)

# of CDs Threaded

Threading efficiency

(%)

MW (kg/mol) PDI

(Mw/Mn) # of EBE

conjugated NMR GPC

5a 3.4 8 20.7 12.6 5.64 1.586 5

5b 10 23 20.2 34.4 10.6 1.652 10

5c 20 31 13.6 5 76.6 1.337 28

Table 2.4 Characterization of N3-α-CD threaded polyrotaxane end capped with 2,4,6-trinitrobenzene

Entry MW of PEG

(kg/mol) # of N3 α-CD threaded Threading efficiency (%)

MW

(kg/mol)

18 10 8.3 7.06 18.46

16

Scheme 2.5 Synthesis of nitriloacetic acid-isocyanate (provided by Kyle J. Wright).

Scheme 2.6 Synthesis of NTA modified polyrotaxanes by post modification with fluorescein end capped NH2-α-CD polyrotaxane.

17

Scheme 2.7 Synthesis of BCN-EBE-NTA modified polyrotaxane by copper free click reaction.

2.3 Conclusion and Future Plans

In this study, the first generation of NTA-polyrotaxanes threaded with α-CD was

synthesized and characeterized by NMR and ITC. ITC data proved that the binding of

ligand protein with polyrotaxane. In an attempt to gain better control of the stoichiometry

and utilize simpler post modification steps, the polyrotaxane was modified with EBE-

NTA in a second generation polyrotaxane. Also, the α-CD was modified with azide and

successfully threaded on the polymer to enable click reaction onto the polyrotaxane

scaffold.

In future studies, the development of a protocol for efficiently making pure azido

-α-CD and its reduction to amino-α-CD is needed. In addition to these cyclodextrins, the

EBE-NTA PRTx synthesis should be optimized and the product should be characterized.

The polyrotaxane should be additionally characterized with 2D-NOE to confirm that α-

cyclodextrin was threaded to PEG, not just merely heterogeneously adjacent to each other,

as illustrated by evidence for a cross peak between the C-H of α-CD and the protons of

PEG. For an additional characterization method other than NMR and GPC, analytical

18

ultracentrifugation (AUC) can also help compare the molecular weights of the product.

Finally, the amount of the Ni2+ content in the polyrotaxane should be analyzed by ICP-

MS for their stoichiometric ratio after activating the NTA with Ni2+. Imaging with cryo-

EM after binding His-tagged proteins to the NTA-polyrotaxane should be investigated to

confirm the ability of the polyrotaxane for capture of the target molecules. Also, washing

Ni2+ with imidazole can determine specific capturing ability of NTA on the polyrotaxane.

2.4 Experimental Details

tert-Butyl N6-((benzyloxy)carbonyl)lysinate (12).

N6-((Benzyloxy)carbonyl)lysine 11 (9.724 g, 34.69 mmol) was added to a 250 mL rb

flask. t-Butyl acetate (120 mL, 888.4 mmol) and perchloric acid (4.5 mL, 52.14 mmol)

were added to the flask. The reaction mixture was stirred overnight with a glass stopper.

The solution was extracted with H2O (240 mL), 5% HCl (240 mL), H2O (x2, 200 mL).

Diethyl ether (150 mL) was added to the combined aqueous layers and 30% NaOH

solution was added until it became pH ~13. The solution was extracted with diethyl ether

(x3, 300 mL) and the combined organic layer was dried over Na2SO4 for 30 min. The

solution was filtered and the ether was removed in vacuo to afford a product in 63.3%

(7.387 g, 21.96 mmol). 1H NMR (400 MHz, CDCl3) δ 7.33 (d, J = 4.7 Hz, 5H), 5.06 (s,

19

2H), 3.67 (d, J = 7.0 Hz, 0H), 3.26 (d, J = 6.4 Hz, 1H), 3.21 – 3.10 (m, 2H), 1.70 – 1.58

(m, 3H), 1.51 (d, J = 11.8 Hz, 2H), 1.43 (d, J = 4.3 Hz, 9H), 1.24 (s, 4H).

Di-tert-butyl 2,2'-((6-(((benzyloxy)carbonyl)amino)-1-(tert-butoxy)-1-oxohexan-2-

yl)azanediyl)diacetate (13).

2-Amino-6-benzyloxycarbonyl-amino hexanoic acid 12 (H-Lys-OtBu) (7.39 g, 22.0

mmol) was dissolved in dimethylformamide (80 mL) and t-butyl bromoacetate (9.71 mL,

65.9 mmol) and triethylamine (10.7 mL, 76.8 mmol) were added to the flask. The

reaction mixture was stirred at 70°C for 3 days before removing the DMF in vacuo. Ethyl

acetate (EtOAc) was added and the suspension was filtered to give a white solid. The

orange colored filtrate was extracted with diethyl ether (x4, 300 mL) and the combined

organic layers were dried over Na2SO4 before removal of the ether in vacuo. The crude

product was purified by silica column chromatography (5:5 EtOAc/Hexane) to afford a

product in 46.4% yield (5.76 g, 10.2 mmol). 1H NMR (400 MHz, CDCl3) δ 7.57 – 6.99

(m, 5H), 5.05 (s, 3H), 3.34 – 2.85 (m, 5H), 1.71 – 0.67 (m, 28H).

20

Di-tert-butyl 2,2'-((6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)azanediyl)diacetate

(14)11.

Di-tert-butyl 2,2'-((6-(((benzyloxy)carbonyl)amino)-1-(tert-butoxy)-1-oxohexan-2-

yl)azanediyl)diacetate 13 (0.95 g, 1.68 mmol) was dissolved in methanol (30 mL) in a

100 mL rb flask and Pd/C (wet) was added to the solution. The reaction flask was

evacuated and H2 was purged with a flushing adapter (x2). The reaction mixture was

stirred for 8 hr at rt under H2. The solution was filtered through a pad of Celite and

methanol was removed in vacuo to recover the product in 87.3% (0.628 g, 1.46 mmol)

yield. 1H NMR (400 MHz, CDCl3) δ 5.48 (s, 3H), 3.51 – 3.36 (m, 4H), 3.29 – 3.17 (m,

3H), 2.96 – 2.92 (m, 2H), 1.50 – 1.34 (m, 34H).

Mono-6-azido-6-deoxy-cyclodextrin, N3 α-CD (17)37,41.

To a 250 mL rb flask, α-cyclodextrin (10 g, 10.29 mmol) was dissolved in dry DMSO (80

mL) before addition of sodium azide (6.68 g, 102. 8 mmol) and triphenylphosphine

(10.236 g, 30. 87 mmol). The reaction mixture was sonicated to help dissole the materials

21

before addition of tetrabromomethane (10.236 g, 30.87 mmol). The solution turned

yellow in color and was exothermic. The rb flask was evacuated and purged with Ar (x3)

and reaction mixture was stirred for 48 hr at rt under Ar before reducing the solution in

vacuo and precipitation with cold acetone (x2, 1000 mL). The crude product was filtered,

and dried under vacuum before purification by silica flash column chromatography (4:1

Acetonitrile/water) to yield a pure product as a white powder. (1.3 g, 1.3 mmol) The

presence of azide was confirmed by staining with triphenylphosphine solution and

ninhydrine solution1H NMR (500 MHz, DMSO-d6) δ 5.54 (d, J = 7.0 Hz, 6H), 5.44 (d, J

= 2.8 Hz, 6H), 4.79 (d, J = 3.4 Hz, 6H), 4.52 (t, J = 5.7 Hz, 6H), 3.76 (d, J = 2.8 Hz, 3H),

3.65 – 3.55 (m, 4H), 3.42 – 3.34 (m, 2H), 2.51 (d, J = 3.7 Hz, 4H). IR (KBr): 3274.93,

2029.70 cm-1, ESI-MS: expected 1020 found 1020 as [M+Na]+

Mono-6-(p-toluenesulfonyl)-6-deoxy-cyclodextrin, Ts α-CD (16)42.

α-Cyclodextrin (20.0 g, 0.02 mol) was dissolved in water (700 mL) and NaOH (11.68 g,

0.29 mol) was added. The reaction mixture was stirred at 0°C for 2 hr after adding p-

toluene sulfonyl chloride (p-TsCl) (8.0 g, 0.04 mol). p-TsCl (6.0 g, 0.03 mol) was added

to the reaction mixture and stirred for 3 hr at rt. After filtering, the clear filtrate was

neutralized using concentrated HCl and 4.0 M NaOH solution. The solvent was then

reduced to 50 mL in vacuo and precipitated in the cold acetone. The crude product was

22

collected by filtration and rinsed with acetone before purification by recrystallization in

water (x4, ~50 mL) to afford the product as a white solid (61.43 % purity) in 17.5 %

yield (4.051 g, 3.59 mmol). 1H NMR (500 MHz, DMSO-d6) δ 7.49 (d, J = 7.7 Hz, 2H),

7.12 (d, J = 7.7 Hz, 2H), 5.51 (s, 10H), 5.45 (d, J = 2.7 Hz, 10H), 4.79 (d, J = 3.4 Hz,

10H), 4.52 (s, 10H), 3.76 (s, 10H), 3.69 – 3.53 (m, 35H), 3.38 (d, J = 18.6 Hz, 17H), 3.27

(s, 14H). 13C NMR (126 MHz, DMSO-d6) δ 128.58, 125.97 , 102.31 , 82.52 , 73.72 ,

72.55 , 72.51 , 60.46. IR (KBr): 3295.25, 1149.57, 1020.30 cm-1 ESI-MS (m/z): expected

1150, found 1150.6 as [M+Na]+

O

OH

HOO

O

OOH

OH

OH

O

O

OH OH

HO

O

O

OOH

OHHO

OOH

HO

HO

O

O

OHHO

OH

O

S

O

O

H2O, 24 hr, 100 oCO

OH

HON3

O

OOH

OH

OH

O

O

OH OH

HO

O

O

OOH

OHHO

OOH

HO

HO

O

O

OHHO

OH

O

NaN3

16 17

Mono-6-azido-6-deoxy-cyclodextrin, N3 α-CD (17)42.

Ts α-CD 16 (64.4 % purity, 4.05 g, 3.59 mmol) was dissolved in water (220 mL) and

sodium azide (4.2 g, 64.7 mmol) was added to the solution before heating at refluxed for

24 hr. The reaction mixture was cooled down to rt and the volume of the solution was

reduced in vacuo. The product was precipitated in the cold acetone, filtered, and rinsed

with acetone to afford the crude product. This crude product was purified by

recrystallization in hot water (20 mL) to get the product.

23

Mono-6-amino-6-deoxy-cyclodextrin, NH2 α-CD 42.

N3 α-CD 17 (2.0 g, 2 mmol) was dissolved in a 50:50 mixture of MeOH/H2O before

addition of Pd/C (wet) (0.5 mmol). A balloon filled with H2 gas was attached to the top of

the flask and the reaction flask was evacuated and purged with hydrogen three times

before stirring the reaction for 48 hr at rt under H2. Pd/C was removed by filteration

through Celite and the volume of the collected filtrate was reduced under vacuum. The

white solid was dried under vacuum overnight.

Poly(ethylene glycol)-bis-carboxylic acid (20)

To a 500 mL rb flask with a stir bar, Jones reagent (2.0 M CrO3 in aqueous H2SO4; a: 15

mL; b: 8 mL; c: 4.6 mL) was added. Poly(ethylene glycol) (10g, a:10 kDa, 1.0 mmol;

b:20 kDa, 0.5 mmol; c:35 kDa, 0.2 mmol) was dissolved in acetone (250 mL) and added

to the rb flask containing Jones reagent dropwise over 2 hr. The reaction mixture was

stirred for another 30 min after the addition was complete. Excess Jones reagent was

quenched by adding 3 mL of isopropanol until the reddish color disappeared. The

solution was transferred to another 500 mL rb flask and rinsed with isopropanol (50 mL).

24

The solution was concentrated in vacuo and re-dissolved in water (250 mL) and MeOH

(360 mL) before extracting the solution with DCM (x4, 120 mL). The combined organic

layers were dried over Na2SO4 and concentrated in vacuo. The concentrated solution was

precipitated into cold diethyl ether (x2, ~800 mL) before gathering the precipitate by

filtration and drying under vacuum to afford the poly(ethylene glycol)-bis-carboxylic acid

product as a white powder. (a: 9.0 g, 90 %; b: 8.3 g, 83 %; c:5.9 g, 84 %) The presence of

the carboxylic acid functional group was confirmed by staining with bromocresol green

reagent.

Poly(ethylene glycol)-di-methanesulfonate (21).

Poly(ethylene glycol) (15 g, a: 10 kDa, 1.5 mmol; b: 20 kDa, 0.75 mmol; c: 35 kDa, 0.43

mmol) was dried azotropically in toluene and dried under vacuum overnight. PEG was

dissolved in dry DCM (100 mL) and cooled to 0°C in an ice bath. Triethylamine (a: 31.2

mL, b: 15.6 mL, c: 8.91 mL) was added to the solution and methanesulfonyl chloride (a:

30.3 mL, b: 15.2 mL, c: 8.65 mL) was added. The reaction mixture was stirred for

additional 25 min at 0°C before filtering and evacuating the DCM in vacuo. The product

was precipitated by adding the concentrated reaction mixture to cold diethyl ether (1000

mL) dropwise and stirring for 30 min in the ice bath. The precipitate was filtered and

dried under vacuum to yield a product as a white powder. (a: 19.12 g, b: 16.24 g, c: 22.30

g).

25

Poly(ethylene glycol)-di-azide (22).

Poly(ethylene glycol)-di-methanesulfonate 21 (a: 19.12 g, 1.88 mmol; b: 16.24 g, 0.81

mmol; c: 22.3 g, 0.63 mmol) was dissolved in DMF (100 mL) in a 250 mL rb flask.

Sodium azide (a: 1.22g, 18.8 mmol; b: 0.52 g, 8.10 mmol; c: 0.41 g, 6.34 mmol) was then

added to the solution and the rb flask was purged with N2. The reaction mixture was

stirred at 70℃ for 72 hr under N2. DMF was evaporated in vacuo and the reaction

mixture was re-dissolved in DCM (~200 mL) and filtered. The filtrate was washed with

H2O (x2, 150 mL) and MeOH (~50 mL) was added to enhance the separation. The

combined aqueous layers were washed with DCM (x2, 150 mL) and the combined

organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The crude

product was re-dissolved in DCM (50 mL) and precipitated in cold diethyl ether (1000

mL), filtered and dried under vacuum to afford a white powder as a product. The

presence of azide was confirmed by staining with a 20% triphenylphosphine solution and

ninhydrin solution. (a: 10.1 g, b: 12.1 g, c: 9.3 g).

Poly(ethylene glycol)-bis-amine (4).

Poly(ethylene glycol)-di-azide 22 (a: 10.1 g, b: 12.1 g, c: 9.3 g) and triphenylphosphine

(a: 1.58 g, 0.94 g, 0.42 g) were dissolved in methanol (100 mL) in a 250 mL rb flask. The

rb flask was evacuated and purged with N2 and the solution was stirred for 48 hr at 75°C

26

under N2. Ammonium hydroxide solution (35 %, 16 mL) and water (16 mL) were then

added to the solution and stirred for an additional 2 hr. The reaction mixture was filtered

through a pad of active carbon and Celite by rinsing the pad with methanol. The filtrate

was dissolved in DCM (20 mL) and reduced in vacuo. The product was precipitated in

cold ether, filtered and dried in vacuum to give the product as a white powder (a: 6.28 g,

b: 6.13 g, c: 6.12 g). The presence of an amine group was confirmed by staining with

ninhydrin solution.

Poly(ethyleneglycol)-bis-alkyne (3).

Poly(ethyleneglycol) (a: 3.4 kDa, 5.0 g, 1.47 mmol; b: 10 kDa, 5.0 g, 0.5 mmol, c: 20

kDa, 5.0 g, 0.25 mmol) was dissolved in DCM (25 mL) and THF (15 mL) in an dried

250mL rb flask with a stir bar. HOBt (5.0 equi, a: 994 mg, b: 337 mg, c: 169 mg) and

propargylamine (16.0 equi, a: 1.507 mL, b: 0.512 mL, c: 0.256 mL) were added and

dissolved followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (8.0 equi, a: 2.26

g, b: 0.77 g, c: 0.38 g). The solution was evacuated and filled with N2. N,N-

Diisopropylethylamine (10.2 equi, a: 1.90 g, b: 0.65 g, c: 0.32 g) was added via gas tight

syringe and additional pyridine (30 mL) was added to be fully solubilized. The reaction

mixture was stirred at rt under N2 for 48 hr. The solution was concentrated in vacuo and

re-dissolved in water (120 mL) and MeOH (250 mL). The solution was rinsed with DCM

(x4, 100 mL) and dried over Na2SO4. The combined organic layers were concentrated in

vacuo and precipitated into cold diethyl ether (x2, ~500 mL). The precipitate was filtered

27

and dried under vacuum to give the product as a white powder (a: 3.988 g, b: 4.129 g, c:

4.197 g). 1H NMR (400 MHz, CDCl3) δ 4.09-3.63 (ethylene glycol chain), 2.84 (alkyne

proton), 2.24.

Modification of PRTx with protected NTA (6, 7).

To a 100 mL rb flask, dried Compound 5 (a: 3.8 kDa, b: 122.83 kDa, 800 mg) and dry

DMSO (10 mL) were added. CDI (10 equiv/CD, a: 0.36 g; b: 0.95 g) in dried DMSO (10

mL) was added directly with a glass syringe. The flask was evacuated and purged with N2

(x3) before attachment of a N2 balloon to the top of the flask and addition of DIEA (10

equiv/CD, a: 0.40 mL; b: 1.04 mL) by glass syringe. The reaction mixture was stirred at

rt for 6 hr under N2 and then Lys-NTA-OTMS (10 equiv/CD, a: 1.31 g; b: 2.29 g) or Lys-

NTA-OtBu3 (10 equiv/CD, a: 0.96 g; b: 2.51 g) was added directly. The flask was

evacuated and purged with N2 again and the solution was stirred at rt for an additional 24

hr under N2. The solution was dialyzed against MeOH (3 days) and H2O (3 days) before

28

lyophilization to afford 6a (0.626 g), 7a (0.146 g), 6b (0.765 g), and 7b (0.702 g) as

orange colored powders. 6a: 1H NMR (400 MHz, DMSO-d6) δ 5.47 (d, J = 32.3 Hz,

11H), 4.78 (s, 6H), 4.06 (d, J = 9.0 Hz, 13H), 3.49 (d, J = 1.3 Hz, 113H), 1.53 (s, 9H),

1.35 (s, 11H), 0.90 (d, J = 10.6 Hz, 14H).;7a 1H NMR (400 MHz, DMSO-d6) δ 5.47 (d, J

= 34.7 Hz, 11H), 4.79 (s, 6H), 3.49 (s, 96H), 2.91 (s, 2H), 1.38 (s, 50H).; 6b:1H NMR

(400 MHz, DMSO-d6) δ 5.48 (d, J = 34.5 Hz, 11H), 4.79 (s, 6H), 4.42 (s, 8H), 4.06 (d, J

= 8.9 Hz, 10H), 3.49 (s, 78H), 2.90 (s, 2H), 1.45 (d, J = 72.8 Hz, 9H), 0.92 (t, J = 8.5 Hz,

11H).; 7b: 1H NMR (400 MHz, DMSO-d6) δ 5.47 (d, J = 34.1 Hz, 11H), 4.79 (s, 6H),

4.48 (s, 6H), 3.49 (s, 141H), 2.90 (s, 3H), 1.38 (s, 59H).

Deprotection of TMS modified NTA-PRTx (8).

To a 50 mL rb flask, TBAF (10 equi/TMS, a: 0.32 mL, b: 0.23 mL) in THF (1 M) was

added. THF was evaporated in vacuo and dried DMSO (5 mL) was added. Compound 6

(a: 16.73 kDa, 0.5 g; b: 117 kDa, 0.5 g) was added and the reaction mixture was stirred

for 2 hr at rt under N2. The reaction mixture was dialyzed against DMSO (2 d), MeOH (3

d), and H2O (3 d) before lyophilization to afford the product (a: 0.216 g, b: 0.263 g). 6a:

1H NMR (400 MHz, DMSO-d6) δ 5.44 (s, 8H), 4.79 (s, 6H), 3.44 (d, J = 47.8 Hz, 368H),

1.56 (s, 37H), 1.31 (d, J = 7.3 Hz, 50H), 0.93 (t, J = 7.4 Hz, 47H); 6b: 1H NMR (400

29

MHz, DMSO-d6) δ 5.52 (s, 12H), 4.79 (s, 8H), 3.43 (d, J = 48.3 Hz, 210H), 2.07 (s, 3H),

1.55 (s, 17H), 1.29 (q, J = 7.4 Hz, 21H), 0.92 (t, J = 7.3 Hz, 21H).

Deprotection of OtBu3 modified NTA-PRTx (8).

Compound 7 (a: 12.1 kDa, 0.162 g, b: 102.9 kDa, 0.5 g) was dissolved in dry DCM (10

mL) before addition of Et3SiH (3.3 equiv/NTA, a: 0.1 mL; b: 0.13 mL) and β-

mercaptoethanol (3.3 equiv/NTA, a: 0.1 mL, b: 0.1 mL) to the solution. The flask was

cooled to 0°C with an ice bath and TEA (10mL) was added dropwise over 10 min. The

reaction mixture was stirred for 24 hr at rt under N2 before dialyzing against DMSO (2 d),

MeOH (3 d), H2O (3 d) and lyophilizing to afford the product (a: 0.063 g, b: 0.208 g). 8a:

1H NMR (400 MHz, DMSO-d6) δ 5.45 (s, 10H), 4.79 (s, 6H), 4.44 (s, 4H), 3.77 – 3.41

(m, 119H), 2.07 (s, 5H), 1.28 (d, J = 30.7 Hz, 13H), 0.86 (s, 6H).; 8b: 1H NMR (400

MHz, DMSO-d6) δ 5.52 (s, 9H), 4.79 (s, 6H), 4.43 (s, 6H), 4.12 (s, 4H), 3.49 (t, J = 2.4

Hz, 130H), 1.35 (s, 13H), 0.87 (s, 3H).

30

Synthesis of polyrotaxanes threaded with unmodified α-cyclodextrin (9).

α-Cyclodextrin (α-CD) (5.33 g, 5.48 mmol) was dissolved in water (35 mL) before

addition of poly(ethylenglycol)-bis-alkyne 3 (0.5 g, a: 3.4 kDa, b: 10 kDa, c: 20 kDa).

The mixture was bath sonicated (PW:100 %, frq: 37 kHz) for 1 hr to form a white gel

within a few minutes. This solution was stirred at rt overnight before addition of 5-

azidoflurescein (dissolved in DMSO), and stirring for 10 min. CuSO4 solution (1.0 M)

and 1.0 M sodium-L-ascorbate solution were added via gas tight syringe and this solution

was stirred for another 48 hr at rt. The crude product was centrifuged (7000 rpm, 5 min),

decanted, and re-suspended in 5% EDTA solution and then re-centrifuged. This process

was repeated twice to remove any remaining Cu2+ and Cu+. The material was dialyzed

against water (MWCO: 12-14 kDa, Fisher) for 7 days to remove any unreacted materials.

9a 1H NMR (500 MHz, DMSO-d6) δ 6.21 (s, 0.17H), 6.11 (s, 0.11H), 5.52-5.43 (m,

11.4H), 4.79 (s, 6H), 4.48 (s, 6.3H), 3.79-3.28 (m, 72.3H).; 9b 1H NMR (500 MHz,

DMSO-d6) δ 5.45 (s, 4.05H), 5.43 (s, 3.8H), 5.43 (s, 6H), 4.79 (s, 5.28H), 3.76-3.28 (m,

72.59H).; 9c 1H NMR (500 MHz, DMSO-d6) δ 5.45 (s, 2.80H), 5.43 (s, 8.17H), 4.79 (s,

6H), 4.78 (s, 5.98H), 3.76-3.28 (m, 64.10H).

31

Modification of Polyrotaxanes with 2, 2’-(Ethylenedioxy)bis(ethylamine) (10).

Polyrotaxane 9 (0.5 g, a: 0.02 mmol; b: 0.007 mmol; c: 0.004 mmol) was dissolved in

dried DMSO (15 mL). The solution was bath sonicated (PW: 100 %, frq: 37 kHz) to

assure complete dissolution. Triethylamine (5.0 equiv, a: 215 mg; b: 223 mg; c: 214 mg)

was added, followed by carbonyldiimidazole (2.5 equiv, a: 172 mg; b: 179 mg; c: 171

mg). The solution was evacuated and purged with N2 three times and stirred for 24 hr at rt

under N2. 2, 2’-(Ethylenedioxy)bis(ethylamine) (50.0 equiv, a: 3.14 g, b: 3.27 g, c: 3.13 g)

was added by gas tight syringe and the reaction mixture was stirred for an additional 24

hr at rt under N2. The solution was dialyzed against water for 7 days to removed

unreacted materials and lyophilized to afford a product as an orange colored powder (a:

0.259 g, b: 0.215 g, c: 0.302 g). 10a 1H NMR (500 MHz, DMSO-d6) δ 6.69 (s, 1H), 5.36

(t, J = 4.9 Hz, 1H), 4.85 (s, 6H), 4.53 (d, J = 5.9 Hz, 3H), 3.86 – 3.42 (m, 143H), 2.79 (s,

2H), 2.67 (d, J = 2.0 Hz, 1H), 2.15 – 1.97 (m, 1H), 1.27 (s, 7H), 0.89 (s, 1H).; 10b 1H

NMR (500 MHz, DMSO-d6) δ 7.58 (dd, J = 12.1, 7.2 Hz), 5.28 (s, 2H), 4.76 (s, 6H), 4.45

(d, J = 5.9 Hz, 2H), 3.80 – 3.38 (m, 105H), 2.71 (s, 3H), 2.60 (s, 1H), 2.33 (s, 1H), 1.95

(s, 2H), 1.41 (s, 1H), 1.19 (s, 7H), 0.81 (s, 2H). 10c 1H NMR (500 MHz, DMSO-d6) δ

8.84 (s), 8.46 (s), 8.29 (s), 7.06 (s), 6.70 (d, J = 8.8 Hz, 0H), 5.66 (s, 1H), 5.50 (s, 1H),

4.80 (s, 3H), 4.61 – 4.23 (m, 2H), 3.51 (s, 39H), 2.75 (s, 1H).

32

Synthesis of Azido α-CD cyclodextrin (18)37.

Compound 3 (1.5 g, 1.5 mmol) was dissolved in water (4 mL) in an Erlenmeyer flask.

For better suspension of the N3-α-CD solute, the solution was stirred for 1 hr at rt and

bath sonicated for 1 hr (PW:100%, frq: 37 kHz). Bis-amine PEG (0.3 g, 0.03 mmol) was

added to the solution and was stirred for 1 hr at rt, bath sonicated for 1 hr (PW: 100 %,

frq: 37 kHz), and stirred overnight at rt. The solution became cloudy as the

pseudopolyrotaxane precipitated from water. The TNBS-ONa solution was neutralized to

pH~8 by adding sodium bicarbonate. The reaction mixture was stirred overnight at rt.

The entire reaction mixture was dialyzed against H2O for a week, and washed with H2O

by centrifugation (8000 rpm, 6 min) until the supernatant became clear. The pellet was

lyophilized to afford the product as a yellowish orange powder. (9.7 mg) 1H NMR (400

MHz, DMSO-d6) δ 8.6 (m), 5.52 (d, J = 7.1 Hz, 6H), 5.44 (d, J = 2.8 Hz, 6H), 4.80 (d, J

= 3.4 Hz, 6H), 4.48 (d, J = 5.8 Hz, 6H), 3.77 (t, J = 9.1 Hz, 7H), 3.71 – 3.55 (m, 21H),

3.54 – 3.48 (m, 26H), 3.33 (d, J = 0.8 Hz, 126H), 1.23 (s, 2H), 0.85 (s, 2H).

33

CHAPTER 3. THE FORMATION OF THE GOLD NANOPARTICLE CROSS-LINKED POLYROTAXANE NETWORK

3.1 Introduction

The polyrotaxane architecture can improve the efficiency of single particle cryo-

EM analysis for protein structure determination because the lateral and rotational

freedom of the α-CD units along the polyrotaxane can provide various orientations of the

captured protein targets. In addition, the affinity architecture encourages high

concentration of the target onto the protein scaffold, which enables analysis of multiple

copies of the target.

Furthermore, cross-linking of polyrotaxane into a network as illustrated in Figure

3.1, can allow much higher concentration of the targeting protein to be captured and

analyzed when compared to a single strand of the polyrotaxane. Meanwhile, use of gold

nanoparticles (AuNP) as an electron dense cross-linking material facilitates finding the

protein target in the TEM experiment at low magnification quickly which can minimize

the degradation of the sample by electron beam. AuNP provides easily identified

materials as a boundary which can guide one to search for the bound proteins. By

modifying its surface thiol groups, gold nanoparticle can also provide multiple binding

sites for grafting cross-linkers for polyrotaxanes.43 44-47

n

ad

B

F

Figure 3.1

There

etwork stru

dsorbate spe

Blodgett tec

Figure 3.2 T

The schema

have been

uctures. So

ecies 49, irr

chniques 51-

he interfacia

atic illustrati

n various ap

far, the agi

adiation wit

-54 have b

al reaction se

ion of gold nnetwork

pproaches to

itation of b

th intense p

been used

etup for goldnetwork

nanoparticle k

o fabricate

biphasic liqu

pulsed laser

for AuNP-

d nanoparticlk

cross-linked

gold nanop

uid mixtures

r sources 50

-network sy

le cross-link

d polyrotaxa

particles to

s 48, additio

, and Langm

ynthesis.55

ked polyrotax

34

ane

form

on of

muir-

Gold

xane

35

nanoparticle networks have been formed using flexible and covalently cross-linked gold

nanoparticle linked by dodecanethiol in hexanes on floating films of the nanoparticles.52

Duan and coworkers reported the use of amphiphilic gold nanoparticles with mixed

polymer brush coatings which can be spontaneously assembled into 2D arrays at the oil-

water interface.56

In this study, an AuNP cross-linked PRTx 2D network was synthesized using an

interfacial reaction approach with an aqueous gold nanoparticle solution in the top layer

and the pseudopolyrotaxane solution in dichloromethane in the bottom layer as illustrated

in Figure 3.2. Previously, the synthesis of gold nanoparticle films at the water-organic

solvent interface was successfully performed by Malm and coworkers in 2006. 57

3.2 Result and Discussion

To enhance of the efficiency in protein structure determination capacity of SPA

cryo-EM, gold nanoparticle cross-linked polyrotaxane network was synthesized. Both

ends of bis-amine PEG were modified with NHS-thioctic acid, and the thioctic acid-

pseudopolyrotaxane intermediate was produced by threading this polymer with multiple

α-CDs. These pseudopolyrotaxanes were cross-linked with gold nanoparticles at the

interface of immiscible solutions. Modification of the gold nanoparticles by surface-

deposition of pseudopolyrotaxanes to form a gold nanoparticle film was achieved by

depositing a pseudopolyrotaxane-containing solution onto the AuNP solution and letting

it dry.57 In order to stably keep positioning and retaining the tiny TEM grid within the

interface, tabbed TEM grids were chosen. Each tab was carefully bent into 90° and held

by

gr

F

w

o

th

w

W

fi

p

pr

ex

fo

so

su

y tweezers q

rid was coat

Figure 3.3 UV

As a

without pseud

f cross-linke

he samples p

was placed in

While the pu

ield of view

articles in t

roduct still

xistence of

orm the AuN

olutions is U

urface modi

quite steadily

ted with form

V-Vis data f

a control exp

dopolyrotaxa

ed AuNPs w

produced by

n contact wit

ure DCM sam

w, the sample

the field of

needs mor

the pseudop

NP cross-lin

UV-Vis spe

ifications an

y with a clam

mvar and car

for gold nano

periment, the

ane in the di

was counted

y interfacial

th a DCM la

mple showe

es produced

view at the

re character

polyrotaxane

nked network

ectroscopy. T

nd sizes of

mp ring. Als

rbon.

oparticle lay

e bottom lay

ichlorometha

and compare

reaction of

ayer containi

ed irregularly

using pseud

e same mag

rization, it

e scaffold en

k. A commo

This techniq

f AuNP pro

so, to preven

ers of AuNP

yer of the bip

ane layer. Th

ed with the

f gold nanop

ing the pseu

y spaced 34

dopolyrotax

gnification.

was provis

nabled gold

on analysis m

que allows

oduct.58 In

nt the loss of

P cross-linke

phasic mediu

hen, the app

number of A

particles in t

udopolyrotax

481 gold nan

xane in DCM

Therefore,

sionally con

nanoparticl

method for g

the investig

this experi

f the network

ed PRTx netw

um was prep

proximate nu

AuNPs prese

the top layer

xane (Figure

noparticles i

M solution 1

even though

ncluded tha

le crosslinkin

gold nanopa

gation of pa

iment, Nano

36

k, the

work.

pared

umber

ent in

r that

e 3.4).

in the

18690

h the

at the

ng to

article

article

odrop

37

analysis which is a micro-volume UV-Vis spectrometry instrument appropriate for low

sample volume experiments, was attempted by washing out the network product from the

TEM grid with water. Unfortunately, this approach did not work due to an inadequate

sample concentration. Therefore, an indirect method that measured the changes in

concentration of gold nanoparticle in the top layer as a function of time by UV-vis

spectroscopy was utilized. This is based on the assumption that as more AuNP were

exploited for pseudopolyrotaxane crosslinking, the concentration of the gold nanoparticle

on the top layer would be decreased. Over a 60 min period, it was found that the gold

nanoparticle absorbance intensity at 523 nm decreased. Since the intensity of the UV-Vis

spectra is related to the concentration of gold nanoparticle, 58 the decrease in intensity of

the peak indicated the decrease in the bulk concentration of gold nanoparticles as they

were consumed in the interfacial reaction to form the AuNP cross-linked PRTx network.

Also, the arrangement of gold nanoparticles of AuNP cross-linked α-CD threaded PRTx

in the TEM image was similar to the TEM image of hexane thiol capped gold

nanoparticle network formed by closely packed with Langmuir trough.52 Therefore, the

network formation by interfacial approach enabled the formation of the network although

it needs to be confirmed.

T

w

li

th

Figure 3.4nanopartic

TEM image o

For

with AuNP to

igands. This

he reaction o

4 TEM imagcle cross-linkof the hexan

the next stu

o form the n

can be man

on EM grid.

ges of cross-lked polyrotaxnethiol-cappe

udy, a N3-α-

network in o

nipulated via

linked gold nxane networed gold nano

trough 52

-CD threade

order to bett

a copper free

nanoparticlerk formed byoparticles aft2

ed pseudopo

ter control it

e click reacti

es; A: TEM iy interface mter crosslink

olyrotaxane

ts stoichiom

ion, which c

image of golmethod; B: Tking by Lang

was cross-li

metry with pr

can also faci

38

ld The gmuir

inked

rotein

ilitate

a

ar

g

N

ta

la

m

ab

d

im

Figure 3.5conditions;

added to the nanoparticl

image of

The

rrangement

old nanopar

NTA of the

agged T7 ph

ayer as a neg

meaning that

bsence of th

issolved in D

mages showe

5 TEM imageA: TEM imbottom layees cross-linkf T7 phages

network w

of gold nano

rticle networ

AuNP-PRT

hages. (Figu

gative contro

t there was

he pseudopol

DCM and ad

ed the forma

es of AuNP age of the goer (i.e. pseudked with α-ccaptured on

was formed

oparticles of

rks formed u

Tx network w

ure 3.5) As a

ol, only a fe

neither a PR

lyrotaxane. A

dded to the b

ation of the n

from the intold nanopart

dopolyrotaxayclodextrinthe gold nan

at the int

f the TEM im

using a Lang

was activate

a result, wh

ew well-sepa

RTx networ

Also, when

bottom layer

network, but

erfacial netwticles when dane-free contthreaded psenoparticle cr

terface of i

mages was s

gmuir trough

ed with Ni2

hen only DC

arated gold n

rk formed no

α-CD threa

r, the arrange

t the absence

work setup udichloromettrol); B: TEMeudopolyrotross-linked p

immiscible

similar to th

h as reported

2+ to enable

CM was add

nanoparticle

or any virus

ded pseudop

ement of Au

e of azide lin

under differethane alone wM image of axane; C: TEpolyrotaxane

layers, and

he TEM ima

d by Dahl. 52

e capture of

ded at the bo

es were obse

s captured i

polyrotaxane

uNPs on the

nked NTA o

39

ent was gold EM e

d the

age of

2 The

f His-

ottom

erved,

in the

e was

TEM

on the

40

α-CD did not promote capture of T7 phage particles. However, when NTA-N3-α-CD

threaded pseudopolyrotaxane was dissolved in DCM, the network was formed by cross-

linking with gold nanoparticles as the density of gold nanoparticles observed in the same

magnification with EM image of α-CD threaded pseudopolyrotaxane was similar. Also,

captured T7 phages were observed in this case. Therefore, N3 α-CD threaded

pseudopolyrotaxane formed a network by crosslinking with gold nanoparticles via

biphasic reaction set up, such that copper free click reaction and Ni2+ activation on the

EM grid worked to capture the His-tagged protein targets.

3.3 Conclusion and Future Plans

In the extension of synthesis of single stranded PRTx for efficient SPA Cryo-EM

analysis for protein structure determination, AuNP cross-linked PRTx network was

formed and the azide was conjugated with NTA through copper free click reaction and

T7 phage was captured specifically on the EM grid.

In future studies, an imidazole wash of the network will allow the confirmation of

Ni2+ capturing of the targeting proteins on the affinity polymers. Also, substitution of

thioctic acid with an excess amount of mercaptoacetic acid will be performed to analyze

the polyrotaxane composition of the network. In addition, the different types of network

can be designed with trinitrophenyl-antibodies for better selective and efficient biding of

TNBS-capped polyrotaxanes for the network synthesis.

41

3.4 Experimental Details

2,5-Dioxopyrrolidin-1-yl 5-(1,2-dithiolan-3-yl)pentanoate (19)59.

To a 250 mL rb flask, lipoic acid (2.5 g, 12.12 mmol) and N-hydroxysuccinimide (1.53 g,

13.33 mmol) were dissolved in DMF (60 mL). 1-Ethyl-3-(3-dimethylamineopropyl)

carbodiimide•HCl (2.06 g, 13.33 mmol) was added to the solution and the flask was

evacuated and purged with Ar. The reaction mixture was stirred for 4 hr at rt under Ar.

The solution was diluted with EtOAc (300 mL) and washed with H2O (250 mL),

saturated NaHCO3 (250 mL), and H2O (x2, 250 mL). The combined organic layers were

dried over MgSO4, filtered, and concentrated in vacuo to give a yellowish solid as a

product. 1H NMR (400 MHz, CDCl3) δ 2.84 (s, 3H), 2.66 – 2.40 (m, 3H), 1.98 – 1.88 (m,

1H), 1.84 – 1.65 (m, 4H), 1.63 – 1.51 (m, 2H).

Poly(ethylene glycol)-di-lipoic acid (20).

To a 50 mL rb flask, poly(ethylene glycol)-bis-amine 4 (10 kDa, 2.0 g) and NHS-thioctic

acid 19 (0.606 g, 2.0 mmol) were dissolved in DCM (10 mL). The flask was purged with

N2 and N, N-diisopropylethylamine (0.360 mL, 0.002 mmol) was added to the solution

dropwise. The reaction mixture was stirred for 6 d at rt under Ar. The solvent was

42

evaporated in vacuo and re-dissolved in DCM (10 mL). The solution was precipitated in

cold ether (200 mL), filtered, and dried under vacuum to afford a product as a pale

yellowish powder (1.95g). 1H NMR (400 MHz, CDCl3) δ 3.59 (s, 420H), 3.15 – 3.03 (m,

4H), 2.80 (s, 2H), 2.58 (s, 4H), 1.38 (s, 7H).

Bicyclo[6.1.0]non-4-yn-9-ylmethyl (4-nitrophenyl) carbonate (21) 60.

In a 5 mL conical vial, bicyclo[6.1.0]non-4-yn-9-ylmethyl (2-(2-(2-

aminoethoxy)ethoxy)ethyl)carbamate (50 mg, 0.33 mmol) was dissolved in DCM (2.5

mL) before addition of 4-nitrophenyl chloroformate (74 mg, 0.36 mmol) and pyridine (68

μL, 0.84 mmol). After stirring the reaction for 30 min at rt, the reaction mixture was

quenched by adding sat. ammonium chloride (2.5 mL). The solution was extracted with

DCM (x3, 2.5 mL) and the combined organic layers were dried over Na2SO4, filtered,

and concentrated in vacuo. The crude product was purified by a silica column flash

chromatography (4:1 Hex/EtOAc) and dried to afford a pure product in 53% (26.5 mg,

0.09 mmol). 1H NMR (400MHz, CDCl3) δ 8.28-8.26 (m. 2H), 7.39-7.37 (m, 2H), 4.41-

4.39 (m, 2H), 2.33-2.03 (m, 6H), 1.27-1.24 (m, 2H), 1.23-0.85 (m, 6H).

43

Bicyclo[6.1.0]non-4-yn-9-ylmethyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate

(22)61.

To a solution of bicyclo[6.1.0]non-4-yn-9-ylmethyl (4-nitrophenyl) carbonate (0.313 g,

0.281 mmol) in DMF (8.85 mL) was added 2, 2’-(ethylenedioxy)bis(ethylamine) (0.246

mL, 1.684 mmol) followed by triethylamine (0.117 mL, 0.843 mmol). The reaction

mixture was stirred for 1 hr at rt before removal of the solvent in vacuo and solubilization

of the residue with DCM (8.85 mL). The solution was extracted with 1N NaOH solution

(x2, 2.21 mL) followed by H2O (2.21 mL) and the combined aqueous layers extracted

with EtOAc (x3, 2.21 mL). The combined organic extracts were washed with brine (2.21

mL), dried over MgSO4, concentrated in vacuo, and dried under vacuum overnight to

afford the product (0.032 g, 0.099 mmol). There was no further purification step.

Bicyclo[6.1.0]non-4-yn-9-ylmethyl (2-(2-(2-((((2,5-dioxopyrrolidin-1-

yl)oxy)carbonyl)amino)ethoxy)ethoxy)ethyl)61.

To a 5 mL conical vial, N,N'-disuccinimidyl carbonate (0.03 g, 0.119 mmol) followed by

dry triethylamine (48.4 μL, 0.339 mmol) was added to a stirring solution of

bicyclo[6.1.0]non-4-yn-9-ylmethyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (0.032

g, 0.099 mmol) in dry THF (1 mL). The vial was evacuated and purged with Ar three

ti

in

st

23

1

tr

T

d

an

(0

m

1

so

st

G

In

3

b

imes. The re

n vacuo to a

tep.

314, DIEA

THF, 17 hr, rt

-(Bicyclo[6.

rioxa-4,13,1

To a stir

ioxopyrrolid

nd di-tert-

0.036 g, 0.1

mmol) was ad

7 hr at rt u

olvent was r

tep.

Gold nanopa

n a 10 mL E

mL of DCM

ath sonicate

action mixtu

afford a pro

OO

NH

.1.0]non-4-y

15,19-tetraaz

rred soluti

din-1-yl)oxy

-butyl 2,2

146 mmol) i

dded. The co

under Ar and

removed in

article polyr

Erlenmeyer f

M. The solu

ed for 30 m

ure was stirre

duct (0.068

O OHN

O

yn-9-yl)-18-c

zahenicosan

on of b

)carbonyl)am

2'-((6-amino-

in dry THF

onical vial w

d monitoring

vacuo to ob

rotaxane ne

flask, N3 α-C

ution was sti

min (pw: 1

ed for 17 hr

g, 0.146 m

HN N

O

OOH

OHO

O OH

carboxy-19-

n-21-oic acid

icyclo[6.1.0

mino)ethoxy

-1-(tert-buto

, dry N,N-d

was evacuate

g the reactio

btain the pro

twork synth

CD or α-CD

irred at rt fo

00%, frq: 3

at rt under A

mmol). There

-(carboxym

d (23) 62.

0]non-4-yn-9

y)ethoxy)eth

oxy)-1-oxohe

diisopropylet

ed and purge

on by TLC

oduct. There

hesis (AuNP

D (0.036g, 0.

or 30 min to

37 MHz). T

Ar. The solv

e was no fur

methyl)-3,14-

9-ylmethyl

hyl) (0.068

exan-2-yl)az

thylamine (0

ed with Ar b

(5% MeOH

e was no fur

P PR netwo

0375mmol)

o disperse th

Thioctic aci

vent was rem

rther purific

-dioxo-2,7,1

(2-(2-(2-(((

g, 0.146 m

zanediyl)diac

0.025 mL, 0

before stirrin

H in DCM).

rther purific

rk)63.

was dissolv

he α-CD and

id PEG (0.0

44

moved

cation

10-

((2,5-

mmol)

cetate

0.146

ng for

. The

cation

ved in

d then

035g,

0

T

so

v

p

fo

gr

n

w

in

by

th

C

T

n

ex

p

.00075mmo

The reaction

olution. Out

ial and ly

seudopolyro

ormvar and

rid was pl

anoparticle

was left ove

nterface of th

y controlling

he network w

Click reactio

The BCN-EB

etwork TEM

xcess BCN-

aper and rin

l) was added

n mixture b

of 3 mL of

yophilized.

otaxane disso

carbon coat

aced in the

solution (10

rnight to fo

he biphasic s

g the height

was confirme

on on the go

BE-NTA in H

M grid. The

EBE-NTA r

sed with dei

d to the solu

became clou

pseudopoly

To a clea

olved in DCM

ed) was ben

e pseudopo

0 nm, 5.6 x 1

orm the gol

solutions. Th

of the lab ja

ed by TEM.

old nanopar

H2O (50 μL

grid was lef

residue was

ionized wate

ution, sonica

udy as the

yrotaxane sol

an, dry 10

M (3 mL). T

nt to 90° rela

olyrotaxane

1012 particl

ld nanoparti

he grid was

ack. The grid

ticle cross-l

) was added

ft in the 4 °

removed by

er (x2, 20 μL

ated for 1 hr

pseudopoly

lution, 0.1 m

0 dram vi

The tab of th

ative to the

solution la

les/mL) was

icle cross li

lifted to tak

d was dried

linked polyr

d on top of t

°C saturated

y wicking ou

L) which was

r, and stirre

yrotaxane p

mL was trans

ial was ad

he tabbed EM

surface of t

ayer and th

s added on t

inked PRTx

ke off the net

for 2 hr and

rotaxane ne

the AuNP cr

environmen

ut with a sm

s then wicke

d overnight

precipitated

sferred to a

dded the

M grid (500 m

the grid. The

he aqueous

the top of it.

x network a

twork on the

d the formati

etwork.

ross linked P

nt overnight

mall piece of

ed away.

45

at rt.

from

small

dried

mesh,

e EM

gold

This

at the

e grid

ion of

PRTx

t. The

f filter

N

N

n

w

sm

w

T

H

o

en

w

im

Ni2+ chelatio

Ni2+ solution

etwork on th

with deionize

mall piece o

wicking.

The capturin

His10 tagged

f the Ni2+ a

nvironment.

wicked out w

mage was tak

n on the NT

(2 mM, 50

he TEM grid

ed water. Af

of filter pap

ng of T7 pha

T7 phage (1

activated Au

Non-captur

with a small

ken.

TA.

μL) in PBS

d. To preven

fter 2 hr, th

per, followe

age on the a

1x1011 partic

uNP cross-li

red T7 phag

l piece of fi

S buffer was

nt the solutio

he excess Ni

ed by water

affinity grid

cles/mL, 20

nked PRTx.

ge was rinse

lter paper. T

s added to th

on from dryin

i2+ was rem

r addition (x

d.

μL) in PBS

. This was

d with deion

The grid wa

he AuNP cr

ng, a petri d

moved by wi

x3, 20 μL)

buffer was

left at 4 °C

nized water

as dried ove

ross-linked P

dish was satu

cking out w

and remova

added on th

C in the satu

(x3, 20 μL

ernight and

46

PRTx

urated

with a

al by

he top

urated

) and

TEM

47

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47

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45

APPENDICES

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VITA

82

VITA

Minji Ha was born in Busan, South Korea. She received a B.S. in chemistry from

The Pennsylvania State University in University Park, Pennsylvania in 2012. She joined

the Department of Chemistry at Purdue University in West Lafayette, Indiana in 2012.

She chose to pursue Master studies under the excellent mentorship of Professor David H.

Thompson.