Targeted Cancer Therapy Systems:: an in Silico Study Of

Targeted Cancer Therapy Systems: An In Silico Study of Radiohalogenated Ligands in the Estrogen Receptor and the Synthesis of a Molecular Toolkit for the Fabrication of Customizable Nanoparticles

by Kelton K. Barnsley

B.S. in Chemistry, Worcester Polytechnic Institute

A thesis submitted to

The Faculty of the College of Science of Northeastern University in partial fulfillment of the requirements for the degree of Master of Science

December 5, 2014

Thesis directed by

Robert Hanson Professor of Chemistry and Chemical Biology

Dedication

Dedicated to Jennifer Wetherby, who provided encouragement and emotional support when I needed it most. I love you.

Acknowledgements

I would like to thank my advisor, Dr. Hanson, for taking me on as a student and for giving me the opportunity to work on these fascinating projects. I would like to thank Dr. Hanson, Dr.

Ondrechen and Dr. Amiji for serving on my thesis committee and for the time and effort they put into reviewing this document. I would also like to thank Dr. Ondrechen for her help and guidance with the computational study that informed the first half of this thesis. I would like to thank Dr. Amiji and the members of his group, especially Arun Iyer, for helping me to purify and freeze-dry the 4-iodophenyl dextran carbamate on page 49. My thanks also go to John Bottomy for always being willing to set up an infrared spectrometer when I needed one. I would also like to thank my fellow lab group members for their friendship and help: Emily Corcoran, James

Teh, Philip Gauthier, Anna Williams, Nisal Gajadeera, and Lenny Dao.

iii

Abstract of Thesis

Chemotherapy is often limited by off-target toxicity and the development of multi-drug resistance in response to treatment. Strategies which reduce off-target toxicity by passively or actively targeting cancer cells may improve the efficacy of chemotherapy. Herein, two projects relating to targeted therapy are described. In the first project, the binding modes of 1,1-bis(4- hydroxyphenyl)-2-phenylethylenes (THPEs), a class of synthetic estrogens previously developed by our group, in the human estrogen receptor α-ligand binding domain were studied using molecular modeling programs YASARA AutoDock and Schrodinger Glide. The results were internally consistent and supported the observation that a bromine or iodine atom at the 2- position of the THPEs contributes positively to their binding in the estrogen receptor. In the second project, a “molecular toolkit” approach to the synthesis of multifunctional nanoparticles was envisioned. Our hypothesis was that the physical and chemical properties of the final product could be defined by controlling the types and relative amounts of prefunctionalized polymer units (PPUs) as well as the emulsification conditions. The design and syntheses of heterobifunctional linkers and other components for a preliminary molecular toolkit are reported, and the literature on select α,ω-heterobifunctional aliphatic linkers is examined.

Table of Contents

Dedication ii

Acknowledgements iii

Abstract of Thesis iv

Table of Contents v

List of Figures vii

List of Tables viii

List of Schemes ix

List of Symbols x

Glossary of Terms xi

Introduction 1

Chapter 1: A computational study of the binding modes of 2 1,1-bis(4-hydroxyphenyl)-2-(3-hydroxphenyl)ethylenes in the binding site of ERα-LBD.

Background & Goals 2

Methodology & Experimental Design 5

Results 14

Discussion 25

Summary & Conclusions for Chapter 1 33

Chapter 2: Synthesis of Heterobifunctional Linkers as 35 Components of a Molecular Toolkit for the Fabrication of Customizable Nanoparticles

Background: Functionalized Nanoparticles 35 for Drug Targeting and Drug Delivery

Background: Heterobifunctional Linkers for 37 Drug Targeting and Delivery

Synthetic Goals 47

Results & Discussion 49

Summary & Conclusions for Chapter 2 60

Experimental: Synthetic Procedures for Chapter 2 61

Overall Summary & Conclusions 77

Bibliography 79

Appendix A: Binding Modes of Ligands in the ERα-LBD 92

Appendix B: Chemical Spectra for Chapter 2 144

List of Figures

Figure 1: IBHPE and THPEs 4

Figure 2: Ligands included in computational study 6

Figure 3: Starting poses for ligands 11

Figure 4: Ligplot+ binding mode comparisons 27

Figure 5: Azido and alkynyl linkers for molecular toolkit 48

Figure 6: 35-45 kDa Dextran and 4-iodophenyl dextran 50

carbamate (DoS = 4.42%)

Figure 7: Mechanism of isocyanation by Banwell’s reagent 57

vii

List of Tables

Table 1: Relative binding affinities of BHPEs and THPEs in 4

competitive binding assays with [3H]estradiol

Table 2: YASARA/AutoDock Docking Experiment Results 14

Table 3: AutoDock vs. Glide Docking Experiment Results 16

Table 4: Halogen-contacting Residues and Their Distances 17

from the Halogen/Vinyl Proton

Table 5: Hydrogen Bonds Between Ligand and Receptor 23

Table 6: Dihedral Angles of Rotatable Functional Groups 24

Before and After Docking

viii

List of Schemes

Scheme 1: Synthesis of hydrophilic linkers 51

Scheme 2: Initial proposed pathway to 1-azido-10-isocyanatodecane 53

Scheme 3: Pathways to 1-azido-10-isocyanatodecane 55

Scheme 4: Isocyanation practice pathway 58

Scheme 5: Synthesis of hydrophobic alkynes 59

Scheme 6: Synthesis of 10-azidodecyl 1H-imidazole-1-carboxylate 60

List of Symbols

Å: Angstrom (1*10-10 meters) nm: Nanometers (1*10-9 meters)

°C: Degrees Celsius kDa: KiloDaltons (1 Dalton = 1 gram/mole)

N: Normality (equivalents/liter)

M: Molarity (moles/liter)

δ: Chemical shift ppm: Parts per million

MHz: MegaHertz (1*106 cycles/second) s: Singlet br. s.: Broad singlet d: Doublet t: Triplet m: Multiplet cm-1: Wavenumbers (inverse of the wavelength of infrared light in centimeters) g: Grams ml: Milliliters (1*10-3 liters)

Mn: Number-average molecular weight of a polymer (g/mol) mmol: Millimoles (1*10-3 moles)

J: J-coupling interaction between similar protons in 1H-NMR spectra

Glossary of Terms

MDR: Multi-drug resistance

ERα-LBD: Estrogen receptor alpha ligand binding domain

125IVME2: 17α-E-[ [125I]iodovinyl-11β-methoxyestradiol

123IBHPE: 2-[123I]iodo-1,1-bis(4-hydroxyphenyl)-2-phenylethylene

BrBHPE: 2-bromo-1,1-bis(4-hydroxyphenyl)-2-phenylethylene

BHPE: 1,1-bis(4-hydroxyphenyl)-2-phenylethylene

ITHPE: 2-iodo-1,1-bis(4-hydroxyphenyl)-2-(3-hydroxyphenyl)ethylene

BrTHPE: 2-bromo-1,1-bis(4-hydroxyphenyl)-2-(3-hydroxyphenyl)ethylene

THPE: 1,1-bis(4-hydroxyphenyl)-2-(3-hydroxyphenyl)ethylene

RBA: Relative binding affinity

All-para-ITHPE: 2-iodo-1,1-bis(4-hydroxyphenyl)-2-(4-hydroxyphenyl)ethylene

All-para-BrTHPE: 2-bromo-1,1-bis(4-hydroxyphenyl)-2-(4-hydroxyphenyl)ethylene

All-para-THPE: 1,1-bis(4-hydroxyphenyl)-2-(4-hydroxyphenyl)ethylene

11β4HPE2: 11β-(4-hydroxyphenyl) estradiol

E2: 17β-estradiol (a.k.a. estradiol)

DES: Diethylstilbestrol

PDB: Protein Data Bank

3ERD: PDB code for crystal structure of ERα-LBD in complex with

diethylstilbestrol

1A52: PDB code for crystal structure of ERα-LBD in complex with 17β-estradiol

LPC: Ligand Protein Contact (server)

YASARA: Yet Another Scientific Artificial Reality Application; a suite of integrated

molecular modeling programs

AutoDock: The module within YASARA that simulates docking between ligands and

receptor binding sites

Schrodinger: Another suite of integrated molecular modeling programs

Maestro: The graphical interface of Schrodinger

Glide: The module within Schrodinger that simulates docking between ligands

and receptor binding sites

NaCl: Sodium chloride

PM3: Parameterized Model number 3; a semi-empirical method of calculating

the electronic structure of molecules using quantum mechanics

Yamber 3: A force field used by YASARA/AutoDock in its calculations

Asp: The amino acid aspartate (aspartic acid)

Glu: The amino acid glutamate (glutamic acid)

Trp: The amino acid tryptophan

Leu: The amino acid leucine

Arg: The amino acid arginine

Phe: The amino acid phenylalanine

His: The amino acid histidine

Thr: The amino acid threonine

Ala: The amino acid alanine

Met: The amino acid methionine

Ile: The amino acid isoleucine

xii

RMSD: Root-mean-square distance

Van der Waals radius: The radius of an imaginary hard sphere which represents the volume

occupied by an atom

Hydrogen bond: An interaction between two electronegative atoms in which a hydrogen

atom covalently bonded to one also interacts with a lone pair of electrons

on the other

PPU: Prefunctionalized polymer unit

MFNP: Multifunctional nanoparticle

NP: Nanoparticle

RES: Reticuloendothelial system

MPS: Mononuclear phagocyte system (another term for RES)

PEG: Poly(ethylene glycol)

OEG: Oligo(ethylene glycol)

TsCl: 4-toluenesulfonyl chloride (a.k.a. tosyl chloride)

DMF: N,N-dimethylformamide

DCM: Dichloromethane (a.k.a. methylene chloride)

NMR: Nuclear magnetic resonance

EtOAc: Ethyl acetate

NaH: Sodium hydride

THF: Tetrahydrofuran

RT: Room temperature

FT-IR: Fourier transform infrared spectroscopy

ATR: Attenuated total reflectance

xiii

Pyr.: Pyridine

CH3CN: Acetonitrile

NaN3: Sodium azide

TPG: Triphosgene

HNBoc2: Di-tert-butyl-iminodicarboxylate (a.k.a. di-boc-amine)

Cs2CO3: Cesium carbonate

TFAA: Trifluoroacetic acid

DIEA: N,N-diisoproopylethylamine

Mg(ClO4): Magnesium perchlorate

Tf2O: Trifluoromethanesulfonic anhydride (a.k.a. triflic anhydride)

DMAP: 4-dimethylaminopyridine

Boc2O: Di-tert-butyl dicarbonate

HCl: Hydrogen chloride (hydrochloric acid when in aqueous solution)

TPP: Triphenylphosphine

DIAD: Diisopropyl azodicarboxylate

CDI: 1,1’-carbonyldiimidazole

DI water: Deionized water

MW: Molecular weight

DMSO-d6: Dimethylsulfoxide (hexadeuterated)

CDCl3: Chloroform (deuterated)

Erlenmeyer: A type of glassware with a wide base that tapers to a narrow mouth

RBF: Round-bottomed flask

xiv

Hirsch filtration: A method of filtering off an insoluble solid by the use of vacuum suction

and a specialized filtration device called a Hirsch funnel

Introduction:

There were 14,090,000 cases of cancer and 8,201,000 deaths caused by cancer worldwide in

2012.[1] Although treatment has improved greatly in recent decades, treatment of tumors by chemotherapy often fails in the long term due to two factors: toxicity of the chemotherapeutic compounds which limits the dosage and duration of treatment, and development of multi-drug resistance (MDR) which renders subsequent chemotherapy ineffective.[2-4] What is needed is a way to deliver the desired chemotherapeutic to the target cells such that off-target effects are greatly minimized. Targeted delivery, whether active, passive or a combination of the two, is a strategy that improves the distribution of the active compound, decreases off-target toxicity, and thereby increases the chance of curing the patient before MDR becomes an issue. Some targeted delivery systems may even bypass or overcome the mechanisms of MDR.[5-8] In response to this challenge, many sophisticated targeted drug delivery systems are under development. This thesis describes two projects aimed at the development of tools designed to overcome the challenges facing chemotherapy.

Chapter 1: A computational study of the binding modes of 1,1-bis(4-hydroxyphenyl)-2-(3- hydroxphenyl)ethylenes in the binding site of ERα-LBD.

Background & Goals:

One promising approach for the selective destruction of cancerous cells involves the use of intracellular radiation combined with active targeting of receptors that are overexpressed in cancer cells. Many ovarian and breast cancers overexpress the human estrogen receptor and require its activation for proliferation. In theory, ER-positive cancers should be vulnerable to methods of therapy which target the estrogen receptor. Endocrine therapy is one approach, the basic mechanism of which is to use antiestrogens to influence ER-expressing cancer cells to stop producing estrogen, thereby slowing or halting their growth. However, endocrine therapy, also known as hormone therapy, does not have a high rate of success, even in patients with ER- positive breast cancer.[9-11] A radiotherapeutic approach would depend only on getting the payload to the ER, not on the estrogenic or antiestrogenic activity of the drug.

Auger radiation is a form of radiation consisting of low-energy electrons which have a very short range of damage, making it ideal for intranuclear destruction of the DNA of ER-positive cancer cells.[12, 13] Happily, estrogen receptors have been shown to be located in the nucleus of ER- positive cells.[10, 14, 15] Upon binding an estrogen molecule, the ER-ligand complex binds to

DNA.[10, 16, 17] If the ligand in question is labeled with an Auger-emitting isotope, the resulting proximity between the ER-ligand complex and the DNA of the ER-positive cancer cell

2 will result in damage to the DNA and the death of the cell. The radioiodinated estrogens 17α-E-

[125I]iodovinyl-11β-methoxyestradiol (125IVME2) and 2-[123I]iodo-1,1-bis(4-hydroxyphenyl)-2- phenylethylene (123IBHPE) were evaluated as candidates for radioimaging and/or radiotherapy applications in immature female rats.[10] The purpose for using a different isotope of iodine in each compound was so that their biodistributions could be studied simultaneously in the same rat to eliminate the contribution of differences between subjects to the results. IBHPE had a higher off-target concentration in the blood compared with IVME2, meaning that IBHPE would be a less favorable choice as an imaging agent (since the signal from the blood would obscure the signal from the target tissues). However, IVME2 showed higher off-target tissue concentrations in non-peritoneal ER-expressing tissues (vagina and pituitary), as well as in muscle and brain tissue, than IBHPE. This may be due to nonspecific binding of IBHPE with blood proteins, which keeps it in circulation longer and prevents it from going into off-target tissues.[10] For these reasons IBHPE is a better choice for Auger-based radiotherapy than the steroidal estrogen.

125I-labeled estrogens have been studied as potential radiotherapeutic agents in vitro.[12, 18-20]

However, the half-life of 125I is too long (60 days) to deliver a sufficient number of radioactive decays to cause remission in vivo.[16] By contrast, the half-life of 123I is much shorter (13.2 hours)[21] and lines up well with the biological half-life of these sorts of ligands.[16] 80mBr also has a suitable half-life (4.4 hrs)[22] for radiotherapy in vivo.

The tri-hydroxyphenyl ethylene 2-iodo-1,1-bis(4-hydroxyphenyl)-2-(3-hydroxyphenyl)ethylene

(ITHPE), which was synthesized by members of the Hanson group,[23] has been shown to have significantly better retention in the uterus and ovary than the di-hydroxyphenyl ethylene

IBHPE.[24] As part of a planned study of THPEs by our group, the H- and Br- substituted

THPEs were also synthesized.[23]

Figure 1: IBHPE (left) and THPEs (right).

Compound Relative Binding Affinity (Relative to E2)

BHPE 38%[9]

BrBHPE 44%[9]

IBHPE 14%[9]

THPE 5.5%[23]

BrBHPE 53%[23]

ITHPE 79%[23]

Table 1: Relative binding affinities of BHPEs and THPEs in competitive binding assays with [3H]estradiol. [THPE data is from a study by our group that is currently pending publication].

Having synthesized the THPEs and determined their relative binding affinities, our group was interested in doing an in silico examination of their binding modes in the estrogen receptor. As

Table 1 shows, the RBAs of BrTHPE and ITHPE are at least an order of magnitude higher than the unhalogenated THPE. One of our goals for the in silico study was to see if we could rationalize and understand the data in terms of the orientations of the ligands inside the binding site. Another goal was to compare the predicted binding energies with the relative binding affinities.

Methodology & Experimental Design:

Ligand selection:

The purpose of this study is to examine the binding modes of THPE, BrTHPE, and ITHPE, so those three ligands were naturally included. The di-hydroxyphenyletheylene IBHPE was included since it is the precursor to the THPEs. The THPEs our group synthesized have a hydroxyl group at the 3-position of the phenyl ring at the 2-position of the ethylene core because having it at the 4-position as in the other phenyl rings would make the compound chemically unstable. However, simulated ligands are not limited by real-world chemical stability, so we took this opportunity to also examine the binding modes of the all-para-hydroxy THPEs. We also included 11β-(4-hydroxyphenyl)-estradiol, a steroidal estrogen from a previous study,[25] because we thought that the 4-hydroxyphenyl group at its 11β position may be analogous to the

4-hydroxyphenyl group trans to the halogen/vinyl proton on the THPEs and IBHPE, just as the other two hydroxyphenyl groups roughly resemble the A- and D-rings of the steroid. Estradiol

(E2) was naturally included in our set, as was diethylstilbestrol (DES), a nonsteroidal estrogen with structural similarities to the THPEs.

Figure 2: Ligands included in the in silico study.

Receptor crystal structure sources:

There are two classes of estrogenic compounds under consideration in this study: nonsteroidal, diethylstilbestrol-like compounds, and 11β-(4-hydroxyphenyl)-estradiol, a steroidal estrogen.

Therefore, we chose the crystal structures of the ERα-LBD in complex with diethylstilbestrol

(PDB ID: 3ERD)[26] and 17β-estradiol (PDB ID: 1A52)[27]. Both files were downloaded from the Protein Data Bank.[28] A list of ligand-protein contacts for both starting complexes (3ERD and 1A52) was generated with the Ligand Protein Contact (LPC) server.[29]

Modeling ligand-receptor interactions:

Two different docking programs were used to study ligand binding to the receptors, the

AutoDock[30] module embedded in the YASARA[31] suite of programs and Glide,[32, 33] supplied by Schrodinger, Inc. AutoDock was chosen because I had prior experience with the program, and because docking experiments can be run quickly enough in order to test multiple starting positions of the ligand in a reasonable space of time. This allowed me to determine whether the program consistently returned a similar lowest-energy binding mode for a given ligand, regardless of its starting position. The data were largely consistent across different starting positions. After completing our study with YASARA/AutoDock, Dr. Ondrechen, our collaborator on this project and expert on molecular modeling, suggested that we repeat the experiments using a different modeling program and see if it returned similar results. The

Schrodinger suite of programs was also available to us, and it offered similar tools as YASARA: a protein preparation wizard, a molecule-building interface (which was actually more chemist- friendly than YASARA’s) and a docking program (Glide). Additionally, Glide allows for the user to create a packet of different ligands, then submit all of them at once to be docked in a given

7 receptor.

YASARA: Ligand Preparation and Minimization:

Ligands were first prepared using YASARA molecular modeling software.[31] Each ligand besides DES and E2 was built in silico by modifying the molecules of DES and E2 found in the

PDB structure files 3ERD and 1A52, respectively. All carbon-carbon bonds in the aromatic rings were given bond orders of 1.5. The olefinic (C=C) bond orders were set at 2. All other bonds were assigned an order of 1. Each ligand was then subjected to energy minimization to refine the bond lengths and bond angles. The resulting structures were minimized again from three different starting positions. The minimized conformation with the lowest energy for each ligand was used in the docking experiments. Solvation of the ligands prior to minimization was carried out in a solvent box at physiological pH (7.4) with 0.9% NaCl concentration (the default values).

The wisdom of using physiological pH was considered carefully, because the acidity inside of tumor tissues and cancer cells can diverge greatly from that of normal physiological pH.

Although the pH in tumor tissue and cells is often more acidic than normal, this is not always true. Intratumoral pH can vary widely, from 6.4 to 7.7.[34] Because of the uncertainty regarding tumor pH, the default value of 7.4 was used for all experiments. Geometry optimization on each ligand was performed quantum mechanically using PM3.[35] The simulation cell was defined as extending 10 Å around all atoms, and the Yamber 3 force field was used.

YASARA: Receptor preparation:

The crystal structures of ER with bound agonists diethylstilbestrol (PDB ID: 3ERD) and estradiol (PDB ID: 1A52) were imported into YASARA from the Protein Data Bank. The ER

8 crystal structure is a dimer containing two pairs of subunits, where each pair contains a ligand- binding subunit and a nonbinding subunit. Every subunit besides the “A” chain, which contains the binding site, was deleted in order to simplify the computation. The 1A52 structure contains three gold ions, incorporated for purposes of diffraction data collection. These Au ions are not relevant to ligand binding and were also deleted. The native ligand (DES or E2) was also deleted, although an alternate scene was saved with the native ligand intact for the purpose of pre- positioning the ligand to be studied as closely as possible to the native ligand using the superposition function. This is discussed further under the heading “Docking”, below.

Crystallographic water molecules in both structures and the chloride ion in the 3ERD structure were left intact. The simulation cell was defined as extending 6 Å around specified atoms in residues of the binding site. These atoms used to define the simulation cell boundaries are: Asp

351 OD1 and OD2 (carboxylate oxygen atoms), Glu 353 OE1 and OE2 (carboxylate oxygen atoms), Trp 383 CB (β carbon on the side chain), Leu 387 O (backbone carbonyl oxygen), Arg

394 NH2 (an η nitrogen), Phe 425 CB (β carbon), His 524 ND1 (imidazole δ nitrogen), and HOH

11/HOH 558 (crystallographic water molecule near Glu 353 and Arg394 in 3ERD/1A52, respectively). All of the residues listed above were identified as ligand-contacting residues by the

LPC server, with the exception of Asp 351, which we included in the simulation cell so as not to exclude a priori the possibility of antagonistic binding by our ligands.[26, 36] However, it should be noted that the ligands we investigated are unlikely to engage in antagonistic binding due to their lack of a long, hydrophobic side chain capable of forming a hydrogen bond with Asp

351.

AutoDock/YASARA: Docking:

For the AutoDock calculations, each ligand was manually pre-docked into the simulation cell of the prepared receptor in four different starting positions, which were labelled as A, B, C, and D

(Figure 3). A fifth starting position was also obtained by superimposing the nonsteroidal ligands onto the bound diethylstilbestrol molecule of the 3ERD complex structure and by superimposing the steroidal ligands onto the estradiol of the 1A52 structure. This was accomplished using the

“superpose” function in YASARA, which minimizes the RMSD (root-mean-square distance) between the ligand to be docked and the native ligand. The reason each ligand was docked from multiple starting positions was to make sure that the results were reliable and not dependent on a specific initial position of the ligand. The superpose function was used in order to obtain a starting position which was as close as possible to the crystallographic position of diethylstilbestrol (for the nonsteroidal ligands) or estradiol (for the steroidal ligands).

After positioning each ligand, the YASARA macro “dock_runlocal.mcr” was executed. In each docking run, AutoDock/YASARA tries 250 possible positions for the ligand, then collects similar resulting poses (determined by RMSD) into average “clusters”. The best clusters from each docking experiment are reported in Table 2 in the Results section (below). The result with the best predicted binding energy out of all four or five experiments for each ligand-receptor pair is compared with the results obtained with Glide in Table 3.

Figure 3: Starting poses A-D for the nonsteroidal ligand ITHPE (left) and the steroidal ligand E2 (right) in the binding site of 3ERD. Screenshots were taken in YASARA.

Schrodinger/Maestro/Ligprep: Ligand Preparation:

The ligands were built using the Ligprep feature of Maestro, the graphical interface for the

Schrodinger suite of programs. Dihedral angles for each ligand were adjusted so that they match those of the lowest-energy conformations that were used as starting points in the YASARA-based docking experiments. This was done so that the starting conformations of the ligands used in both sets of experiments would be as close as possible. This process did not present any significant difficulties. The ligands were saved together as a Maestro file (“.mae”).

Schrodinger/ProteinPrepWiz: Receptor Preparation:

The “A” chains of 3ERD and 1A52 were imported and prepared using the Protein Preparation

Wizard[37] in the Schrodinger software suite. The gold atoms in 1A52 were deleted, but water

11 molecules and the chloride in 3ERD were left intact, as was done in YASARA. Restrained minimization of the protein was performed to relieve steric clashes. The prepared receptor files were used to generate the receptor grids. Grid generation was performed using default settings and no constraints were used.

Schrodinger Glide: Docking:

Docking was performed using Glide with Standard Precision and default settings. The Maestro file containing the ligands was docked with the receptor grid files generated during receptor preparation. Since Glide docking does not allow for pre-positioning of the ligand within the receptor, multiple experiments were not performed for different starting positions of each ligand- receptor pair in Glide.

Analytical methodology:

The best pose generated for each ligand-receptor pair by each program was classified as “A”,

“B”, “C”, or “D” according to the scheme used to define the starting positions (Figure 3). The predicted binding energies (AutoDock) or GlideScores (Glide) were recorded. When inspecting ligand-receptor contacts, hydrogen bonds as well as contacts between the vinyl halogen or hydrogen in the nonsteroidal ligands and the receptor were considered. Glide defines “good” contacts as those whose cutoff ratios (defined by the equation C = D1,2/(R1+R2), where C = cutoff ratio, D1,2 = distance between atomic centers, R1 = Van der Waals radius of first atom and R2 =

Van der Waals radius of second atom) are between 1.3 and 0.89; smaller cutoff ratios qualify a contact as “bad” or “ugly”. YASARA simply calculates contacts based on distance; all atoms within 5 Å of the halogen which are not occluded by another atom in between are considered to

12 be in contact with it. In both cases, all interatomic contacts are within 5 Å of each other.

Potential hydrogen bonds were evaluated manually to determine if they satisfied criteria for distance and bond angles (Table 5).

Results:

YASARA/AutoDock Docking Experiment Results: Receptor Ligand Starting Pose Final Pose Binding Energy Binding Constant (kcal/mol) (pM) 3ERD IBHPE A A 10.11 38660 3ERD IBHPE B A 10.21 32550 3ERD IBHPE C A 9.56 98400 3ERD IBHPE D B 9.11 210720 3ERD IBHPE Superposed on A 9.86 58990 native DES 3ERD ITHPE A A 9.61 89630 3ERD ITHPE B B 8.52 568650 3ERD ITHPE C A 9.24 168550 3ERD ITHPE D A 8.3 823380 3ERD ITHPE SP A 9.38 132050 3ERD BrTHPE A A 10.64 15790 3ERD BrTHPE B A 10.04 43990 3ERD BrTHPE C A 10.07 41350 3ERD BrTHPE D A 9.36 136820 3ERD BrTHPE Superposed on A 11.37 4650 native DES 3ERD THPE A A 8.15 1050000 3ERD THPE B A 7.79 1960000 3ERD THPE C A 7.99 1380000 3ERD THPE D A 8.09 1170000 3ERD THPE Superposed on A 8.86 321730 native DES 3ERD All-para-ITHPE A A 9.75 71020 3ERD All-para-ITHPE B B 9.48 111800 3ERD All-para-ITHPE C C 10.06 42230 3ERD All-para-ITHPE D A 9.25 166640 3ERD All-para-ITHPE Superposed on C 10.22 32090 native DES 3ERD All-para-BrTHPE A A 9.91 54710 3ERD All-para-BrTHPE B C 9.56 97760 3ERD All-para-BrTHPE C A 9.85 59900 3ERD All-para-BrTHPE D A 10.13 37810

3ERD All-para- Superposed on A 10.18 34560 BrTHPE native DES 3ERD All-para-THPE A A 10.61 16670 3ERD All-para-THPE B A 10.41 23240 3ERD All-para-THPE C A 9.46 116280 3ERD All-para-THPE D A 10.2 33310 3ERD All-para-THPE Superposed on A 10.07 41720 native DES 3ERD DES A A 9.95 50500 3ERD DES B A 9.79 66670 3ERD DES C A 9.71 76850 3ERD DES D A 9.85 60530 3ERD DES Superposed on A 9.45 119310 native DES 3ERD E2 A B 11.37 4670 3ERD E2 B B 11.34 4900 3ERD E2 C B 10.89 10440 3ERD E2 D B 11.1 7360 3ERD 11β4HPE2 A B 9.6 91700 3ERD 11β4HPE2 B B 8.6 495140 3ERD 11β4HPE2 C B 9.18 185330 3ERD 11β4HPE2 D B 8.82 341910 1A52 IBHPE A B 10.62 16510 1A52 IBHPE B B 10.52 19560 1A52 IBHPE C A 9.07 223920 1A52 IBHPE D B 10.73 13670 1A52 ITHPE A B 10.21 32620 1A52 ITHPE B B 10.54 18840 1A52 ITHPE C B 10.48 20640 1A52 ITHPE D B 10.29 28880 1A52 BrTHPE A B 10.77 12680 1A52 BrTHPE B B 10.41 23510 1A52 BrTHPE C B 10.86 10940 1A52 BrTHPE D B 10.73 13680 1A52 THPE A B 9.28 158260 1A52 THPE B B 10.21 32940 1A52 THPE C D 8.15 1060000 1A52 THPE D B 9.97 49040 1A52 All-para-ITHPE A B 10.58 17620

1A52 All-para-ITHPE B B 10.64 15920 1A52 All-para-ITHPE C B 9.99 47590 1A52 All-para-ITHPE D B 10 46930 1A52 All-para- A B 10.85 11140 BrTHPE 1A52 All-para-BrTHPE B D 10.42 23060 1A52 All-para-BrTHPE C D 10.13 37510 1A52 All-para-BrTHPE D B 10.47 21020 1A52 All-para-THPE A B 9.63 87170 1A52 All-para-THPE B B 9.45 119320 1A52 All-para-THPE C B 8.94 281910 1A52 All-para-THPE D B 9.41 127250 1A52 DES A C 9.08 220190 1A52 DES B B 8.85 323350 1A52 DES C C 8.69 426180 1A52 DES D B 8.81 351220 1A52 E2 A B 10.69 14640 1A52 E2 B B 11.01 8530 1A52 E2 C B 10.6 16930 1A52 E2 D B 10.32 27190 1A52 E2 Superposed on B 10.21 33040 native E2 1A52 11β4HPE2 A B 12 1600 1A52 11β4HPE2 B B 11.92 1840 1A52 11β4HPE2 C Between A and B 11.89 1920 1A52 11β4HPE2 D Between A and B 11.32 5020 1A52 11β4HPE2 Superposed on B 11.59 3190 native E2 Table 2: Results of every AutoDock docking experiment. Letters of starting poses/final poses refer to the orientations depicted in Figure 3. Bold results are those with the best predicted binding energies for each ligand in each receptor crystal structure. These are the results used to compare with the Glide-generated results in Table 3, below.

AutoDock vs. Glide Docking Experiment Results: Receptor Ligand AutoDock Best Glide Best Final AutoDock GlideScore Final Pose Pose Binding Energy (kcal/mol) 3ERD IBHPE A A 10.21 -8.99 3ERD ITHPE A A 9.61 -9.12 3ERD BrTHPE A A 11.37 -9.95 3ERD THPE A A 8.86 -9.99 3ERD All-para-ITHPE C A 10.22 -9.81 3ERD All-para- A A 10.18 -10.49 BrTHPE 3ERD All-para-THPE A A 10.61 -9.97 3ERD DES A A 9.95 -9.85 3ERD E2 B B 11.37 -9.67 3ERD 11β4HPE2 B n/a* 9.6 n/a* 1A52 IBHPE B B 10.73 -10.62 1A52 ITHPE B B 10.54 -10.6 1A52 BrTHPE B B 10.86 -10.8 1A52 THPE B B 10.21 -10.86 1A52 All-para-ITHPE B B 10.64 -11.08 1A52 All-para- B D 10.85 -11.57 BrTHPE 1A52 All-para-THPE B B 9.63 -11.4 1A52 DES C A 9.08 -8.87 1A52 E2 B B 11 -10.37 1A52 11β4HPE2 B B 12 -10.94 Table 3: Best results of AutoDock-based docking experiments compared with Glide-based docking experiments. GlideScores are an estimation of the ligand binding free energy, similar to the AutoDock-predicted free ligand binding energy, but expressed as a negative number instead. *Glide did not return results for this ligand in this receptor.

Halogen-contacting Residues and Their Distances From the Halogen/Vinyl Proton*: 3ERD 1A52 AutoDock Glide AutoDock Glide Ligand Contacting Distance (Å) Contacting Distance Contacting Distance Contacting Distance Residue Residue (Å) Residue (Å) Residue (Å) Atom Atom Atom Atom IBHPE Thr 347 4.078 Ala 350 CB 3.32 Leu 384 4.394 Met 388 4.13 HG2 (3 of 3) HD1 (1 of CE 3) IBHPE Ala 350 CB 3.262 Trp 383 CH2 4.1 Met 388 3.419 Met 388 4.33 HE (3 of CG 3) IBHPE Ala 350 HB 2.909 Trp 383 CZ3 3.81 Met 388 3.816 Leu 391 3.53 (1 of 3) HG (1 of CD2 2) IBHPE Ala 350 HB 2.985 Leu 384 4.38 Leu 391 3.884 Phe 404 3.79 (2 of 3) CD1 CD2 CE2 IBHPE Trp 383 HZ3 3.131 Leu 387 4.46 Phe 404 3.735 Phe 404 4.21 (1 of 1) CD1 CE2 CZ IBHPE Leu 384 4.759 Leu 525 4.06 Phe 404 2.948 Leu 428 3.37 HD1 (3 of 3) CD2 HE2 (1 of CD1 1) IBHPE Leu 384 3.644 Leu 540 3.67 Ile 424 4.826 Leu 428 4.76 HD1 (2 of 3) CD1 HG2 (1 of CD2 3) IBHPE Leu 387 4.300 Leu 540 4.29 Ile 424 4.609 Leu 428 4.68 CD1 CD2 HG2 (3 of CG 3) IBHPE Leu 525 4.388 Leu 540 CG 4.6 Leu 428 3.642 CD2 CD1 IBHPE Leu 525 3.620 Leu 428 2.991 HD2 (3 of 3) HD1 (1 of 3) IBHPE Leu 525 4.980 Leu 428 4.923 HD1 (2 of 3) HD2 (1 of 3) IBHPE Leu 540 4.513 CD2 IBHPE Leu 540 3.819 HD2 (1 of 3) IBHPE Leu 540 4.281 CD1 IBHPE Leu 540 3.545 HD1 (1 of 3) ITHPE Leu 346 CB 4.653 Leu 346 O 4.42 Leu 384 4.483 Met 388 4.47 CD1 CE

ITHPE Leu 346 HB 3.894 Thr 347 CA 4.38 Leu 384 3.622 Leu 391 3.67 (2 of 2) HD1 (1 of CD2 3) ITHPE Leu 347 CA 3.538 Thr 347 CG2 4.37 Met 388 3.460 Phe 404 3.52 CE CE2 ITHPE Leu 347 3.562 Ala 350 CB 3.26 Met 388 2.615 Phe 404 3.86 CG2 HE (3 of CZ 3) ITHPE Ala 350 CB 3.486 Trp 383 CH2 4.4 Met 388 3.579 Leu 428 3.44 CG CD1 ITHPE Ala 350 HB 3.011 Trp 383 CZ3 4.17 Met 388 2.689 (1 of 1) HG (1 of 2) ITHPE Trp 383 4.984 Leu 525 4.74 Met 388 4.769 HH2 (1 of 1) CD1 CA ITHPE Trp 383 HZ3 4.645 Leu 525 4.03 Leu 391 3.947 (1 of 1) CD2 CD2 ITHPE Leu 525 4.654 Leu 540 3.62 Leu 391 4.906 CD2 CD1 HB (2 of 2) ITHPE Leu 525 3.922 Leu 540 3.94 Phe 404 4.618 HD2 (3 of 3) CD2 CE2 ITHPE Leu 525 4.845 Leu 540 CG 4.4 Phe 404 3.853 HD1 (1 of 3) HE2 (1 of 1) ITHPE Leu 525 4.366 Ile HG2 (1 4.793 HD1 (2 of 3) of 3) ITHPE Leu 540 4.861 Ile HG2 (3 4.627 CD1 of 3) ITHPE Leu 540 4.198 Leu 428 3.528 CD2 CD1 ITHPE Leu 540 3.399 Leu 428 2.733 HD2 (1 of 3) HD1 (1 of 3) ITHPE Leu 428 4.685 CD2 BrTHPE Ala 350 CB 3.272 Ala 350 CB 3.19 Leu 346 4.803 Met 388 4.48 HB (2 of CE 2) BrTHPE Ala 350 HB 2.906 Trp 383 CH2 4.29 Leu 346 3.989 Leu 391 3.73 (1 of 3) HD1 (2 of CD2 3) BrTHPE Trp 383 CH2 4.536 Trp 383 CZ3 3.9 Met 388 4.265 Phe 404 3.52 HE (3 of CE2 3) BrTHPE Trp 383 HZ3 3.375 Leu 384 4.25 Met 388 4.904 Phe 404 3.86

(1 of 1) CD1 HG ( 1 of CZ 2) BrTHPE Leu 384 4.647 Leu 387 4.26 Leu 391 4.255 Leu 428 3.48 CD1 CD1 CD2 CD1 BrTHPE Leu 384 3.913 Leu 540 3.93 Phe 404 3.175 HD1 (2 of 3) CD1 CE2 BrTHPE Leu 387 4.558 Leu 540 4.55 Phe 404 2.400 CD1 CD2 HE2 (1 of 1) BrTHPE Leu 525 4.325 Phe 404 3.413 CD2 CZ BrTHPE Leu 525 3.560 Phe 404 2.908 HD2 (3 of 3) HZ (1 of 1) BrTHPE Leu 525 4.782 Ile 424 4.995 HD1 (2 of 3) HG2 (1 of 3) BrTHPE Leu 540 4.290 Ile 424 4.778 CD1 HG2 (3 of 3) BrTHPE Leu 540 3.557 Phe 425 4.991 HD1 (1 of 3) CE1 BrTHPE Leu 540 4.362 Phe 425 4.289 CD2 HE1 (1 of 1) BrTHPE Leu 540 3.638 Leu 428 4.088 HD2 (1 of 3) CD1 THPE* Ala 350 CB 4.062 Ala 350 CB 3.25 Leu 391 4.121 Phe 404 3.76 CD2 CE2 THPE* Trp 383 HZ3 3.955 Leu 428 4.256 (1 of 1) CD1 THPE* Leu 387 4.434 CD1 THPE* Leu 540 4.999 HD1 (1 of 3) All-para- Thr 347 CA 4.948 Leu 346 O 4.33 Leu 384 4.460 Met 388 3.94 ITHPE CD1 CE All-para- Thr 347 4.187 Leu 346 C 4.79 Leu 384 3.644 Met 388 4.84 ITHPE HG2 (3 of 3) HD1 (1 of SD 3) All-para- Ala 350 CB 3.329 Thr 347 CA 4.48 Met 388 3.837 Met 388 3.92 ITHPE CE CG All-para- Ala 350 HB 2.947 Thr 347 CG2 4.47 Met 388 3.032 Leu 391 3.49 ITHPE (1 of 3) HE (3 of CD2 3) All-para- Leu 384 4.266 Ala 350 CB 3.33 Met 388 2.968 Phe 404 4.01

ITHPE CD1 HG (1 of CE2 2) All-para- Leu 384 3.541 Trp 383 CH2 4.6 Met 388 4.904 Phe 404 4.54 ITHPE HD1 (2 of 3) CA CZ All-para- Leu 387 4.257 Trp 383 CZ3 4.34 Leu 391 3.921 Leu 428 3.52 ITHPE CD1 CD2 CD1 All-para- Trp 383 HZ3 3.134 Leu 384 4.71 Leu 391 4.982 Leu 428 4.64 ITHPE (1 of 1) CD1 HB (1 of CD2 2) All-para- Leu 525 4.438 Leu 525 4.21 Leu 391 4.897 Leu 428 4.72 ITHPE CD2 CD2 HB (2 of CG 2) All-para- Leu 525 3.652 Leu 540 3.99 Phe 404 4.300 ITHPE HD2 (3 of 3) CD1 CE2 All-para- Leu 540 4.399 Leu 540 4.3 Phe 404 3.490 ITHPE CD1 CD2 HE2 (1 of 1) All-para- Leu 540 3.655 Leu 428 3.916 ITHPE HD1 (1 of 3) CD1 All-para- Leu 540 4.660 Leu 428 3.174 ITHPE CD2 HD1 (1 of 3) All-para- Leu 540 3.964 Leu 428 4.620 ITHPE HD2 (1 of 3) HD2 (1 of 3) All-para- Thr 347 CA 4.781 Leu 346 O 4.27 Leu 346 4.666 Leu 384 4.59 BrTHPE HD1 (2 of CD1 3) All-para- Thr 347 4.138 Thr 347 CG2 4.38 Met 388 3.664 Met 388 3.52 BrTHPE HG2 (3 of 3) HE (3 of CE 3) All-para- Ala 350 CB 3.140 Thr 347 CA 4.36 Met 388 4.189 Met 388 4.51 BrTHPE HG (1 of SD 2) All-para- Ala 350 HB 2.726 Ala 350 CB 3.24 Leu 391 4.090 Met 388 3.72 BrTHPE (1 of 3) CD2 CG All-para- Ala 350 HB 2.905 Trp 383 CH2 4.61 Phe 404 3.573 Leu 391 3.98 BrTHPE (2 of 3) CE2 CD2 All-para- Trp 383 HZ3 3.262 Trp 383 CZ3 4.35 Phe 404 2.759 Phe 404 4.45 BrTHPE (1 of 1) HE2 (1 of CE2 1) All-para- Leu 384 4.422 Leu 525 4.21 Ile 424 4.846 Leu 428 3.68 BrTHPE CD1 CD2 HG2 (1 of CD1 3) All-para- Leu 384 3.713 Leu 540 3.88 Ile 424 4.672 BrTHPE HD1 (2 of 3) CD1 HG2 (3 of 3)

All-para- Leu 387 4.227 Leu 540 4.14 Phe 425 4.948 BrTHPE CD1 CD2 HE1 (1 of 1) All-para- Leu 525 4.626 Leu 428 3.869 BrTHPE CD2 CD1 All-para- Leu 525 3.846 BrTHPE HD2 (3 of 3) All-para- Leu 540 4.462 BrTHPE CD1 All-para- Leu 540 3.753 BrTHPE HD1 (1 of 3) All-para- Leu 540 4.639 BrTHPE CD2 All-para- Leu 540 3.958 BrTHPE HD2 (1 of 3) All-para- Ala 350 CB 3.828 Ala 350 CB 3.52 Leu 391 4.000 Phe 404 3.7 THPE* CD2 CE2 All-para- Trp 383 HZ3 4.127 Leu 428 3.503 THPE* (1 of 1) CD1 All-para- Leu 387 4.722 Leu 428 2.772 THPE* CD1 HD1 (1 of 3) All-para- Leu 540 4.808 Leu 428 4.616 THPE* HD1 (1 of 3) HD2 (1 of 3) All-para- Leu 540 4.809 THPE* HD2 (1 of 3) Table 4: Residue atoms in contact with the vinyl halogen or hydrogen* for the top binding conformations (Table 3). Residue atom names are standard names used to identify them in both YASARA and Schrodinger programs. All contacts are within 5 Å of the halogen/vinyl proton.

Hydrogen Bonds Between Ligand and Receptor: Program Receptor Ligand Ligand Residue Residue Distance Ligand Residue Atom Atom (Angstroms) Bonding Bonding Angle (°) Angle (°) AutoDock 3ERD IBHPE Hydroxyl O Glu353 OE1 2.544 111.973 111.694 cis to Iodine AutoDock 3ERD BrTHPE Hydroxyl O Glu353 OE1 2.515 111.943 113.440 cis to Bromine AutoDock 3ERD All-para- Hydroxyl O Glu353 OE1 2.610 103.619 110.218 THPE cis to vinyl proton AutoDock 1A52 E2 A-ring O Glu353 OE1 2.999 113.381 116.571 AutoDock 1A52 11β4HPE2 A-ring O Glu353 OE1 2.691 110.960 108.450 Glide 3ERD IBHPE Hydroxyl O Glu353 OE1 2.73 108.2 111.9 cis to Iodine Glide 3ERD ITHPE Hydroxyl O Glu353 OE1 2.68 109.2 114.0 cis to Iodine Glide 3ERD All-para- Hydroxyl O Glu353 OE1 2.84 107.5 110.6 ITHPE cis to Iodine Glide 3ERD All-para- Hydroxyl O Glu353 OE1 2.52 105.8 111.9 BrTHPE cis to Bromine Glide 3ERD DES Hydroxyl O Glu353 OE1 2.84 108.7 105.7 Glide 1A52 IBHPE Hydroxyl O Glu353 OE1 2.49 108.1 114.8 cis to Iodine Glide 1A52 ITHPE Hydroxyl O Glu353 OE1 2.46 109.1 114.5 cis to Iodine Glide 1A52 All-para- Hydroxyl O Thr 347 OG1 2.76 109.8 104.5 ITHPE trans to Iodine Glide 1A52 BrTHPE Hydroxyl O Glu353 OE1 2.69 107.7 108.4 cis to Bromine Glide 1A52 All-para- Hydroxyl O Glu353 OE1 2.41 106.8 105.4 BrTHPE geminal to Bromine Glide 1A52 THPE Hydroxyl O Glu353 OE1 2.69 107.2 108.3 cis to vinyl proton Glide 1A52 All-para- Hydroxyl O Glu353 OE1 2.41 105.2 105.4 THPE cis to vinyl proton Glide 1A52 DES Hydroxyl O Glu353 OE1 2.41 107.4 105.4 Glide 1A52 E2 A-ring O Glu353 OE1 2.64 108.2 110.8

Glide 1A52 11β4HPE2 A-ring O Glu353 OE1 2.55 123.5 119.3 Table 5: Hydrogen bonding interactions for the top binding conformations (Table 3). Each

23 potential hydrogen bond was manually inspected. Parameters used were as follows: Distance between H-bonding atoms must be between 2.3 and 3.3 Angstroms. The angles between the H- bonding atoms and the covalently bonded atom in both the ligand and residue must both fall between 104 and 125 degrees, after rounding to the nearest integer. In other words, the bonding angles must be within approximately 5 degrees of either a tetrahedral or trigonal bond angle.

Dihedral Angles of Rotatable Functional Groups Before and After Docking: Ligand/Dihedral Angle Before Angle After Angle After Angle After Angle After Atoms Docking Docking (3ERD, Docking (1A52, Docking Docking YASARA) YASARA) (3ERD, Glide) (1A52, Glide) IBHPE A-ring 54.070° 56.982° 35.871° 60.4° 49.4° 11β-ring 46.283° 32.810° 67.890° 63.5° 37.8° D-ring 40.716° 80.118° 70.168° 86.5° 70° ITHPE A-ring 141.908° 97.460° 52.359° 70.9° 47.4° 11β-ring 119.884° 21.676° 103.835° 66.2° 85.9° D-ring -100.380° 114.285° -48.554° -84.5° 132.2° BrTHPE A-ring 42.411° 58.786° 53.884° 70.4° 45.8° 11β-ring 60.422° 57.342° 83.219° 79.5° 81.0° D-ring -141.213° -84.141° 69.201° -74.2° 128.2° THPE A-ring 49.453° 38.751° 33.383° 49.4° 38.4° 11β-ring 99.950° 93.236° 58.265° 84.8° 71.4° D-ring 142.708° 81.127° 48.413° -62.3° 9.9° All-para-ITHPE A-ring 137.699° 70.055° 68.168° 54.3° 43.1° 11β-ring 130.603° 122.801° 98.828° 29.1° 67.8° D-ring 137.764° 105.748° 17.138° 94.6° 55.8° All-para-BrTHPE A-ring 52.812° 38.389° 39.339° 68.5° 57.3° 11β-ring 37.247° 111.878° 83.581° 84.3° 68.7° D-ring 44.346° 118.366° 58.624° 93.3° 24.8° All-para-THPE A-ring 32.121° 43.536° 25.054° 56.8° 36.3° 11β-ring 50.207° 22.895° 77.641° 28.1° 72° D-ring 27.621° 107.911° 21.555° 100.0° 34.1° DES A-ring 114.768° 103.193° 160.038° 54.7° 50.3° Ethyl Geminal to A- 86.276° 81.489° 115.038° -79.7° 74.2° ring D-ring 66.711° 97.210° 46.511° 114.8° 77.3° Ethyl Geminal to D- -158.804 -129.448° 112.766° 135.1° 154.0° ring 11β4HPE2 11β-ring 60.422° 58.689° 59.42° N/A 56.5° Table 6: Dihedral angles of rotatable functional groups of minimized ligands before and after docking. Dihedral angles of docked ligands are from the best poses reported in Table 3. E2 was not included because it is relatively rigid.

Discussion:

The binding modes of 2-substituted-1,1-bis(4-hydroxyphenyl)-2-aryl(alkyl)-ethylenes as estrogen receptor ligands have been examined before.[38-41] However, this is the first comparable computational analysis of 2-substituted-1,1-bis(4-hydroxyphenyl)-2-(3- hydroxyphenyl)ethylenes (THPEs). As stated above, a goal of this computational study was to investigate the binding modes of the THPEs which were synthesized by our group in order to rationalize the observed relative binding affinities. Specifically, we wanted to see if we could explain the fact that the 2-Bromo and 2-Iodo-THPEs were at least an order of magnitude better than the unhalogenated THPE.

The results of the docking experiments were very consistent. The predicted binding modes from each experiment are shown in Table 2 of the Results section (above). The predicted binding modes associated with the best predicted binding energies from the AutoDock experiments are compared with those from the Glide experiments in Table 3. Table 7 (below) shows only the binding mode results in order to illustrate their consistency.

Analysis of Predicted Binding Modes: AutoDock AutoDock AutoDock AutoDock AutoDock Receptor Ligand Starting Pose Starting Pose Starting Pose Starting Pose Superposed Glide Mode A Result B Result C Result D Result Start Result Result 3ERD IBHPE A A A B A A A (5/6) 3ERD ITHPE A B A A A A A (5/6) 3ERD BrTHPE A A A A A A A (6/6) 3ERD THPE A A A A A A A (6/6) 3ERD All-para- A B C A C A A ITHPE (3/6) 3ERD All-para- A C A A A A A BrTHPE (5/6) 3ERD All-para- A A A A A A A THPE (6/6) 3ERD DES A A A A A A A (6/6) 3ERD E2 B B B B n/a B B (5/5) 3ERD 11β4HPE2 B B B B n/a n/a* B (4/4) 1A52 IBHPE B B A B n/a B B (4/5) 1A52 ITHPE B B B B n/a B B (5/5) 1A52 BrTHPE B B B B n/a B B (5/5) 1A52 THPE B B D B n/a B B (4/5) 1A52 All-para- B B B B n/a B B ITHPE (5/5) 1A52 All-para- B D D B n/a D D BrTHPE (3/5) 1A52 All-para- B B B B n/a B B THPE (5/5) 1A52 DES C B C B n/a A B (2/5) & C (2/5) 1A52 E2 B B B B B B B (6/6) 1A52 11β4HPE2 B B B B B B B (6/6) Table 7: Analysis of binding mode results. *Glide did not return a result for this ligand in this receptor.

The most common pose for the nonsteroidal ligands when docked with 3ERD was Pose A, while the steroids preferred Pose B. This is not surprising, as these are the native poses of DES and E2 found in the crystal structures 3ERD and 1A52, respectively. In most ligand-receptor pairings, one or zero out of the five or six docking experiments resulted in a different binding mode. The exceptions are all-para-ITHPE docked in the 3ERD receptor, all-para-BrTHPE docked in 1A52, and DES in 1A52. All-para-ITHPE still results in a binding mode of A in three out of the six docking experiments in the 3ERD receptor. Of the other three results, two are pose C and one is pose B. As illustrated in Figure 3, poses A and C both place the 4-hydroxyphenyl group trans to the halogen atom in the hydrophobic subpocket which contains many hydrophobic residues including Phe 404, Phe 425, Leu 346 and Leu 428. This is known as the 7α-subpocket because it is located in the direction in which the 7α-hydrogen of estradiol points when in complex with the

ER.[36] Because of this, and the fact that the THPEs contain hydroxyl groups on all three rings, poses A and C are very likely functionally similar. This might not be the case for IBHPE, which lacks a hydroxyl group on its 2-phenyl ring, meaning that rotating it from Pose A to Pose C would change the residues with which it might be able to form hydrogen bonds.

The second exception is that of all-para-BrTHPE docked in the 1A52 receptor. In this case, a majority (three out of five) of the experiments resulted in a binding mode different from the norm. While most of the other nonsteroidal ligands had a binding mode of B in 1A52, three of all-para-BrTHPE’s experiments resulted in a binding mode of D (the other two resulted in B). As with Pose A and C, Pose B and D are functionally similar in the case of the THPEs. Both place the 4-hydroxyphenyl group trans to the halogen atom in the hydrophobic pocket opposite the 7α- subpocket. This pocket contains the hydrophobic residues Trp 383, Ala 350, and Leu 525, and it

27 is known as the 11β-subpocket.

The third exception is that of DES in 1A52. Two of the AutoDock experiments resulted in binding mode B while two resulted in C. The GLIDE experiment resulted in binding mode A. As with all-para-BrTHPE, three out of the five poses were not pose B. However, due to diethylstilbestrol’s symmetry, there is very little functional difference between the four poses.

In all of the docking experiments that were performed, estradiol and 11β4HPE2 resulted in a binding mode of B. This was true in both crystal structures of the receptor. Since Pose B is the native pose of estradiol in complex with the ER, the 4-hydroxyphenyl group at the 11β-position of 11β4HPE2 inhabits the 11β-subpocket.

Figure 4: A) THPE, BrTHPE (foreground) and ITHPE docked in the 3ERD crystal structure in binding mode A; B) THPE, BrTHPE (foreground) and ITHPE in the 1A52 crystal structure in binding mode B; C) ITHPE and 11β4HPE2 docked in the 1A52 crystal structure in binding mode B, illustrating a possible functional parallel between the 4-hydroxyphenyl group at the 11β- position of the steroid and the 4-hydroxyphenyl group trans to the halogen of ITHPE. Images generated from AutoDock results using Ligplot+.[42] Ligands and H-bonding residues are shown as ball-and-stick models. Hydrophobic residues are represented as a red semicircle with radiating lines. Residues which overlap between the complexes of the results being compared are circled in red.

Figure 4 (above) illustrates the trends found in the results. The high number of overlapping residues (circled in red) between experiments shows that THPE, BrTHPE, and ITHPE all dock in highly similar orientations in the same crystal structure of the receptor. These results were consistent with the orientations of some similar compounds determined in the literature.[39]

When the ligands are docked in the crystal structure 1A52, the 2-halo group of BrTHPE and

ITHPE occupies the same site as the C-7 of 11β4HPE2 and the dipole of the halogen is oriented toward the 7α-pocket. In this pose, the 1-(4-hydroxyphenyl) group trans to the 2-halo moiety also occupies the hydrophobic pocket similar to, but not identical with, the 11β-(4- hydroxyphenyl) group of 11β4HPE2 (Figure 4C). The phenolic group at the 11β-position in this pose is in the proximity of Asp-351 but does not appear to be close enough to establish any interactions that might lead to antiestrogenic activity.

The results with respect to binding modes of the ligands, discussed above, tell us a few things.

First, the overall consistency of the results between multiple docking experiments from different starting positions and between different molecular modeling programs provides confidence in the quality of the results. Second, the fact that the nonsteroidal ligands tend to favor pose A in one crystal structure of the receptor and pose B in another suggest that the ER binding site may be able to accommodate multiple binding modes for these ligands and that both the 7α and 11β subpockets can accept a bulky hydrophobic group such as a phenyl ring. Perhaps it is this flexibility of binding modes that explains why diethylstilbestrol has four times the binding affinity for the ER relative to estradiol.[43] By contrast, estradiol and 11β4HPE2 always oriented themselves in pose B in both receptor crystal structures.

Given the superior relative binding affinities of BrTHPE and ITHPE compared with the unhalogenated THPE (Table 1), contacts between the vinyl halogen/hydrogen atom and the receptor were analyzed. The results of this analysis are listed in Table 4. One standout feature of these data is that there are many fewer contacts between the vinyl proton and the receptor than there are between the bromine and the receptor or between the iodine and the receptor. This makes sense, that the larger, more polarizable halogens are able to make more contacts and form more energetically favorable interactions than a hydrogen atom can. This could partly explain the better binding affinities of the halogenated compounds. Another feature is that, just as the binding modes between ligands docked in the same crystal structure of the receptor overlap well in Figure 4, the lists of residues in contact with the vinyl halogen/hydrogen are highly similar for ligands docked in the same crystal structure. Although YASARA generally identifies more contacts between the ligand and receptor than Schrodinger does, most of the residues identified as contacts by Schrodinger are also identified by YASARA. This high level of agreement is encouraging.

A close visual analysis of the binding modes provided significant details pertaining to the interactions between the ligands and specific residues within the ER binding pocket. At first glance, it appears as though the 2-(3-hydroxyphenyl) group of THPE, BrTHPE and ITHPE is located in the vicinity of where it may be able to establish hydrogen bonding interactions with

His 524. However, careful inspection revealed that the distance and angle between the oxygen atom of the ligand and the nitrogen atom of His 524 did not meet criteria for hydrogen bond formation in any of the results (Table 5). By contrast, the criteria for hydrogen bonding between the ligand and the Glu 353 residue were met in many cases. Curiously, the oxygen atom of the 1-

(4-hydroxyphenyl) group trans to iodine on all-para-ITHPE had the correct distance and bonding angles to form a hydrogen bond with the hydroxyl group oxygen of Thr 347 (Glide, 1A52). This was the only example of a valid hydrogen bond between the ligand and receptor which did not involve the residue Glu 353. These results support the idea that the interaction between a hydroxyl of the ligand analogous to the A-ring hydroxyl of E2 and the residue Glu 353 of the receptor is important to the binding of ligands by the ER.[26, 27, 36] It should be kept in mind that the pose results are only a static snapshot of the ligand in the calculated lowest-energy position within the receptor. In reality the ligand and the receptor, like all molecules, are in a state of constant thermal motion. It is conceivable that hydrogen-bonding interactions whose criteria are not met by the static poses could exist in equilibrium with those interactions which are identified in Table 5.

In addition to investigating the binding modes, we also used the docking experiments to investigate whether a correlation existed between the calculated binding energies and relative binding affinities obtained for the THPE ligands reported in Table 1. The calculated binding energies of the preferred poses of each ligand in each receptor structure are reported in Table 3.

Looking at both the RBAs (Table 1) and the calculated binding energies (Table 3) of the 2-(3- hydroxyphenyl) ethylenes, there does not appear to be any strong correlation among them.

Ranked in order from highest to lowest binding affinity, the THPEs are: ITHPE (79%), BrTHPE

(53%), and THPE (5.5%). However, when ranked from highest to lowest predicted binding energy (AutoDock, 3ERD), they are: BrTHPE (11.37 kcal/mol), ITHPE (9.61 kcal/mol), and

THPE (8.86 kcal/mol). When docked in the 1A52 crystal structure using AutoDock, the same rank order holds true, but the differences are less dramatic: BrTHPE (10.86 kcal/mol) > ITHPE

(10.54 kcal/mol) > THPE (10.21 kcal/mol). The same rank order also holds true for the hypothetical 2-(4-hydroxyphenyl) ethylenes when docked in 1A52 by AutoDock [All-para-

BrTHPE (10.85 kcal/mol) > All-para-ITHPE (10.64 kcal/mol) > All-para-THPE (9.63 kcal/mol)] but not when docked in 3ERD [THPE (10.61 kcal/mol) > ITHPE (10.22 kcal/mol) > BrTHPE

(10.18 kcal/mol)]. The calculated binding energies generated by Glide (GlideScores) did not support the trend of the 2-halogenated ligands both being better ligands than their unhalogenated counterpart. For both the 2-(3-hydroxyphenyl) and the 2-(4-hydroxyphenyl) ethylenes and for both receptor crystal structures the nonhalogenated ligands had higher (absolute value)

GlideScore than one or both of their 2-halo-counterparts.

These discrepancies between the biological data and calculated predictions are not entirely surprising. Relative binding affinities and binding energies are not exactly the same thing. The former is a measure of how well a compound can displace another ligand (in this case, estradiol) in the estrogen receptor, while the latter is the amount of energy which would theoretically be required to convert the bound ligand-receptor complex to the free ligand and receptor. Although a high binding energy means a stable complex and therefore should contribute positively to the

RBA, there may be many other factors which contribute to RBA in vitro or in vivo which the docking simulations cannot take into account. Furthermore, predicting the binding energies of ligands in receptors is notoriously difficult, even for the best molecular modeling software.

Although comparisons of different results for the same ligand are generally reliable, and predicting binding energies of multiple ligands is a useful high-throughput analysis tool for discovering hits, the significance of any comparison between the software-predicted binding energies of different compounds should be taken with a grain of salt.

Caveats aside, the relative binding affinities and AutoDock-predicted binding energies of the 2-

(3-hydroxyphenyl) ethylenes in the 3ERD receptor structure agree on one thing: ITHPE and

BrTHPE are better ligands than THPE by about one order of magnitude. Binding energy is inversely related to binding constant, so an increase in binding energy by 1 kcal/mol corresponds to a 10-fold decrease in the binding constant, or ten times better binding. Binding constants are shown in Table 2 next to the corresponding predicted binding energies.

Summary & Conclusions for Chapter 1:

The predicted binding modes of the ligands in the receptor were highly consistent across multiple starting positions and two different molecular modeling programs. This consistency suggests that the results of the study are likely to represent reliably the actual binding modes of the ligands in the estrogen receptor. The predicted binding modes of the nonsteroidal ligands differed between the two different crystal structures of the receptor (3ERD and 1A52), while the steroids appeared to bind in similar orientations regardless of the crystal structure. This fact might reflect flexibility on the part of the nonsteroidal ligands. As with the binding modes, there was a large amount of agreement in the identities of the residues which are in contact with the vinyl halogen/hydrogen of the nonsteroidal ligands, although Schrodinger was generally more selective than YASARA. A possible explanation for the observed higher RBAs for BrTHPE and ITHPE as opposed to the unhalogenated THPE is provided by the fact that the vinyl proton had many fewer contacts with the receptor than did the bromine and iodine. The only hydrogen bond interaction which satisfied

33 the criteria (with one exception) was that between the ligand and Glu 353, despite the presence of a hydroxyl in the general vicinity of the basic residue His 524 on most of the ligands.

Although the binding mode results were highly consistent with each other, the calculated binding energies did not correlate well with the observed relative binding affinities of THPE, BrTHPE, and ITHPE. However, the predicted binding constants of BrTHPE and ITHPE were about an order of magnitude better than that of THPE when docked in the 3ERD crystal structure of the receptor by AUTODOCK, which agrees with the RBAs.

Chapter 2: Synthesis of heterobifunctional linkers as components of a molecular toolkit for the fabrication of customizable nanoparticles.

Background: Functionalized Nanoparticles for Drug Targeting and Drug Delivery:

Tumors possess a vasculature distinct from non-cancerous tissue. While the glomerulus pores in a non-tumorous kidney have diameters of about 5 nanometers[44] and even the very large interendothelial junctions in healthy tissue are only 8 nanometers in diameter, tumor pore diameters typically range between 40 and 80 nanometers and the very large interendothelial junctions of tumors are 500 nanometers across[45]. Therefore, nanoparticles with a diameter up to a few hundred nanometers can enter into tumors through their interendothelial junctions, but are excluded from healthy tissue.[46] In response to the challenge of multi-drug resistance, various macromolecules and nanoparticles have been investigated as possible drug delivery vehicles.[7, 47-50]

There are many types of nanoparticles, from liposomes to micelles to polymers to those made of metal or metal oxides. The former three can encapsulate drugs, making them useful for targeted chemotherapy applications. Polymeric nanoparticles have been shown to have improved circulation times and activities with respect to free drugs.[51, 52] Nanoparticles can be decorated with functional groups to impart various desired properties such as increased hydrophilicity or hydrophobicity or a moiety designed to interact with a specific receptor for active targeting.

Usually, the surface of an existing nanoparticle core is decorated with whatever functional groups are desired. One of our aims with this project was to develop a system in which

35 nanoparticles could be synthesized in one step from prefunctionalized polymer units (PPUs). We envisioned a “molecular toolkit” consisting of a selection of PPUs, each containing a functional group tailored to specific purpose such as amphiphilicity, targeting, or imaging.

In this approach, different combinations and ratios of PPUs can be combined and emulsified to quickly yield multifunctional nanoparticles (MFNPs) with the desired properties. Our hypothesis is that the physical and chemical properties of the final product can be defined by controlling the types and relative amounts of PPUs as well as the emulsification conditions. If this hypothesis is valid, the molecular toolkit approach would make it possible for a lab technician in a clinical setting to quickly and easily synthesize nanoparticles with properties optimized for an individual patient’s case.

The group of Dr. Amiji, with whom we were collaborating with on this project, first proposed the idea of using pre-functionalized and well-characterized polymers to synthesize multifunctional nanoparticles.[53] In recent years, Amiji et al. synthesized a small library of dextran nanoparticles encapsulating various anticancer drugs using this combinatorial approach.[54] The results showed that the properties of the formulations seemed to depend on the length of the lipid chains which were attached to dextran. This promising development laid the groundwork for further combinatorial systems based on a molecular toolkit.

Dextran is a homopolysaccharide of glucose composed primarily of α-(1,6) -linked glucose units with occasional α-(1,3)-linked branches. It is water-soluble, biocompatible, and widely available.

It is also highly functionalizable.[55] Dextran has been decorated with a wide variety of

36 hydrophilic and hydrophobic functional groups, including acetals,[56-59] esters,[60] lipids[61-

63] and other groups[64-67]. Dextran is being investigated as a material in the design of nanoparticles for targeted chemotherapy.[54, 56, 58-60, 63-65, 68-73] For all of these reasons, we decided to use dextran as the polymer in our approach. In theory the same approach could be applied to any functionalizable and biocompatible polysaccharide, such as hyaluronic acid or chitosan.

Background: Heterobifunctional Linkers for Drug Targeting and Delivery:

Inspired by Cui’s work[65] we planned to synthesize heterobifunctional linkers which would be connected to dextran on one end, and to a functional-group-containing alkynyl linker on the other, using the azide-alkyne Huisgen cycloaddition.[74] While Cui used 1,1’- carbonyldiimidazole to form carbonate linkages between the hydroxyl groups of dextran and the hydroxyl group of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol, we decided to also investigate using a carbamate linkage because we hypothesized that it would offer greater stability than a carbonate group due to the nitrogen atom. We decided to form our carbamate group by coupling an isocyanate group directly with the hydroxyl groups of dextran. The method of activating dextran with p-nitrophenyl carbonyl chloride before displacing the p-nitrophenyl leaving group by substitution with an amine is well-reported in the literature.[75-80] However, this approach was considered undesirable because it is likely that not all of the p-nitrophenyl groups introduced to the dextran in the first step would be replaced in the subsequent substitution reaction with the amine. Even if the substitution step proceeded to completion, an extra step of characterization

37 would be required to prove that this was so, and even then a small number of unsubstituted p- nitrophenyl groups might remain but be difficult to detect. Reacting unfunctionalized dextran with an isocyanate avoids this problem. Whatever the degree of substitution, we can be certain that the only thing on the dextran is the linker with which it was reacted.

Having decided on using isocyanate-hydroxyl chemistry to link other molecules to dextran, our next concern was to decide what we wanted those molecules to be. In other words, what functional groups do we want our dextran PPUs to have? Arguably the most important property for the PPUs to have is that they must be able to self-assemble into nanoparticles in an aqueous environment and also encapsulate cancer drugs, which are usually hydrophobic. An amphiphilic

PPU consisting of dextran coupled to a hydrophobic alkyl chain would self-assemble into a micelle with the hydrophobic drug molecules in the center.[63] Another important property to consider is the ability of the PPUs to evade detection by the reticuloendothelial system (RES).

Also known as the mononuclear phagocyte system (MPS), the RES includes white blood cells and is responsible (among other functions) for removing foreign bodies from the bloodstream.

The presence of poly(ethylene glycol) (PEG) chains on liposomes has been shown to improve their circulation times in vivo.[81] Dextran itself has also been shown to reduce uptake by the

RES.[82] It is reasonable to suppose that a dextran nanoparticle functionalized with PEG chains might have better RES-avoidance than either a dextran-coated or PEG-coated nanoparticle would on its own.

The chemical environment within tumors and cancer cells is often distinct from the chemical environment within healthy tissue. Acidity can vary widely and the pH within cancerous

38 tissue/cells is often lower than the normal physiological value.[34] The environment within tumors is also more strongly reducing than in healthy tissue, possibly due to lower oxygen concentrations in tumors compared to healthy tissue.[83, 84] These differences can be exploited to design nanoparticles which are stable in normal physiological conditions but which become unstable and release their cargo under conditions characteristic of cancerous tissue. Some investigators have functionalized dextran with acetals to design dextran nanoparticles that hydrolyze and lose their amphiphilicity, causing them to disassemble in an aqueous environment, upon being exposed to low pH.[56, 59, 65] For more examples of nanoparticles designed to react to various external stimuli, interested readers are encouraged to consult this review.[85] We were interested in using reducible disulfide bonds to give our nanoparticles some extra stability in normal conditions which would disappear when the reducing conditions in the tumor triggered the conversion of the disulfide bonds to thiols. This strategy has been used successfully to deliver doxorubicin to breast cancer cells in vitro.[86]

Other possibilities for functional groups which could be incorporated into PPUs include radioactive, fluorescent, or otherwise detectable isotopes or molecules for imaging. Imaging capacity would allow clinicians to track the distribution of the NPs after they are administered.

This would then give them the opportunity to fine-tune the properties of the NPs if the distribution is not optimal. Groups designed to actively target certain receptors could also be incorporated to complement the passive targeting of the nanoparticles themselves. For example, a maleimide group on the end of a PEG chain attached to dextran could be used to form a covalent bond with a thiol-containing peptide. Alternatively, a molecule with estrogenic activity could be attached to target the overexpressed estrogen receptor for active targeting of certain

39 ovarian and breast cancers.

Ultimately, we would have liked for imaging and active targeting elements to be combined into a single nanoparticle to make a truly “theranostic” multifunctional nanoparticle. However, our initial goal was to synthesize a simple molecular toolkit, containing the necessary hydrophobic and PEG components, so that we could begin fabricating nanoparticles and test our hypothesis.

Although there are many potential functional groups with which we could have decorated dextran, we ultimately wanted the linker to be either hydrophobic or hydrophilic, depending on the purpose of the linker and whether we want it to inhabit the core or the outer surface of the nanoparticle. Therefore, it made sense to have two standard linkers, one hydrophobic and one hydrophilic, which could attach to dextran via an isocyanato group at one end and bear a different functional group which won’t react with dextran but will react with another linker bearing a desired functional group (PEG, alkyl, or other). The Huisgen 1,3-cycloaddition reaction is a highly selective reaction in which an azide and an alkyne react to form a triazole.[74] The usefulness of this reaction in the context of dextran functionalization has been demonstrated.[54,

65] These types of reactions, in which two functional groups with high selectivity for one another are used to create a covalent bond between two molecules without a leaving group, are known as “click” reactions, and the field of chemistry dealing with them is called “click chemistry”. Click reactions other than azide-alkyne include thiol-maleimide[87] and thiol- isocyanate.[88] Click chemistry offers the advantage of being able to perform a highly-selective covalent-bond-forming reaction in the presence of other functional groups. This is important for our purposes and for related applications such as drug conjugation, where a linker bearing a

40 necessary functional group must be attached to another linker or molecule without compromising its functionality.

There are two ways in which one could proceed with the assembly of a PPU: click-first or dextran-coupling-first. In the click-first approach, the azido-containing linker is reacted with the functional-group-bearing alkyne via 1,3-cycloaddition to give a single compound which is then coupled with dextran. Due to the tendency of ioscyanates to form dimers or trimers with themselves or react with oxygen, this step may have to be carried out with a precursor to the azido-isocyanate linker. Both an amino group and a boc-protected amino group can be synthons for the isocyanate, and neither should react with alkyne during the cycloaddition step. The dextran-functionalization-first approach presents the same problem as using p-nitrophenyl chloroformate to introduce a carbamate bond to dextran does. That is, we won’t know how many of the azido groups were successfully clicked together with the alkyne and how many were not.

However, because of the selectivity of the azido group, it is unlikely that unreacted azido groups left on the PPUs would react with either the drug cargo or with compounds present in the body, whereas p-nitrophenyl groups could potentially react with many different nucleophiles. Another concern for the click-second approach is that the 1,3-cycloaddition carried out at room temperature requires the use of a copper catalyst which must be removed from the final product.

This purification could be more difficult in the presence of a polymer rather than just a small molecule, although dialysis could be an effective way of removing the metal. And in either case, the copper catalyst will have to be removed in the presence of another polymer – poly(ethylene glycol) (PEG) – either after clicking the isocyanato-azido linker to the PEG-alkyne or after coupling the complete isocyanato-triazole-PEG compound to dextran. Therefore, it is generally

41 desirable to perform the click chemistry first and the dextran coupling step second, but the opposite strategy could also be employed if necessary. An example of when the dextran- coupling-first strategy might be useful is in a hypothetical future generation of our toolkit, in which we decorate a single PPU with multiple different clickable groups (for example, azide and maleimide) in order to click on different functional groups in a stepwise fashion.

To summarize, the PPUs will be constructed from:

-Dextran

-A heterobifunctional linker with an azido group at one end and an isocyanto group (or its synthon) at the other. There will be two variants of these: hydrophobic (alkyl) and hydrophilic

(oligo[ethylene glycol] or OEG).

-A compound with an alkynyl group at one end, and a desired functional group at the other.

For the sake of simplicity we decided that our initial toolkit should contain a hydrophobic alkyne

(for drug encapsulation and assembly of a micelle-like structure), a PEG-alkyne (to avoid uptake by the RES and improve circulation time) and a hydrophobic alkyne with a thiol (or its synthon) at one end (for the purpose of forming crosslinks within the nanoparticle which will degrade in the presence of the tumor’s reducing environment).

As will be described, the synthesis of the hydrophobic heterobifunctional linker turned out to be a major obstacle in this research. The literature provided many examples of the tosylation or ditosylation of α, ω-akanediols and similar compounds, so we decided to use an α, ω-alkanediol as our starting compound, and then use the tosylate groups as leaving groups for subsequent

42 substitution reactions with sodium azide and the isocyanato group synthon.

The tosylation of an alcohol involves the base-promoted nucleophilic attack of the alcohol’s hydroxyl oxygen on the electrophilic sulfur atom of p-toluenesulfonyl chloride (tosyl chloride,

TsCl). The two most commonly used bases in tosylation reactions are triethylamine[89-92] and pyridine[93-102]. Alternative reagents include N-methylmorpholine[103] and a combination of benzenedimethylamine and potassium hydroxide[104]. Chain lengths of α, ω-alcohols that have been reportedly ditosylated in the literature range from six to sixteen carbons. Yields range from low (31%)[92] to high (96%)[96]. Most reported yields seem to be in the neighborhood of 70-

90%.

There were fewer examples in the literature to guide us on how to proceed from an α,ω- ditosylate to the desired α-azido-ω-isocyanatoalkane. Linear α-azido-ω-isocyanatoalkanes have not been reported in the literature, but one group did report the synthesis of three analogs with one and two methyl groups branching from the alkyl section.[105] Hettche, Reib, Hoffman et al. synthesized (S)-1-azido-4-isocyanotopentane, (2S,4R)-1-azido-4-isocyanato-2-methylpentane and 1-azido-4-isocyanato-2-methylbutane via Curtius rearrangement from the carboxylic acid precursors, but did not report yields for any of the three reactions because they immediately cyclotrimerized their isocyanato groups to form the core of a three-armed anion receptor and they didn’t isolate the isocyanate compounds.

The azido group can be installed via a simple substitution reaction with sodium azide (replacing a leaving group), and isocyanates can be synthesized from amines using triphosgene.[106]

Therefore, we thought that the way to proceed might be to install the azido group on one end of the ditosylate and an amino synthon on the other end. Looking into the literature, we found a few references describing the synthesis of an α-tosylated-ω-phthalimide alkane. Three of these[107-

109] reported the synthesis of the product starting with the corresponding α-amino-ω-alcohols, which was reacted with phthalic anhydride to convert the amino group into a phthalimide group followed by tosylation of the alcohol. Harjani, et al. synthesized the six-carbon linear α-tosyl-ω- phthalimide in one pot from the α-amino-ω-alcohol in 54% overall yield. Nagata, et al. and

Zhou, et al. both synthesized the four-carbon α-tosyl-ω-phthalimide in two separate steps from the α-amino-ω-alcohol in 65% and 51% overall yields, respectively. Bartholoma, et al. reported the synthesis of the eight-, ten-, and twelve-carbon α-tosyl-ω-phthalimides starting with the corresponding α-halo-ω-alcohols, which were reacted with potassium phthalimide in a substitution reaction, again followed by tosylation of the hydroxyl in a second step.[110] We decided to adapt this approach to substitute one of the tosyl groups of our ditosylate rather than substituting a halide to synthesize the α-tosylated ω-phthalimide alkane in a single step.

At the time of our literature search, we found only two references for α-azido-alkyl-ω- phthalimides.[111, 112] Both synthesized their products from the corresponding α-bromo-alkyl-

ω-phthalimides by substitution reaction with sodium azide in DMF. This is a classic azide substitution method which has also been used on compounds other than α,ω-alkanes.[113]

As will be elaborated upon in the Results & Discussion section (below), we ultimately had to pursue alternate pathways to obtain our α-azido-ω-isocyanatoalkane. What we found during our exploration of the literature surrounding α,ω-heterobifunctional alkanes was a general lack of

44 reported syntheses for these compounds. Many of our methods had to be adapted from procedures which were carried out on compounds structurally dissimilar from α,ω- heterobifunctional alkanes.

There was only one reference in the literature for any α-amino-ω-tosylalkane in which the amino group is protected with two tert-butyoxycarbonyl (Boc) groups. Yue, Kiesewetter, Chen et al. monosubstituted 1,2-ethaneditosylate, 1,3-propane ditosylate, and 1,5-pentane ditosylate with di- tert-butyl-iminodicarboxylate (HNBoc2) with yields of 64%, 62%, and 45% respectively. They used 1 equivalent of HNBoc2, 1.1 equivalents of n-butylammonium hydroxide (in 40% aqueous form), ethanol and acetonitrile as solvents, and carried out the reactions at room temperature over

1 hour.[114]

We found no examples in the literature of the synthesis of any α-di-Boc-amino-ω-azidoalkane.

There were a few examples of syntheses of α-mono-Boc-amino-ω-azidoalkanes, however, none of them proceeded from the di-Boc compound. Brady, Zhang, Baell et al. synthesized tert-butyl

(5-azidopentyl)carbamate from tert-butyl (5-hydroxypentyl)carbamate in 76% yield over two steps by first mesylating the alcohol and then substituting with sodium azide.[115] Rowland,

Bostic, Best et al. reported a similar procedure for the synthesis of tert-butyl (6- azidohexyl)carbamate in 97% yield over two steps.[116] Lamanna, Smulski, Bianco et al. synthesized tert-butyl (5-azidopentyl)carbamate in 88% yield from the tosylate by substitution with sodium azide.[117] Kaschani, Clerc, Kaiser et al. achieved the same in 89% yield.[118] All of these groups either started with the Boc-protected amine (Brady, Kaschani) or started with the unprotected amine and Boc-protected it using di-tert-butyl dicarbonate prior to carrying out the

45 tosylation and azide substitution steps (Rowland, Lamanna).

We found no literature precedent for the synthesis of α-azido-ω-isocyanato oligo(ethylene glycols), but one group did report the synthesis of an isothiocyanato analog. Aguilar-Moncayo,

Garcia-Moreno, and Mellet et al. reported the synthesis of 11-azido-3,6,9- trioxaundecylisothiocyanate in 40% yield from the 11-azido-3,6,9-trioxaundecylamine precursor, using 4 equivalents thiophosgene, 8 equivalents calcium carbonate, and a 1:1 DCM-H2O solvent system at 0° C followed by stirring for one hour.[119] Also, Brauch, Henze, and Westermann et al. reported the synthesis of analogous isocyanides (with 5, 6, and 7 ethylene groups).[120]

Unfortunately, neither of these procedures was directly applicable for to our endeavors.

A few things stood out in the literature. As was mentioned above, there is not much published work on linear heterobifunctional compounds with functional groups like Boc-protected amines, azides, and isocyanates. Although α,ω-ditosylates with carbon chain lengths ranging from six to sixteen atoms have been reported, most of the reported syntheses of the other bifunctional compounds we looked at had chains containing four to six carbon atoms. Although yields were generally good (in the 70 – 100% range) for some of these reactions, it is probable that as the chain gets longer, the reactivity might decrease, as more of the molecule’s surface area is taken up by the inert aliphatic carbon chain. However, we wanted a linker that was ten carbon atoms long in order to roughly match the length of our tetra(ethylene glycol)-based hydrophilic linker.

Although none of the papers cited above listed significant problems in the synthesis of their linkers, this is not uncommon. Furthermore, syntheses that do not work are rarely published. As we learned over the course of our attempts at synthesizing a molecular toolkit, the synthesis and

46 purification of aliphatic heterobifunctional linkers is not trivial, which might explain the relative lack of representation of this class of compounds (especially at longer internal chain lengths) in the literature.

The intended applications of these heterobifunctional linkers were diverse. Most were used in the construction of a larger and more complex molecule for various applications. Some unique examples include a three-armed anion-receptor,[105] a pentadentate cobalt ligand with applications in redox catalysis,[108] a cyanine-peptide adduct which fluoresces upon enzyme- catalyzed hydrolysis,[109] a rhenium-containing anticancer drug,[110] and an “azide-dendron” based on an adamantane core with three azide-containing arms designed to click onto other alkyne-containing azide-dendrons in order to grow a dendrimer using click chemistry.[117] None of the references for heterobifunctional linkers that we looked at synthesized them for the purpose of preparing multifunctional nanoparticles.

Synthetic Goals:

As was discussed in the previous section, the components of our molecular toolkit would include:

-Dextran

-Heterobifunctional linkers with an azido group at one end and an isocyanto group (or its synthon) at the other. There will be two variants of these: hydrophobic (alkyl) and hydrophilic

(oligo[ethylene glycol] or OEG).

-Compound with an alkynyl group at one end, and a desired functional group at the other.

Possible functional groups to incorporate include aliphatic chains, hydrophilic PEG chains, thiols, imaging and active targeting agents. However, for the first-generation toolkit, we decided to focus on only the first three.

Figure 5: Azido and alkynyl linkers which form components of the imagined molecular toolkit. 1) 1-azido-2-(2-(2-(2-isocyanatoethoxy)ethoxy)ethoxy)ethane; 2) Propargyl PEG-methyl ether (Mn = 550); 3) 1-azido-10-isocyanatodecane; 4) 1-(prop-2-yn-1-yloxy)dodecane; 5) S-(10-(prop- 2-yn-1-yloxy)decyl) benzothioate.

11-azido-3,6,9-trioxaundecan-1-amine was readily available and can be converted to the isocyanate 1 in a single step using triphosgene. We decided to make the hydrophobic linker of a similar length and so we settled on 1-azido-10-isocyanatodecane (3) for our hydrophobic azide linker. Alkynyl linkers 2 and 4 could be easily synthesized by Williamson ether synthesis using propargyl bromide/alcohol and PEG-methyl ether or dodecyl bromide, respectively. The protected thiol 5 could be synthesized using Mitsunobu conditions from thiobenzoic acid and 10-

(prop-2-yn-1-yloxy)decan-1-ol, the latter of which could be synthesized by Williamson ether synthesis between propargyl bromide and 1,10-decanediol. This was convenient for us since we had also decided to use the same diol as a starting point to synthesize 3.

Results & Discussion:

As a preliminary “proof of concept”, we attempted an isocyanate-dextran coupling reaction using

4-iodophenyl isocyanate. This compound was chosen for two reasons: 1) it was commercially available and 2) the aromatic protons would stand out in an NMR spectrum of the product, making it easy to determine whether the reaction was successful. The reaction was carried out using pyridine as both the solvent and base,[121] on the reaction of 4-iodophenyl isocyanate with

1,4-benzenediol as a guide in addition to the aforementioned 2011 Frechet group paper.[65] This experiment was successful, yielding a material both visually and spectrally distinct from unfunctionalized dextran, with the expected peaks in the aromatic region of the 1H-NMR spectrum in addition to a strong peak at 3.89 ppm indicating the presence of a dextran backbone.

The ratio between the integrals of the aliphatic proton signals (which are contributed by the dextran backbone) and those of the aromatic signals (which the 4-iodophenyl group contributes) was 158.5 : 4.0 which means that on average, one out of every 22.64 monomers was substituted.

This works out to a 4.42% degree of substitution. The fact that the product was dialyzed rules out the possibility of the aromatic signals originating from unreacted 4-iodophenyl isocyanate. The material which was obtained after lyophilization was off-white and had a habit of clinging to the sides of its plastic container, whereas pure dextran is white and tends to stick to itself more.

Figure 6: 35-45 kDa Dextran (left) and 4-iodophenyl dextran carbamate (DoS = 4.42%)

There was a precedent in the literature for introducing the carbamate group to dextran by reaction with an isocyanate.[122] That case was distinct from what we have done, however, because Taniguchi et al. grafted dextran onto another polymer (ethylene-vinyl alcohol copolymer) whereas we have decorated dextran with a small molecule using the carbamate functional group as a linkage.

The hydrophilic linker 1 has been reported in the literature previously, although it was synthesized by a different route from what we used.[120] We synthesized the linker in 91% yield by self-catalyzed isocyanation of 11-azido-3,6,9-trioxaundecan-1-amine in dry ethyl acetate, following the general procedure for isocyanation of amines reported by Charalambides, et al.[106] Because the reaction is self-catalyzed, there is no base to remove. The only other reagent is triphosgene, which decomposes to phosgene gas, so that no purification is necessary.

Because 1 and its amino precursor are structurally similar, it was difficult to confirm the identity of the product and consumption of the precursor using NMR methods. Therefore, the primary method for determining whether and to what extent an isocyanation reaction has occurred is infrared spectroscopy. In order to demonstrate the transformation of the amino group, the synthesis of 1 was repeated, only this time a small aliquot of the reaction solution was carefully removed from the flask and transferred to a 20-dram vial each hour. After removing the ethyl

51 acetate under reduced pressure, the remaining oil was quickly analyzed by FT-IR on an ATR machine. This was done every hour for 4 hours and then a final time after 5.5 total hours had elapsed. A strong isocyanate signal (2258 cm-1) was observed after the first hour, and the amino group signal (3348 cm-1) had only one peak, suggesting that all of the amino groups in the flask had reacted with triphosgene to form the R-N(H)-C(O)-O-CCl3 intermediate. By hour 4, the amino group signal had shrunk noticeably, and by hour 5.5, it had disappeared altogether. These data suggest that the reaction of the amine with triphosgene happens quickly, whereas the subsequent deprotonation of the carbamate intermediate and decomposition of the –O-CCl3 group to another equivalent of phosgene to give the isocyanate occurs over takes at least 3 hours.

This is not surprising, given that the amine (or perhaps the carbamate intermediate) is standing in as the base. Use of a base might speed up the reaction, but would almost certainly complicate the purification. Since isocyanato groups are not particularly stable at room temperature, unnecessary purification must be avoided whenever possible

Hydrophilic PEG-alkyne 2 was synthesized according to a literature procedure.[123] The

Williamson Ether method was used to couple propargyl bromide with PEG-monomethyl ether with number average molecular weight of 550 g/mol. Although the product was obtained as a white waxy solid, its mass exceeded the theoretical yield, and per cent yield was not determined.

The 1H-NMR spectrum clearly contained peaks indicating the presence of the propargyl methylene group (d, 4.20 ppm, 2H) terminal methyl group (s, 3.37 ppm, 3H), and a large signal at 3.64 ppm that most likely corresponds to the internal ethylene groups of the PEG chain.

The synthesis of the hydrophobic linker 3 proved to be much more challenging than we had suspected. Initially, we planned to synthesize the isocyanate by self-catalyzed triphosgenation as we did with 1. The amino group would be installed as a phthalimide and then converted using

Gabriel synthesis (Scheme 2). After a few attempts, we found that the best method for obtaining the ditosylate 6 from the starting diol was by a procedure reported by Luscombe, et al. in which pyridine is used as both the base and the solvent.[93] We obtained the ditosylate 6 in 74% yield.

The phthalimide group was installed by substitution reaction with potassium phthalimide according to a literature procedure[110] to yield 7 in 44% yield. We used a 3:1 excess of ditosylate compared with potassium phthalimide in acetonitrile in order to limit disubstitution,

53 with the expectation that unreacted ditosylate could be recovered and re-used. The azide 8 was obtained by substitution of 7 with sodium azide in N,N-dimethylformamide (DMF). 8 was treated with hydrazine in isopropyl alcohol in order to try to obtain 9, the amine precursor to 3.

However, during purification the product stuck to the silica gel and would not come off, likely due to the basicity of primary amines and the acidity of silicon dioxide. To avoid this problem, we doped a silica column with triethylamine by washing it with 1:19 triethylamine:hexane before attempting to purify the amine a second time. Unfortunately, the product still did not come off.

We attempted to purify 9 using an alumina column and even a reverse-phase column, but these efforts were also unsuccessful.

After the 10-azido-decan-1-amine proved difficult to purify, we attempted to synthesize 3 via a pathway that avoided dealing with the free amine. Instead of potassium phthalimide, the ditosylate was reacted with di-Boc-protected amine and cesium carbonate (to increase the nucleophilicity of the amine and accept the extra proton). As with the synthesis of 7, the synthesis of 10-[bis(tert-butoxycarbonyl)amino]decyl 4-methylbenzenesulfonate (10) was performed using a 3:1 ratio of ditosylate to the HNBoc2 in order to promote monosubstitution. 10

55 was obtained in 71% yield. The next step was substitution of the remaining tosyl group of 10 with sodium azide in DMF as before. The azide 11 was obtained in 89% yield. Treating 11 with trifluoroacetic acid gave the triflate salt (12) of the amine in 100% yield. Attempts to synthesize

3 directly from 12, as was reported (with the isocyanate as an unisolated intermediate) by

Biitseva et al.,[124] were not successful.

At this point, we went back into the literature to look for an alternate pathway to 3 which avoided the primary amine and its salt altogether. We found a method[125] by which Boc- protected amines can be converted directly to isocyanates using Banwell’s reagent (triflic anhydride and 4-dimethylaminopyridine).[126] However, this process only works on amines with one Boc-group so one of the Boc groups of 11 had to be selectively removed while preserving the other one.

Figure 7: A plausible mechanism of isocyanation of a boc-protected amine by Banwell’s reagent.

Another search of the literature offered up a method[127] whereby this selective Boc-removal can be accomplished using magnesium perchlorate as a catalyst. Because the magnesium perchlorate forms temporary H-bonds with the carbonyl oxygen atoms of both of the precursor’s

Boc-groups, the catalyst is unable to act on an amine which has only one Boc-group attached to it. Following this method we synthesized the mono-Boc-protected amine 13 from its di-Boc- protected precursor 11 in 80% yield. We then used Banwell’s reagent to attempt to convert 13 to the desired isocyanate 3 using according to the aforementioned literature procedure.[125]

Although we obtained the crude product in 100% yield, due to the number of steps required to reach this point, we did not have enough material to verify the product’s purity or purify it if needed. For this reason, this step was performed using commercially available di-Boc-protected dodecyl amine so that we could practice and work out workup conditions that would yield the

57 pure product without requiring further purification.

We chose dodecyl amine, which we Boc-protected using di-tert-butyl dicarbonate according to an established literature procedure.[128] This yielded the Boc-protected amine 14, although the per cent yield could not be determined due to some product spillage. We then used Banwell’s reagent to convert the carbamate 14 to dodecyl isocyanate (15). After two unsuccessful attempts, we found a workup procedure which removed most of the impurities to give the dodecyl isocyanate 15 in 100% yield. After quenching the reaction with saturated aqueous sodium bicarbonate, the organic layer was washed with 10 ml of 0.1N HCl. This was calculated to be enough to protonate the remaining 1 equivalent of 4-dimethylaminopyridine and hydrolyze the remaining 3 equivalents of triflic anhydride from the Banwell’s reagent.

The syntheses of the alkynyl functional group linkers 4 and 5 were relatively straightforward. 1-

(Prop-2-yn-1-yloxy)dodecane (4) was synthesized from dodecyl bromide and propargyl alcohol using the Williamson ether method according to literature procedure,[129] and was obtained in

55% yield after purification on silica. 10-Hydroxydecyl phenyl sulfanylformate (5) was synthesized in two steps. First, decane-1,10-diol was reacted with propargyl bromide under

Williamson ether conditions to obtain 10-prop-2-ynoxydecan-1-ol (16) in 34% yield after purification, according to literature procedure.[130] 16 then underwent Mitsunobu reaction with thiobenzoic acid, triphenylphosphine, and diisopropyl azodicarboxylate (DIAD) to yield 5, which after purification was obtained in 81% yield.

One of the initial goals of this project was to develop functional linkers that would form a carbamate linkage to dextran, which we believe will be stronger than the carbonate linkage which is reported widely in the literature. To this end, we synthesized 10-azidodecyl 1H- imidazole-1-carboxylate (18) in two steps from 10-bromo-1-decanol. First, the latter was substituted with sodium azide to give 10-azidodecan-1-ol (17) in 53% yield, followed by reaction with 1,1’-carbonyl diimidazole (CDI) to give the product in 93% yield. No column chromatography was necessary.

Summary & Conclusions for Chapter 2:

Our original goal for this project was to see if multifunctional nanoparticles could be fabricated from prefunctionalized polymer units (PPUs) in a reproducible manner which would allow researchers and eventually clinicians to control the physicochemical properties while also being able to synthesize new nanoparticles quickly and easily from a limited set of PPUs. Although we did not meet this goal, the components for assembling a preliminary toolkit of dextran-based

PPUs have been synthesized. Furthermore, we have been demonstrated that organic isocyanates

60 can be reacted with dextran to form carbamate-linked functionalized dextrans with our synthesis of 4-iodophenyl dextran carbamate.

During our attempts at reaching our target hydrophobic linker 3, one of the most time-consuming aspects was the purification of the long, oil-like compounds, which often took several dozen fractions to elute. The difficulty of purifying and working with these longer aliphatic chains may be a reason for the general dearth of literature on these α, ω-heterobifunctional linkers, and also for why the literature that does exist mostly reports on shorter analogues having around six internal carbon atoms.

Experimental: Synthetic Procedures for Chapter 2:

All reactions were performed in flame-dried, argon-flushed flasks unless stated otherwise.

Dextran was provided for us by the Amiji group. Pyridine, poly(ethylene glycol) methyl ether

(Mn = 550), 4-iodophenyl isocyanate, sodium hydride, decane-1,10-diol, triphosgene, 11-azido-

3,6,9-trioxaundecan-1-amine, potassium phthalimide, di-tert-butyl-iminodicarboxylate, 4- dimethylaminopyridine, di-tert-butyl dicarbonate, propargyl alcohol, triphenylphosphine, trifluoroacetic acid, magnesium perchlorate and 1,1’-carbonyldiimidazole were purchased from

Aldrich or Sigma-Aldrich. Propargyl bromide, anhydrous dichloromethane, anhydrous acetonitrile, anhydrous N,N-dimethylformamide, sodium azide, diethyl ether, and sodium bicarbonate were purchased from Acros. P-toluenesulfonic sulfonyl chloride, magnesium sulfate, ethyl acetate, acetonitrile, methanol, hexane, and 10-bromo-1-decanol were purchased from

Fisher. Tetrahydrofuran was purchased from Acros and dried on site.

Dextran 4-iodophenyl carbamate:

Dextran with a molecular weight of 35-45 kDa (0.042 g, ~0.00105 mmol dextran or ~0.259 mmol glucose monomers) was added to a 20 ml vial. Pyridine was added to the vial in 1 ml increments until it was half full (~10 ml total). The dextran dissolved readily. 4-Iodophenyl isocyanate (0.136 g, 0.555 mmol) was added to the vial. The solution was stirred. After a few minutes, it was clear that not all of the reactants had dissolved, so the contents of the vial were transferred to a larger round-bottom flask (250 ml) and more pyridine (20 ml) was added. The reactants fully dissolved. The solution was stirred for two days. The pyridine was evaporated under reduced pressure. The remaining solid was dissolved in dimethyl sulfoxide (140 ml). After the solid had fully dissolved, 20 ml of this solution was taken and dialyzed against DI water. The dialysis tube was permeable only to molecules with MW < 12 kDa. The water was changed every half hour for the first few hours. After one day, the product had precipitated inside the dialysis tube. The contents of the tube were centrifuged and the majority of the remaining solvent was removed. The product was chilled in a freezer, then lyophilized (freeze-dried) overnight. The lyophilized product was a dry, flaky, beige-colored solid.

1H NMR (399 MHz, DMSO-d6) δ ppm 3.82 - 3.91 (m, 77 H) 7.28 (d, J=8.06 Hz, 1 H) 7.54 (d,

J=8.79 Hz, 1 H)

IR cm-1 635, 751, 769, 815 (strong), 1003, 1357, 1236, 1279, 1482, 1550, 1588, 1634, 3295

(hydroxyl)

1-Azido-2-(2-(2-(2-isocyanatoethoxy)ethoxy)ethoxy)ethane (1):

1-Azido-2-(2-(2-(2-isocyanatoethoxy)ethoxy)ethoxy)ethane has been reported in the

62 literature.[120] It was synthesized according to a different procedure.[106] A 100 ml two-necked round-bottomed flask was attached to a reflux condenser and then flame-dried under argon. The flask was then charged with triphosgene (0.604 g, 0.94 mmol) dissolved in 10 ml freshly dry ethyl acetate (freshly dried over molecular sieves). While stirring, 11-azido-3,6,9-trioxaundecan-

1-amine (372 ml, 1.87 mmol) dissolved in dry ethyl acetate (10 ml) was slowly added to the flask. The flask was heated until reflux was achieved. After 3 hours the reaction was cooled to room temperature. The solvent was evaporated under reduced pressure, yielding the product as a reddish brown oil in 91% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 3.34 (dt, J=7.69, 4.95 Hz, 3 H) 3.55 - 3.59 (m, 2

H) 3.62 (d, J=2.93 Hz, 6 H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 43.36, 50.84, 70.19, 70.55, 70.69, 70.83,

70.85, 125.31

-1 IR cm 556, 585, 634, 819, 934, 113 (strong, C-O), 1284, 1347, 1444, 1775, 2097 (strong, -N3),

2221, 2259 (strong, -N=C=O), 2869

Propargyl-PEG-methyl ether (2):

A propargyl-PEG-methyl ether (Mn = 4600 g/mol) has been reported previously and we synthesized ours (Mn = 550) according to literature procedure.[123] A flame-dried round- bottomed flask was charged with sodium hydride (60% by weight in paraffin oil; 139 mg, 3.158 mmol) and freshly dried tetrahydrofuran (15 ml) under argon. Poly(ethylene glycol) methyl ether

(Mn = 550; 1.158 g, 2.105 mmol) was dissolved in 2 ml dry THF, then the solution was added to the flask with the sodium hydride. The solution fizzed upon addition of the PEG-methyl ether, then gradually turned yellow-green over the next 90 minutes. After this time, propargyl bromide

(80% by volume in toluene; 0.5 ml, 4.211 mmol) was added in a dropwise manner. The solution became cloudy and took on a brownish color. After stirring for two days at room temperature, glacial acetic acid was added to quench any unreacted sodium hydride. Aqueous calcium carbonate was added until the mixture had a pH of 7.4. The solvents were removed under reduced pressure, yielding a white, waxy solid. The crude yield exceeded the theoretical yield, suggesting that the product was not completely dry. The product was not lyophilized, and the pure per cent yield was not determined.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 1.75 (br. s., 4 H) 3.37 (s, 3 H) 3.52 - 3.56 (m, 3

H) 3.62 - 3.67 (m, 71 H) 4.20 (d, J=2.20 Hz, 2 H)

Decane-1,1-diyl bis(4-methylbenzenesulfonate) (6):

Decane-1,1-diyl bis(4-methylbenzenesulfonate) has been reported previously, and was synthesized according to literature procedure.[93] Decane-1,10-diol (5.204 g, 29.86 mmol) was crushed into a fine powder and then dissolved in anhydrous pyridine (20 ml). P-toluenesulfonyl chloride (14.621 g, 76.69 mmol) was added while stirring. After half an hour, the mixture had become a white solid. Glacial acetic acid (20 ml) and dichloromethane (200 ml) were added to the flask to dissolve the solid. A metal spatula was used to help break the solid up into smaller pieces. Once the solid had completely dissolved, the solution was transferred to a wide-mouthed

Erlenmeyer and washed with deionized water (100 ml, twice), 2N HCl (100 ml, twice) and brine

(100 ml). The aqueous layers were decanted, and the combined aqueous layer was back-extracted with dichloromethane (100 ml). The organic layers were combined and concentrated under reduced pressure. The concentrated solution was poured into a stirring solution of 4:1 hexane:ethyl acetate. The solution immediately turned opaque and white. The white solid

64 precipitate was filtered and washed with 4:1 hexane:EtOAc, then collected and re-dissolved in

DCM. After concentrating to dryness again under reduced pressure, the crude product was nearly pure. The crystallization procedure was repeated to yield pure decane-1,1-diyl bis(4- methylbenzenesulfonate) in 74% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 1.17 (br. s., 8 H) 1.26 (d, J=6.60 Hz, 6 H) 1.61

(dt, J=13.74, 6.69 Hz, 5 H) 2.44 (s, 5 H) 4.00 (t, J=6.23 Hz, 4 H) 7.34 (d, J=8.06 Hz, 3 H) 7.78

(d, J=8.06 Hz, 3 H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 21.85, 25.52, 29.03, 29.06, 29.41, 70.88,

128.09, 130.02, 133.50, 144.85

Melting Point = 107.4 °C

10-(1,3-dioxoisoindolin-2-yl)decyl 4-methylbenzenesulfonate (7):

10-(1,3-dioxoisoindolin-2-yl)decyl 4-methylbenzenesulfonate has been previously reported and was synthesized according to literature procedure.[110] A 500 ml round-bottom flask was charged with potassium phthalimide (0.089 g, 0.48 mmol), 6 (0.654 g, 1.3260 mmol), and 40 ml acetonitrile. The mixture was refluxed with stirring for 3.5 hours. After cooling to room temperature, the flask was placed into an ice bath. The white solid that precipitated was collected via Hirsch funnel filtration. The remaining solution was concentrated under reduced pressure to yield a yellow substance. This material was purified on silica using 1:49 methanol:dichloromethane. The product was obtained in 44% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 1.14 - 1.31 (m, 13 H) 1.55 - 1.67 (m, 4 H) 2.42

(s, 2 H) 3.64 (t, J=7.33 Hz, 2 H) 3.98 (t, J=6.60 Hz,

2 H) 7.32 (d, J=8.06 Hz, 1 H) 7.69 (dd, J=5.50, 2.56 Hz, 1 H) 7.76 (d, J=8.06 Hz, 1 H) 7.81 (dd,

J=5.86, 2.93 Hz, 1 H)

13C NMR (100.37 MHz, CHLOROFORM-d): δ ppm 21.87, 25.51, 27.02, 28.79, 29.00, 29.08,

29.31, 29.48, 29.51, 38.24, 70.93, 123.38, 128.10, 130.02, 132.36, 133.38, 134.10, 144.84,

168.70

2-(10-Azidodecyl)isoindoline-1,3-dione (8):

2-(10-Azidodecyl)isoindoline-1,3-dione has not been reported in the literature. It was synthesized according to a literature procedure for a different compound.[113]

7 (0.204 g, 0.446 mmol) was dissolved in N,N-dimethylformamide (6 ml). A two-necked 250 ml flask was charged with sodium azide (0.096 g, 1.48 mmol) and the solution of 7. The mixture was stirred at 100 °C for 20 hours. The amber-brown solution was diluted with deionized water

(60 ml) after cooling to room temperature. The solution immediately turned milky white. The aqueous layer was extracted with dichloromethane (3 x 40 ml). The organic layer was then combined and washed with brine (2 x 40 ml) followed by deionized water (2 x 40 ml). The organic layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The aqueous layer was back-extracted with DCM, and the extract was dried over magnesium sulfate and concentrated under reduced pressure as well. The combined concentrated crude oil was purified on silica with DCM as the mobile phase. The pure product was collected in 40% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 1.11 - 1.36 (m, 17 H) 1.45 - 1.61 (m, 4 H) 1.61

- 1.71 (m, 2 H) 3.12 - 3.27 (m, 2 H) 3.53 - 3.70 (m, 3 H) 7.70 (dd, J=5.50, 3.30 Hz, 1 H) 7.81 -

7.86 (m, 1 H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 26.90, 29.31, 29.56, 38.27, 51.69, 123.38,

132.39, 134.07, 168.71

-1 IR cm 1697 (C=O), 2097 (-N3), 2848, 2909

10-[bis(tert-butoxycarbonyl)amino]decyl 4-methylbenzenesulfonate (10):

10-[bis(tert-butoxycarbonyl)amino]decyl 4-methylbenzenesulfonate has not been reported in the literature. A 500 ml round-bottomed flask was charged with acetonitrile (30 ml), 6 (0.216 g, 2.89 mmol), di-tert-butyl-iminodicarboxylate (1.397 g, 0.965 mmol) and cesium carbonate (0.480 g,

1.47 mmol). The solution was stirred at 80 °C for 5 hours. After cooling to room temperature, the acetonitrile was evaporated under reduced pressure. The crude product (a solid) was dissolved in dichloromethane, leaving the insoluble cesium salts behind. The solution was washed with deionized water (2 x 100 ml) followed by brine (2 x 100 ml). The aqueous layers were back- extracted with DCM and the combined organic layers were dried over magnesium sulfate. The magnesium sulfate was removed via Hirsch funnel. The solvent was evaporated under reduced pressure. The crude product was purified on silica with 10 – 20% ethyl acetate in 90 – 80% hexane as the mobile phase. The pure product was obtained as a clear oil in 71% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 1.22 (d, J=15.39 Hz, 12 H) 1.49 (s, 16 H) 1.59 -

1.66 (m, 2 H) 2.44 (s, 3 H) 3.52 (t, J=7.33 Hz, 2 H)

4.00 (t, J=6.60 Hz, 2 H) 7.34 (d, J=7.33 Hz, 2 H) 7.78 (d, J=8.06 Hz, 2 H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 21.84, 25.51, 26.98, 28.28, 28.98, 29.09,

29.22, 29.44, 29.48, 29.61, 46.66, 70.90, 82.17, 128.07, 130.01, 133.34, 144.85, 152.91

IR cm-1 658, 1089, 1121, 1175, 1255, 1385, 1438, 1503, 1661 (strong, C=O), 2857, 2929

Tert-butyl N-(10-azidodecyl)-N-tertbutoxycarbonylcarbamate (11):

Tert-butyl N-(10-azidodecyl)-N-tertbutoxycarbonylcarbamate has not been reported in the literature. Synthesis was performed according to a literature procedure for a different compound.[113] 10 (1.460 g, 2.77 mmol) was dissolved in N,N-dimethylformamide (30 ml).

While stirring at room temperature, sodium azide (1.863 g, 28.7 mmol) was added slowly. The reaction mixture was left stirring at 70 °C overnight. The mixture was poured into ice water (300 ml) and extracted with diethyl ether (4 x 50 ml). The organic layer was washed twice each with deionized water and brine (each portion was equal to half the height of the liquid in the separatory funnel). The organic layer was dried over magnesium sulfate which was then removed via Hirsch filtration. The solvent was evaporated under reduced pressure. The crude oil was purified on silica with a mobile phase gradient from pure hexane to 5% ethyl acetate to 95% hexane to give the product as an oil in 89% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 1.26 (br. s., 9 H) 1.48 (s, 17 H) 1.52 - 1.60 (m,

4 H) 3.23 (t, J=6.96 Hz, 2 H) 3.48 - 3.55 (m, 2 H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 26.90, 26.99, 28.27, 28.30, 29.03, 29.22,

29.31, 29.46, 29.57, 29.64, 46.68, 51.67, 82.15, 152.94

IR cm-1 734, 780, 856, 888, 1034, 1120 (strong), 1174, 1255, 1297, 1346, 1366 (strong), 1392,

1456, 1694, 1716, (strong) 1743, 1788, 2094 (-N3), 2856, 2928, 2979

10-Azidodecan-1-aminium triflate (12):

10-Azidodecan-1-aminium triflate has not been reported in the literature. 11 (0.101 g, 0.253 mmol) was dissolved in dichloromethane (5 ml) and placed in a flame-dried 100 ml round- bottom flask. Trifluoroacetic acid (0.5 ml, 6.273 mmol) was added and the solution was stirred

68 for 2 hours. After all of the starting material had been consumed, the solvent was removed under reduced pressure, giving the triflate salt of 10-azidodecylammonium in 100% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 1.26 (br. s., 17 H) 1.52 - 1.68 (m, 5 H) 2.53 (d,

J=11.72 Hz, 1 H) 2.90 (br. s., 3 H) 3.24 (t, J=6.96 Hz, 2 H) 3.55 (s, 1 H) 3.79 (t, J=8.06 Hz, 1 H)

7.78 (br. s., 4 H) 11.99 (br. s., 1 H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 26.46, 26.87, 27.63, 29.01, 29.11, 29.27,

29.38, 29.50, 40.22, 51.65, 162.08

-1 IR cm 721, 797, 836, 1129 (strong), 1172, 1306, 1429, 1470, 1532, 1665 (strong), 2095 (-N3),

2854, 2929, ~3100 (broad)

Tert-butyl N-(10-azidodecyl) carbamate (13):

Tert-butyl N-(10-azidodecyl) carbmate has been reported in the literature.[131] However, it was synthesized via a different route. We adapted a different literature procedure[127] to synthesize

13. 11 (0.188 g, 0.472 mmol) was dissolved in acetonitrile (5 ml). This solution was added to a flame-dried 25 ml round-bottom flask. While stirring, magnesium perchlorate (0.028 g, 0.127 mmol) was added. The contents of the flask were stirred at 50 °C for 3 hours. After cooling to room temperature, the acetonitrile was evaporated under reduced pressure. The product was partitioned between ether and 0.3 N HCl. The organic layer was washed with brine. The aqueous layers were combined and back-extracted with dichloromethane. The combined organic layer was dried over magnesium sulfate which was then removed via Hirsch filtration. The solvent was evaporated under reduced pressure to yield the pure product in 86% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 1.25 (br. s., 10 H) 1.41 (s, 10 H) 1.52 - 1.60 (m,

2 H) 3.02 - 3.11 (m, 2 H) 3.22 (t, J=6.96 Hz, 2 H) 4.54 (br. s., 1 H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 16.89, 26.97, 28.62, 29.03, 29.31, 29.44,

29.57, 29.62, 30.26, 40.80, 51.66, 79.14, 156.19

-1 IR cm 1167 (strong), 1248 (strong), 1365, 1391, 1456, 1515, 1690 (strong), 2093 (strong, -N3),

2855, 2926, 2976, 3355 (broad, -N-H)

1-azido-10-isocyanatodecane (3):

1-Azido-10-isocyanatodecane has not been reported in the literature. Synthesis was performed according to a literature procedure for conversion of N-tert-butyl carbamates to isocyanates.

[125] To a stirring solution of 13 (0.074 g, 0.248 mmol) in dry dichloromethane (10 ml) was added a 1.0 M solution of triflic anhydride (1.24 mmol) in dry DCM (1 ml) followed by 4- dimethylaminopyridine (0.092 g, 0.744 mmol) at 0 °C. The vial was capped and stirring continued for 30 minutes. The reaction was quenched with a saturated aqueous solution of sodium bicarbonate (10 ml) and then diluted by a factor of ten with DCM. The combined solution was washed twice with brine in a separatory funnel. The combined aqueous layers were back-extracted twice with DCM. The combined organic layers were dried over magnesium sulfate which was removed via Hirsch filtration. The solvent was evaporated under reduced pressure to yield an orange-brown greasy solid in 100 % (crude) yield. Due to the small amount of material (0.057 g), the number of steps involved in getting to this product, and the reactivity of the isocyanato functional group, this step and its workup was practiced using N-tert-butyl dodecyl carbamate synthesized from commercially available dodecylamine.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 1.19 - 1.40 (m, 12 H) 1.53 - 1.65 (m, 3 H) 3.17

- 3.31 (m, 4 H) 6.67 - 6.82 (m, 1 H) 8.11 (br. s., 1H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 26.47, 26.72, 26.81, 26.90, 29.03, 29.17,

29.20, 29.29, 29.54, 30.08, 30.41, 31.48, 40.28, 41.81, 43.20, 44.63, 48.53, 51.69, 149.90

IR cm-1 509 (strong), 573, 604, 631, 800, 997, 1029, 1131-1147, 1190-1212 (strong), 1270, 1372,

1397, 1444, 1538-1564, 1647, 1732, 2093.82 (-N3), 2276 (-N=C=O), 2856, 2927, 3264

Tert-butyl dodecyl carbamate (14):

Tert-butyl dodecyl carbamate has been reported in the literature.[132] Synthesized according to another literature procedure.[128] Dodecylamine (0.924 g, 4.99 mmol) was dissolved in dry dichloromethane (10 ml) and added to a flame-dried 50 ml round-bottom flask. The solution was stirred and cooled to 0 °C. Di-tert-butyl dicarbonate (1.390 g, 6.37 mmol) was dissolved in dry

DCM (5 ml) and added to the flask. A white precipitate immediately formed and appeared to fill the flask. The reaction was stirred overnight. In the morning, the white precipitate appeared to be gone, replaced by a clear solution. The solution became cloudy when disturbed. The solvent was evaporated under reduced pressure leaving behind a white waxy/soapy solid. The crude product was purified on silica using a gradient of hexane followed by 9:1 hexane: ethyl acetate. Per cent yield was not determined.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 0.87 (t, J=6.60 Hz, 2 H) 1.25 (s, 16 H) 1.43 (s,

9 H) 3.10 (q, J=5.86 Hz, 2 H) 4.49 (br. s., 1 H)

Dodecyl isocyanate (15):

Dodecyl isocyanate is commercially available. Due to the number of steps in synthesizing the isocyanate 3, we decided to synthesize 15 as a substitute for practice and to develop workup conditions which would yield us the isocyanate with no need for further purification on a

71 column. 15 was synthesized according to literature procedure.[125] A 50 ml, flame-dried flask was charged with 14 (0.087 g, 0.305 mmol) and 4-dimethylaminopyridine (0.111 g, 0.909 mmol). The flask was sealed and flushed with argon once again. Dry dichlormethane (10 ml) was added, followed by a solution of triflic anhydride (0.25 ml, 1.5 mmol) dissolved in dry DCM (12 ml). The reaction was stirred under argon at 0 °C for 30 minutes, then quenched with a saturated aqueous solution of sodium bicarbonate (20 ml). Hydrochloric acid (0.1 N, 10 ml) was added.

The contents of the flask were transferred to a separatory funnel and shaken. After removing the aqueous layer, the organic layer was washed with more HCl (0.1 N, 15 ml) followed by two portions of brine (15 ml each). When the pH of the organic layer was 7 it was dried over magnesium sulfate. The magnesium sulfate was removed by Hirsch filtration and the solvent was evaporated under reduced pressure to give the product as a yellow oil in 100% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 0.87 (t, J=6.60 Hz, 3 H) 1.25 (br. s., 20 H) 1.43

(br. s., 1 H) 1.55 - 1.64 (m, 2 H) 3.28 (t, J=6.96 Hz, 2 H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 14.34, 22.91, 26.46, 26.75, 28.62, 29.19,

29.22, 29.57, 29.64, 29.70, 29.73, 29.77, 29.83, 30.42, 31.52, 32.14, 43.22, 44.73, 118.31, 121.51

IR cm-1 607, 1073, 1147, 1187 (strong), 1230, 1371, 1459, 2269.72 (-N=C=O), 2854, 2923, 3314

(weak)

1-(Prop-2-yn-1-yloxy)dodecane (4):

1-(Prop-2-yn-1-yloxy)dodecane has been reported previously[133] but was synthesized according to literature procedure for 1-(prop-2-yn-1-yloxy)octane.[129] A 500 ml oblong round- bottomed flask flame-dried under argon was charged with 0.492 g of sodium hydride (60% by weight in paraffin oil; 481 mg, 8.025 mmol). The flask was flushed once more with argon. N,N-

72 dimethylformamide (25 ml) was added to the flask. With stirring, the flask was cooled in an ice bath to 0 °C. Propargyl alcohol (1 ml, ~17 mmol) was added. The flask was removed from the ice bath. After returning to room temperature, the flask was charged with dodecyl bromide (1.9 ml, ~8.0 mmol). The flask was heated to 70 °C and stirred overnight. The reaction was cooled to room temperature and quenched with methanol (10 ml). The DMF was diluted with deionized water (250 ml). The resulting aqueous layer was extracted with dichloromethane (2 x 80 ml). The organic layer was washed with two portions each of brine and deionized water (a portion is half the height of the liquid in the separatory funnel). The organic layer was dried over magnesium sulfate, which was then removed with Hirsch filtration. The solvent was evaporated under reduced pressure. The crude product was purified on silica with a gradient of 1:1 hexane:dichloromethane followed by pure DCM. The product eluted as a clear oil in 55% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 0.84 (t, J=6.60 Hz, 2 H) 1.22 (br. s., 19 H) 1.54

(quin, J=6.96 Hz, 2 H) 2.36 (t, J=2.20 Hz, 1 H) 3.45 (t, J=6.60 Hz, 2 H) 4.07 (d, J=2.20 Hz, 2 H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 14.26, 22.88, 26.31, 29.57, 29.66, 29.72,

29.80, 29.82, 29.86, 29.89, 32.14, 58.11, 70.36, 74.15, 80.17

10-Prop-2-ynoxydecan-1-ol (16):

10-Prop-2-ynoxydecan-1-ol has been previously reported and was synthesized according to literature procedure.[130] A flame-dried, argon-flushed flask was charged with sodium hydride

(60% by weight in paraffin oil; 0.380 g, 9.501 mmol) followed by dry tetrahydrofuran (3 ml).

While the solution was stirring, decane-1,10-diol (1.657 g, 9.508 mmol) was dissolved in dry

THF (2 ml) in a separate vial. N,N-dimethylformamide (6 ml) was added to help the diol dissolve. This solution was added to the stirring solution in the flask. After 30 minutes propargyl

73 bromide (0.84 ml, 14.129 mmol) was added dropwise to the stirring solution. The contents of the flask changed from an opaque off-white color to a darker tan. The next day, the contents of the flask were a light tan or yellow-orange color. After three more days, the solids were filtered out and washed with diethyl ether. The filtrate was re-filtered two more times to remove all solids, then washed with brine (two portions; a portion is half the height of the liquid in the separatory funnel) and deionized water (two portions). The organic layer was dried over magnesium sulfate, which was then removed with Hirsch filtration. The solvent was evaporated under reduced pressure to yield the crude product as a dark, clear, amber-colored viscous liquid with a lighter- colored precipitate. The crude product was purified on silica with a gradient of pure hexane followed by 2:1 hexane:EtOAc to yield the pure product as a yellow oil in 34% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 1.24 (br. s., 14 H) 1.52 (dquin, J=14.11, 6.92,

6.92, 6.92, 6.92 Hz, 4 H) 2.12 (br. s., 1 H) 2.38 (t, J=2.20 Hz, 1 H) 3.45 (t, J=6.60 Hz, 2 H) 3.56

(t, J=6.60 Hz, 2 H) 4.08 (d, J=2.20 Hz, 2 H)

10-Hydroxydecyl phenylsulfanylformate (5):

10-Hydroxydecyl phenylsulfanylformate has not been reported in the literature. It was synthesized according to a literature procedure for a similar compound.[134] A flame-dried, argon-flushed round-bottomed flask was charged with 16 (0.680 g, 3.2 mmol), thiobenzoic acid

(0.414 ml, 3.52 mmol), triphenylphosphine (1.121 g, 4.160 mmol) and anhydrous tetrahydrofuran (6 ml). The flask was cooled to 0 °C. Diisopropyl azodicarboxylate (0.45 ml,

2.285 mmol) was added dropwise. The solution was stirred for two days, after which diethyl ether (30 ml) was added to the flask. The organic solution was washed with saturated aqueous sodium bicarbonate (2 x 20 ml), deionized water (2 x 20 ml), and brine (2 x 20 ml). The aqueous

74 layers were combined into one and back-extracted with ether (40 ml). The combined organic layer was dried with magnesium sulfate, which was then removed by Hirsch filtration. The solvent was evaporated under reduced pressure to yield the orange-colored crude product. The crude product was purified on silica with a gradient of 60:40 hexane: DCM; 60:40 DCM:hexane;

80:20 DCM:hexane; 100% DCM; 1:19 methanol:DCM. The product eluted as a thick, honey- colored oil in 81% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 0.88 (t, J=6.60 Hz, 2 H) 0.96 (d, J=6.60 Hz, 1

H) 1.29 (br. s., 14 H) 1.37 - 1.47 (m, 3 H) 1.54 - 1.61 (m, 4 H) 1.67 (dt, J=14.84, 7.60 Hz, 3 H)

2.41 (br. s., 1 H) 3.06 (t, J=7.33 Hz, 2 H) 3.50 (t, J=6.60 Hz, 2 H) 4.13 (d, J=2.20 Hz, 2 H) 7.41 -

7.48 (m, 6 H) 7.48 - 7.59 (m, 3 H) 7.72 (dd, J=13.19, 8.06 Hz, 4 H) 7.97 (d, J=8.06 Hz, 1 H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 26.29, 29.15, 29.28, 29.35, 29.61, 29.63,

29.70, 29.72, 29.78, 58.23, 70.52, 74.27, 80.29, 127.40, 128.68, 128.80, 131.76, 131.79, 132.44,

132.55, 132.70, 133.43, 133.54, 137.49, 192.40

IR cm-1 634, 687 (strong), 709, 746, 773, 911 (strong), 997, 1024, 1096 (strong), 1175, 1205,

1305, 1432, 1477, 1582, 1660.65 (C=O), 2094, 2853, 2925

10-Azidodecan-1-ol (17):

10-Azidodecan-1-ol has been reported in the literature.[135] 10-Bromo-1-decanol (0.9 ml, 4.22 mmol) was dissolved in N,N-dimethylformamide (35 ml). Sodium azide (0.426 g, 6.324 mmol) was added to the solution. The solution was stirred and heated to 80 °C. The sodium azide dissolved upon heating. The reaction was stirred for 48 hours at this temperature. After cooling to room temperature, the solvent was diluted with deionized water (350 ml). The aqueous layer was extracted with ethyl acetate (150 ml). The extract was washed with a 5% aqueous solution of

75 lithium chloride (120 ml) and 0.5 N hydrochloric acid (120 ml). The combined aqueous washes were back-extracted with ethyl acetate. The combined organic layer was dried over magnesium sulfate, which was then removed via Hirsch filtration. The solvent was evaporated under reduced pressure. The crude product was purified on silica using a gradient of hexane, 1:49

EtOAc:hexane, 1:19 EtOAc:hexane, 1:9 EtOAc:hexane, 1:4 EtOAc:hexane, 1:1 EtOAc:hexane,

3:1 EtOAc:hexane, 100% EtOAc, and finally 1:9 methanol: EtOAc. When this failed to remove the remaining starting material, the crude product was purified again on a column packed with

C18-silica using acetonitrile as the mobile phase. This gave the pure product as a clear oil in 53% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 1.19 (br. s., 12 H) 1.38 - 1.52 (m, 4 H) 3.14 (t,

J=6.60 Hz, 2 H) 3.21 (br. s., 1 H) 3.46 (t, J=6.60 Hz, 2 H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 25.93, 26.90, 29.03, 29.32, 29.58, 29.68,

32.96, 51.68, 63.14

-1 IR cm 557, 636, 722, 922, 1055 (broad), 1258 (broad), 1349, 1464, 2090 (strong, -N3), 2854,

2925, ~3100-3500 (broad, hydroxyl)

10-Azidodecyl 1H-imidazole-1-carboxylate (18):

10-Azidodecyl 1H-imidazole-1-carboxylate has not been reported in the literature. It was synthesized according to a literature procedure for a similar compound.[65] 17 (0.200 g, 1 mmol) was stirred in dry tetrahydrofuran (10 ml) over molecular sieves. 1,1’-carbonyldiimidazole

(0.244 g, 1.51 mmol) was added and the reaction was stirred for 1 hour at room temperature.

After that time, the solvent was removed under reduced pressure. The crude product was dissolved in ethyl acetate (50 ml), then washed with a saturated aqueous solution of ammonium

76 chloride (2 x 50 ml) followed by brine (2 x 50 ml). The organic layer was dried over magnesium sulfate, which was removed via Hirsch filtration. The solvent was evaporated under reduced pressure to yield the pure product as a clear oil in 93% yield.

1H NMR (399 MHz, CHLOROFORM-d) δ ppm 1.23 (br. s., 15 H) 1.47 - 1.56 (m, 2 H) 1.67 -

1.76 (m, 2 H) 1.95 (s, 1 H) 3.17 (t, J=6.96 Hz, 2 H)

4.33 (t, J=6.96 Hz, 2 H) 6.98 (s, 1 H) 7.35 (s, 1 H) 8.05 (s, 1 H)

13C NMR (100.37 MHz, CHLOROFORM-d) δ ppm 25.88, 26.86, 28.65, 29.00, 29.27, 29.50,

29.52, 51.63, 68.66, 117.28, 130.78, 137.27, 148.94

IR cm-1 650, 769, 830, 1001, 1094, 1172, 1237 (strong), 1279 (strong), 1374, 1403, 1468, 1758

(strong, C=O), 2092 (-N3), 2927

Overall Summary & Conclusions:

Herein, work toward two very different approaches to targeted therapy has been presented and discussed. We have investigated the binding modes of synthetic estrogens in the human estrogen receptor ligand binding domain. Our observations rationalized the observed contribution of the halogen atom to relative binding affinity, showing that the halogen establishes many more contacts with the receptor than the equivalent vinyl proton does. We have also synthesized novel heterobifunctional linkers in the pursuit of developing a molecular toolkit for an ambitious interdisciplinary project which may hold great promise for nanomedicine.

It should be noted that these two different approaches to targeted chemotherapy are not

77 necessarily mutually exclusive. One potentially interesting avenue of exploration might be to study the biodistribution of radiolabeled 1,1-bis(4-hydroxyphenyl)-2-(3-hydroxphenyl)ethylenes when encapsulated in a dextran nanoparticle. Alternatively, an active targeting element based on the THPEs or a steroidal estrogen such as 11β-4-hydroxyphenylestradiol might be incorporated into a prefunctionalized polymer unit.

One thing to keep in mind is that cancer is not one disease, but many. There will most likely never be a single magic bullet for all or even most forms of cancer. Therefore, it is important that chemists and other researchers continue to pursue many diverse strategies for the analysis and treatment of cancers. By continuing to broaden both our understanding of the underlying mechanisms of cancer and the medical community’s arsenal of tools for analyzing and treating individual cases, we can continue to reduce the human toll of cancer and improve the quality of life for people who are struggling with it.

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Appendix A: Binding Modes of Ligands in the ERα-LBD

Crystal Structures of ERα-LBD in Complex with Diethylstilbestrol (3ERD) and Estradiol

(1A52):

Top left: 2-D representation of diethylstilbestrol (DES) in the binding site of the human estrogen receptor ligand binding domain’s A chain (PDB ID: 3ERD). Top right: 2-D representation of estradiol (E2) also in the ERα-LBD binding site’s A chain (PDB ID: 1A52). Both images made using Ligplot+.[42] Ligands and H-bonding residues are shown as ball-and-stick models. 92

Hydrophobic residues are represented as a red semicircle with radiating lines. Bottom left: 3-D view of DES in the ERα-LBD binding site’s A chain (3ERD). The water molecule seen to the left of the ligand is HOH 11. Bottom right: 3-D view of E2 in the ERα-LBD binding site’s A chain

(1A52). The water molecule seen to the left of the ligand is HOH 558. Both screenshots were taken in YASARA.[31] Only ligands within 4Å contact distance of the ligand as reported by the ligand-protein contact server[29] are visualized, as well as Asp 351. Basic residues which would be positively charged at physiological pH are colored blue; acidic residues which would be negatively charged at the same pH are red. All other residues are grey. Ligand atoms are colored by element.

Screenshots of Halogen-Residue contacts in YASARA/AutoDock (All poses are the highest- energy poses i.e. those reported in Table 3-6):

Residues within contact distance (5Å) of vinyl halogen/hydrogen are colored yellow. Ligand and all other residues are colored by element. Halogen-contacting residue labels are black

(in 3ERD) or magenta (in 1A52). Arg 394, Glu 353, and His 524 are labeled in white for reference.

Ligand: IBHPE

Receptor crystal structure: 3ERD

“Front” view

Ligand: IBHPE

Receptor Crystal Structure: 3ERD

“Rear” view

Ligand: ITHPE

Receptor crystal structure: 3ERD

“Front” view

Ligand: ITHPE

Receptor crystal structure: 3ERD

“Rear” view

Ligand: BrTHPE

Receptor crystal structure: 3ERD

“Front” view

Ligand: BrTHPE

Receptor crystal structure: 3ERD

“Rear” view

Ligand: THPE

Receptor crystal structure: 3ERD

“Front” view

100

Ligand: THPE

Receptor crystal structure: 3ERD

“Rear” view

101

Ligand: All-para-ITHPE

Receptor crystal structure: 3ERD

“Front” view

102

Ligand: All-para-ITHPE

Receptor crystal structure: 3ERD

“Rear” view

103

Ligand: All-para-BrTHPE

Receptor crystal structure: 3ERD

“Front” view

104

Ligand: All-para-BrTHPE

Receptor crystal structure: 3ERD

“Rear” view

105

Ligand: All-para-THPE

Receptor crystal structure: 3ERD

“Front” view

106

Ligand: All-para-THPE

Receptor crystal structure: 3ERD

“Rear” view

107

Ligand: IBHPE

Receptor crystal structure: 1A52

“Front” view

108

Ligand: IBHPE

Receptor crystal structure: 1A52

“Rear” view

109

Ligand: ITHPE

Receptor crystal structure: 1A52

“Front” view

110

Ligand: ITHPE

Receptor crystal structure: 1A52

“Rear” view

111

Ligand: BrTHPE

Receptor crystal structure: 1A52

“Front” view

112

Ligand: BrTHPE

Receptor crystal structure: 1A52

“Rear” view

113

Ligand: THPE

Receptor crystal structure: 1A52

“Front” view

114

Ligand: THPE

Receptor crystal structure: 1A52

“Rear” view

115

Ligand: All-para-ITHPE

Receptor crystal structure: 1A52

“Front” view

116

Ligand: All-para-ITHPE

Receptor crystal structure: 1A52

“Rear” view

117

Ligand: All-para-BrTHPE

Receptor crystal structure: 1A52

“Front” view

118

Ligand: All-para-BrTHPE

Receptor crystal structure: 1A52

“Rear” view

119

Ligand: All-para-THPE

Receptor crystal structure: 1A52

“Front” view

120

Ligand: All-para-THPE

Receptor crystal structure: 1A52

“Rear” view

121

Screenshots of Hydrogen-bonding contacts in YASARA/AutoDock (All poses are the highest-energy poses i.e. those reported in Table 3-6):

Hydrogen-bonding heteroatoms are colored magenta. Ligand and H-bonding residue are colored by element. All other residues are colored grey. H-bonding residue is labeled.

Ligand: IBHPE

Receptor crystal structure: 3ERD

122

Ligand: BrTHPE

Receptor crystal structure: 3ERD

123

Ligand: All-para-THPE

Receptor crystal structure: 3ERD

124

Ligand: E2

Receptor crystal structure: 3ERD

125

Ligand: 11β4HPE2

Receptor crystal structure: 3ERD

126

Ligand: E2

Receptor crystal structure: 1A52

127

Ligand: 11β4HPE2

Receptor crystal structure: 1A52

128

Screenshots of Hydrogen-bonding contacts in Schrodinger Glide (All poses are the highest- energy poses i.e. those reported in Table 3-6):

All atoms are colored by element. Hydrogen bonds are indicated by thick green lines.

Distance between H-bonding heteroatoms is purple. Dihedral angles are green. See Table 5 for a complete list of these distances and dihedral angles.

Ligand: IBHPE

Receptor crystal structure: 3ERD

H-bonding residue: Glu 353

129

Ligand: ITHPE

Receptor crystal structure: 3ERD

130

Ligand: All-para-ITHPE

Receptor crystal structure: 3ERD

131

Ligand: All-para-BrTHPE

Receptor crystal structure: 3ERD

132

Ligand: DES

Receptor crystal structure: 3ERD

133

Ligand: IBHPE

Receptor crystal structure: 1A52

H-bonding residue: Glu 353

134

Ligand: ITHPE

Receptor crystal structure: 1A52

135

Ligand: BrTHPE

Receptor crystal structure: 1A52

136

Ligand: THPE

Receptor crystal structure: 1A52

137

Ligand: All-para-ITHPE

Receptor crystal structure: 1A52

H-bonding residue: Thr 347

138

Ligand: All-para-BrTHPE

Receptor crystal structure: 1A52

H-bonding residue: Glu 353

139

Ligand: All-para-THPE

Receptor crystal structure: 1A52

140

Ligand: DES

Receptor crystal structure: 1A52

141

Ligand: E2

Receptor crystal structure: 1A52

142

Ligand: 11β4HPE2

Receptor crystal structure: 1A52

143

Appendix B: Chemical Spectra for Chapter 2

Dextran 4-iodophenyl carbamate:

144

145

1-Azido-2-(2-(2-(2-isocyanatoethoxy)ethoxy)ethoxy)ethane (1):

146

147

148

Propargyl-PEG-methyl ether (2):

149

Decane-1,1-diyl bis(4-methylbenzenesulfonate) (6):

150

151

10-(1,3-dioxoisoindolin-2-yl)decyl 4-methylbenzenesulfonate (7):

152

153

2-(10-Azidodecyl)isoindoline-1,3-dione (8):

154

155

156

10-[bis(tert-butoxycarbonyl)amino]decyl 4-methylbenzenesulfonate (10):

157

158

159

Tert-butyl N-(10-azidodecyl)-N-tertbutoxycarbonylcarbamate (11):

160

161

162

10-Azidodecan-1-aminium triflate (12):

163

164

165

Tert-butyl N-(10-azidodecyl) carbamate (13):

166

167

168

1-azido-10-isocyanatodecane (3):

169

170

171

Tert-butyl dodecyl carbamate (14):

172

Dodecyl isocyanate (15):

173

174

175

1-(Prop-2-yn-1-yloxy)dodecane (4):

176

177

10-Prop-2-ynoxydecan-1-ol (16):

178

10-Hydroxydecyl phenylsulfanylformate (5):

179

180

181

10-Azidodecan-1-ol (17):

182

183

184

10-Azidodecyl 1H-imidazole-1-carboxylate (18):

185

186

187