in partial fulfillment of the requirements for

Synthesis of Guanidinylated-Substituted Polymers that bind Trans-activation Responsive Region of Human Immunodeficiency Virus Type-1 RNA

Master’s Thesis

Presented to

The Faculty of the Graduate School of Arts and Sciences

Brandeis University

Department of Biochemistry

Jason Pontrello, Advisor, Department of Chemistry

Melissa Kosinski-Collins, Advisor, Department of Biology

In Partial Fulfillment

of the Requirements for

Master’s Degree

by

Shakara Lavisha Scott

May, 2013

Copyright by

Shakara L. Scott

© 2013

!!!"

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This thesis is dedicated to my Godfather James Scott and my best friend Anushka R. Aqil

who always reassured me that the fuel of failure is lack of enthusiasm. But I always

!"#$!#"%&'()'*$+,%-.#'%$'/#'/0')'%/%-1#'2345'/#67

(89/!":';<-.#'="'><%"'*/#?'%"<%'">="!:&'

nor enthusiasm be stirred by spiritless men.

Enthusiasm in our daily work lightens effort and

#+!-:',<=$!'/-#$'@,"<:<-#'#<:A:6BBB7

CJames A. Baldwin

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Acknowledgements

There are numerous people without whom this thesis might not have been written and

to whom I am deeply indebted.

To my mentors: It is with immense gratitude that I acknowledge the support and help of

my professors and mentors, Dr. Jason K. Pontrello and Dr. Melissa Kosinski-Collins.

This thesis and fascinating research project would have remained a dream had it not been

for them. The life of an undergraduate research student is not trivial and therefore it was

filled with frustration because nothing works the first time, or the second time. Despite

these difficulties Jason and Melissa attitudes remained steadfast. They both continually

and convincingly conveyed a perennial spirit of adventure and excitement in regard to

research and teaching respectively. In retrospect, I have come to appreciate that they were

preparing me for more than research; but for the ambivalent world out there. They

challenged me to ameliorate my diligence as well as apply myself independently pushing

me towards fulfilling my potential. Thank you, Dr. Pontrello and Dr. KC. I had amazing

learning experience and lots of laughter too!

To my lab buddies: I would be remiss if I did not mention, my lab mentors Nate

Shammay, Larry Friedman, Deb Bordne and Anna Vilenchik. They are all excellent

teachers, scientists and genuinely affable people. Individually, they all played an essential

!"#$%&'%(!)'*+"!,&'-%,$%+!",%)%-)./0%1'2$!-!)21)($%&'("%)%3*",$.4)(5%6",7$($'(%

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"

researcher. I admire all of them and I look forward to preserving these life-long

friendships. Thank you, Nate, Larry, Deb and Anna.

To family and friends: Lastly, I want to express my gratitude to my family, and friends

old and new, who have made these four years at Brandeis tolerable and most importantly,

memorable. All of you showered me with your love and support continually. For these

things I am extremely grateful. I would also like to thank Hirvelt Megie for coloring my

life with his candid humor, wealth of hugs and smiles. Thank you Anushka for making

me smile even when you depart from me. I could have never asked for a better best

friend.

!"#$%&'#$(%)&*#+,#-./$0$1&"-2$-(34$#&$#5+,3$%.$)+*4,#6'$7(&-&8(9+-$+,2$&*$

otherwise. Thank you mom for raring a child who is worldly and knowledge driven, I

would not be here without these foundations. Thank you Dad, for always being

optimistic, caring and ever-present! You are the more than a daughter could ever ask for.

I hope you know that I live to make you proud.

Now without further ado, it brings me great joy to present to you my biochemistry

:+'#4*6'$#54'(';$0$9&,'(24*$#5('$1&*3$+$9"-%(,+#(&,$&<$%.$242(9+#(&,$+,2$#5(*'#$<&*$

academics and research.

I hope you enjoy it!

!

!

!

!

ABSTRACT

Synthesis of Guanidinylated-Substituted Polymers that bind Trans-activation Responsive

Region of Human Immunodeficiency Virus Type-1 RNA

A thesis presented to the Department of Biochemistry

Graduate School of Arts and Sciences

Brandeis University

Waltham, Massachusetts

By Shakara Lavisha Scott

Multiple targets exist in the development of HIV-1 anti-viral drugs, one of which

includes the interaction between the transcriptional activator protein (Tat) and the Trans

Activation Response Region (TAR) element of RNA. During transcription, TAR RNA, a

59-base stem-bulge-loop structure, located at the 5’end of all HIV-1 mRNAs, recruits

Tat, which modulates viral gene expression in infected cells. Previous experiments have

shown that the Arginine Rich Motif (ARM) of Tat is integral for the association of Tat to

TAR. Altogether, literature suggests that the inhibition of Tat/TAR RNA interaction is an

attractive route to controlling HIV-1 expression and replication. We sought to design

synthetic polymers that would disrupt the necessary interaction between Tat and TAR-

vi

RNA, hindering HIV replication. To target the TAR-RNA, we sought to replicate the

basic ARM of Tat by functionalizing amine-amenable polymer scaffolds derived from

the Ring-Opening Metathesis Polymerization (ROMP), with the guanidinium derivatives

of arginine and agmatine. To this end, we have successfully synthesized the

guanidinylated polymers. We hypothesize that by amending the polymer scaffolds with

guanidinums, an essential requisite for binding of small molecules to TAR RNA, we may

be able to retard the RNA. To test this hypothesis, we assayed the RNA-binding activities

of the guanidinylated polymers using an Electrophoretic Mobility Shift Assay (EMSA)—

based approach. Our studies indicate that all the synthetic guandiniums are RNA-binding

molecules that recognize and retard the mobility of wild-type TAR RNA in a

concentration range of 1—400 µM. Additionally, we found that the introduction of

magnesium (Mg2+

) in the binding buffer strongly stimulates RNA folding as well as

increases the RNA-binding specificity of the polymeric compounds. To optimize the

binding between the polymers and the RNA we will explore different binding buffers that

may increase the binding affinity, allowing us to characterize the Ka and Kd values. In

addition, it is expected that TAR-RNA binding molecules may inhibit the association of

Tat/TAR; therefore, ongoing work seeks to elucidate the selectivity and specificity of the

guanidinum-conjugated polymers, in addition determining the effects of the polymers on

protein-TAR RNA interactions by EMSA.

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Table of Contents

Dedication iii

Acknowledgements iv-v

Abstract vi-vii

Table of Contents viii-ix

List of Tables x

List of Figures xi-xii

List of Synthetic Schemes xiii

List of Abbreviations xiv-xvi

I. Introduction

Section I: Human Immunodeficiency Virus Type 1 1

HIV-1 Genome 2

The Role of Tat in HIV-1 Replication and Life Cycle 5

Trans-activator of Transcription (TAT) 9

Extracellular Tat 11

Trans-Activator Response (TAR) Element RNA 12

Binding of Tat to Tri-nucleotide Bulge of TAR-RNA 13

Activation of HIV-LTR by Tat 16

Summary 19

Section II: Current Drug Treatments 20

Section III: Motivation

Therapeutic Efforts: Inhibitors Targeting TAR-RNA 22

Small Molecule Inhibitors targeting TAR-RNA 23

Synthetic Polymers Targeting TAR-RNA 26

Summary 29

Section IV: Aims 31

Synthetic Utility of ROMP-derived Polymers 34

Determination of Protein-Nucleic Acid Interactions 34

Summary 35

II. Materials and Methods

Section I: Chemical Synthesis 37

Section II: Biological Assays 49

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III. Results

Section I: Chemical Synthesis of Multivalent Guanidiniums 58

Summary 69

Section II: Electrophoretic Mobility Shift Assay (EMSA) 70

IV. Discussion

Section I: Chemical Synthesis of Multivalent Guanidiniums 83

Section II: Electrophoretic Mobility Shift Assay (EMSA) 86

V. Conclusion 94

VI. Future Directions 95

VII. Bibliography 96

VIII. Appendices 102

1H NMR Spectra 102

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LIST OF TABLES

Page

Table 3.1 Quantified Arginine 10-mer binding experiment 72



Table 3.4 Quantified Polymer Binding Experiment 79

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LIST OF FIGURES

Page

Figure 1.1 Structure of the HIV-1 Virion 3

Figure 1.2 Organization of HIV-1 Genome and Viral Promoter 5

Figure 1.3 The Essential Steps in Life Cycle HIV-1 7

Figure 1.4 Trans-activator of Transcription (Tat) 10

Figure 1.5 Trans-activation Response Element (TAR) RNA 13

Figure 1.6 Interactions of TAR RNA with the ARM of Tat 14

Figure 1.7 The recognition of HIV-1 TAR RNA by Tat and Cyclin T1 16

Figure 1.8 The activation of RNA polymerase II by Tat and cellular co-factors 18

Figure 1.9 Examples of Antiviral Drugs used to treat HIV 21

Figure 1.10 Classical Approaches to Tat-TAR inhibition 22

Figure 1.11 Structures of small molecules that bind TAR 24

Figure 1.12 Modular design for TAR stem-loop and 3-base-bulge inhibitors 25

Figure 1.13 Neomycin B-Hexaarginine Conjugate 26

Figure 1.14 Structure of TAR RNA binding Oligocarbamate 27

Figure 1.15 Schematic of combination library of branched peptides 28

Figure 1.16 Bindings of FL4 to TAR in the presence of tRNA using EMSA 29

Figure 1.17 A General Scheme for Polymer Conjugation and Design 32

Figure 1.18 Approaches of Various Ligand Displays 35

Figure 3.1 Iodolactonization Mechanism for Isolation of Exo-norbornene 60

Figure 3.2 Mechanism for the formation of the amine-reactive ester 62

Figure 3.3 Gel Electrophoresis of control TAR RNA 71

Figure 3.4 Titration of TAR RNA with ROMP-derived Arginine

Peptidomimetics (10-mer) 73





Figure 3.7 Titration of Arginine-Conjugated polymers with HIV-TAR after pre-

incubation in Mg2+ binding buffer 80

Figure 3.8 Titration of TAR RNA with arginine conjugated 10-mer (A),

norbornene-arginine monomer (B) and free arginine (C) 82

Figure 4.1 Binding-modes of RNA-polymer 88

Figure 4.2 Equilibrium Constant for the binding of ligand to single site 92

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LIST OF SCHEMES

Page

Scheme 1 Isolation of Exo-Norbornene 59

Scheme 2 Conversion of Exo-Norbornene to Succimidyl Ester Monomers 61

Scheme 3 Synthesis of Ruthenium Carbene Polymerization Catalyst 63

Scheme 4 Synthesis of Succinimidyl Ester Substituted Polymer Scaffolds 65

Scheme 5 Conjugation of Synthetic Polymer scaffolds with Guanidinium

Derivatives 67

Scheme 6 Synthesis of Arginine Norbornene Control Monomers 68

Scheme 7 General Route for the Synthesis of Agmatine Monomers 69

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LIST OF ABBREVIATIONS

10-mer 10 unit ROMP-derived polymer


50-mer 50 unit ROMP-derived Polymer


AIDS Autoimmune deficiency syndrome

ARM Arginine Rich Motif

ART Antiretroviral therapy

BIV Bovine Leukemia Virus

CCR5 C-C Chemokine Receptor Type-5

CDCl3 Chloroform

CDK-7 Cyclin-Dependent Kinase-7

CDK-9 Cyclin-Dependent Kinase-9

cDNA Complementary DNA

CH2Cl2 Dichloromethane (a.k.a. methylene

chloride)

C NMR Carbon Nuclear Magnetic Resonance

CTD Carboxyl-Terminal Domain

CXCR4 C-X-C Chemokine receptor Type-4

D2O Deuterium oxide

DEPC Diethyl pyrocarbonate

DMF N, N-dimethylformamide

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DSIF DRB Senstivity Inducing Factor

ECM Extracellular Matrix

EDCI N-(3-dimethyl aminopropyl)-!"-ethyl

carbodiimide

EMSA Electrophoretic Mobility Shift Assay

EtO2 Ethyl Ether

Et2O2 Diethyl Ether

EtOAc Ethyl Acetate

FDA Food and Drug Administration

GAGs Glycosaminoglycans

H2O Water

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H2SO4 Sulfuric acid

HAART Highly Active Antiretroviral Therapy

HIV-1 Human Immunodeficency virus type 1 1H NMR Proton Nuclear Magnetic Resonance

HSPGs Heperan Sulfate Proteoglycans

HTLV Human T-Cell Leukemia Virus

I2 Iodine

IN Intergrases

KI Potassium Iodide

KMnO4 Potassium Permanganate

KS Kaposi Sarcoma

LTR Long Terminal Repeat Region

MCH I Major Histocompatibility Class I

MeOH Methanol

MgSO4 Magnesium Sulfate

mRNA Messenger Ribonucliec Acid

N2 Nitrogen

NaHCO3 Sodium Bicarbonate

NaOH Sodium Hydroxide

Na2S2O3 Sodium Thiosulfate

Nef Negative regulator factor

NELF Negative Elongation Factor

NeoR Neomycin B-Hexaarginine

NF-#$%%% Nuclear Factor Kappa B

NHS N-Hydroxysuccinimide

NKT Natural Killer T Cells

NMM N, N-Methylmorpholine

NMR Nuclear magnetic resonance

NRTIs Nucleoside Reverse Transcriptase

Inhibitors

NNRTIs Non-Nucleoside Reverse Transcriptase

Inhibitors

MQ MilliQ

PI Protease Inhibitors

PIC Pre-integration Complex

PPM Parts per million

P-TEFb Positive Transcription Elongation Factor

Complex-b

RNA Ribonucleic Acid

RNA Pol II Ribonucleic Acid Polymerase II

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ROMP Ring-opening-metathesis polymerization

SAHA Suberoylanilidehydroxamic Acid

SIV Simian Immunodeficiency Virus

ssRNA Single-stranded Ribonucleic Acid

TAK Tat-associated Kinase

Tat Trans-activator of transcription

TAR Trans-activation Responsive Element

TFIIH Transcription Factor II H

TLC Thin-Layer Chromatography

RT Reverse Transcriptase

VOR Vorinostat

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I . Introduction:

The synergic interactions of macromolecules in both the extracellular and

intracellular environment are ubiquitous and integral for a variety of pathological and

physiological functions; RNA-protein interactions are a vital class of these protein-nucleic

acid associations. These interactions control some of the most intrinsic biological processes

including activation of cellular genes, transcription, translation, and replication. Owing to the

prevalence of RNA-protein complexations in the cell, regulation of these interactions allows

safeguard and control over the production and proliferation of cellular life. Intensive studies

have shown that RNA-protein mediated interactions play a crucial role in infectious diseases

that are associated with viral replication including cancer, the Human Immunodeficiency

Virus Type-1 (HIV-1), Acquired Immunodeficiency Syndrome (AIDS), and other AIDS

related pathologies such as Kaposi Sarcoma (KS) and Human T-Cell Leukemia Virus

(HTLV) (Dewhurst 1996; Noonan 2000; Mishra 2008; Khalil 2011). As a result, synthetic

methods to selectively control RNA-protein interactions represent attractive ways of

regulating biological functions and can be developed into powerful therapeutic tools.

An archetypal example of protein nucleic acid interactions is the mechanism of trans-

activation in the HIV-&%'()*+,%-%./0'12345%36-3%7-+%8)19(8(3-31:%;4%361%<=>?%8-/:1@(9%0A%

1981 (Zhao 2004; Karn 1999). HIV is a retroviral disease that causes AIDS, a condition that

increases the risk contracting malignant cancers and (/'-+(0/%0A%.0880)3*/(+3(9%(/A193(0/+5%

due to a decimated immune system (Karn 1999; Mishra 2008). In 2012, the global estimate

for people living with HIV/AIDS reached a daunting 34.2 million (UNAIDS 2012). Due to

the lack of a cure, scientists worldwide have been working assiduously to combat this global

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pandemic with the objective of disrupting the HIV-1 replication cycle (Karn 1998; Mishra

2008).

Early studies of HIV-1 virus revealed significant insight for understanding the viral

replication cycle and the functions of the viral gene products (Cullen 1986; Dingwall et al.

1989; Weeks et. al. 1990; Sodroski 1995a). However, large variability in HIV-1 strains has

complicated development and pursuit of effective antiviral drugs. The most sought out

therapeutic strategy targets the RNA-protein interaction between HIV-1 Transactivator of

transcription (Tat) protein and Transactivation responsive region (TAR) RNA that is

essential to the viral replication and pathogenesis (Karn 1999). On route to a cure is the

design of synthetic drugs that selectively inhibit Tat-TAR interaction, for which a detailed

understanding of the HIV genome and replication cycle is required.

Section I : Human Immunodeficiency V irus Type 1

H I V-1 G enome: The HIV virus belongs to the lentivirus genus and retroviridae

family that also includes the Bovine Leukemia Virus (BIV), Simian Immunodeficiency Virus

(SIV) and HTLV. Retroviruses carry with them a single-stranded RNA and an enzyme that

allows for a reversal of genetic transcription from RNA to DNA. The HIV-1 virus is

spherical in shape and contains two copies of single-stranded RNA (ssRNA) (F igure 1.1).

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F igure 1.1: Structure of H I V V ir ion. HIV-1 virions possess two strands of genetic material

(viral RNA); viral enzymes encased in a capsid, and are further protected by a protein matrix.

Each virion is spherically shaped and measures about 1/10,000 th of a millimeter in diameter

(Figure not drawn to scale). The enzymes intergrase (IT) and reverse transcriptase (RT) help

the virus copy itself once in the host cell. The outer coat or viral envelope consists of two

layers: a lipid membrane taken from the human cell that the virus particle budded as well as

fatty acids. Protruding through the envelope are HIV proteins Env. The Env protein is made

up of two glycoproteins; the cap, gp120 and the trans-membrane anchor glycoprotein, gp41.

(This image was extracted from NIAID March 29, 2013

http://www.niaid.nih.gov/topics/hivaids/understanding/biology/Pages/structure.aspx).

Each copy of RNA is approximately 10, 000 nucleotides in length and encodes for nine genes

(gag, pol, vif, vpr, rev, tat, vpu, env and nef) (F igure 1.2). Of the nine genes, six encode for

viral accessory proteins, which assist in the proliferation of the HIV-1 virus. The HIV viral

proteins vif and vpu influence the assembly and budding of new virions. Env encodes for the

viral envelope glycoprotein SU (gp160) that is essential for the binding of host cell receptors

and co-receptors. Nef, the negative regulator factor protein participates in cell activation, T-

cell apoptosis and the down-regulation of host molecules that are critical for the development

of cellular and humoral immune responses (Ranjan Das et al. 2005). Rev mediates the

transportation of unspliced messenger RNA (mRNA) from the nucleus into the cytoplasm.

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The molecular functions of viral protein R (vpr) include nuclear import of viral pre-

integration complex (PIC), modulation of T-cell apoptosis, transcriptional co-activation of

viral and host genes, and regulation of nuclear factor kappa B (NF-#$B%-93('(34%CD0E-/%

2011). Tat is a Trans-activating protein that regulates viral replication and gene expression.

Taken together, though all the viral proteins contribute to the processes that fuel the HIV-1

infection and evasion of the immune system, the role of Tat, Rev, and Vpr are considered to

be the largest contributors to the morbidity and mortality of HIV/AIDS (Karn 1999; Romani

2010; Kogan 2011). The numerous functions of Vpr, Rev and Tat in the viral life cycle

suggest that they would be attractive targets for therapeutic intervention and development of

HIV antiviral agents. In this manuscript, we focus on the Tat protein.

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F igure 1.2: O rganization of H I V-1 G enome (top) and the V iral L T R Promoter (bottom).

The Tat gene is encoded by two exons (labeled in red). The first exon codes for the first 72aa

are sufficient for transactivation. The second exon encodes for amino acids 73-104. A

detailed structure of the organization of the HIV LTR promoter is shown at the bottom of the

picture. The HIV LTR promoter contains many binding site and resembles promoters

activated by RNA Polymerase II. Immediately downstream of the start of transcription is the

transactivation response region (TAR). TAR encodes a stem-loop RNA structure that acts a

switch during HIV replication. Tat recruits transcription factors on the LTR to up regulate the

transcription of the HIV-1 genome (Karn 1999; Romani 2010). (This image was adapted

from Karn 1999).

The Role of Tat in H IV-1 Replication and L ife Cycle : HIV virions predominantly

target immune cells expressing glycoprotein CD4 (cluster differentiation 4) and thus infect a

variety of immune cells such as dendritic cells, CD4+ T lymphocytes and macrophages

(Stevenson and Crowe et al. 2003). In addition, recent evidence suggests that natural killer T

cells (NKTs) are also an important target of HIV-1 virions during the early course of

infection (Fleuridor et al. 2003). The significance of the HIV virusFCD4 interaction is

underscored by studies that have demonstrated that the HIV virus is able to target vital

anthropomorphic cells. Two main phases dominate the pathogenesis of HIV-1 virus (F igure

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1.3) (Karn 1999). In the first phase, the virus enters the cell via a fusion mechanism between

the glycoprotein 120 SU (gp120) envelope of the virion and the CD4 cell membrane receptor.

This fusion between the virus and the host cell membrane also requires chemokine

coreceptors CCR5 (predominant during acute and asymptomatic phases of the HIV-1

infection) and CXCR4 (Crowe 2003; Stevenson et al. 2003; Mishra 2008). Once in the

cytosol, the virus uncoats and uses its inherent reverse transcriptase (RT) to synthesize

double-stranded viral DNA. This is followed by nuclear import of the viral DNA. The

accessory protein Rev transports the viral DNA into the nucleus where intergrase (IN)

catalyzes the integration to the host genome (Mishra 2008). The second phase involves viral

gene expression, replication, assembly, and virion maturation (Karn 1999).

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F igure 1.3: The Essential Steps in L ife Cycle of H I V-1. The process of infection includes

fusion of the HIV envelop with the CD4 receptors on host cell membrane; a mechanism

mediated by viral envelop glycoprotein 160. Subsequently the viral RNA is reverse

transcribed to the corresponding double stranded cDNA using viral RT and integrated into

the host cell genome (red arrows) by the enzyme intergrase. Upon activation of the host cells,

Tat is produced and is shown to simultaneously enhance the processivity RNA polymerase

increasing the production of full-length viral mRNAs (blue arrows). Rev transports the

mRNAs to ribosome where the proteins are transcribed followed by assembly into new

virions at the cell membrane (green arrows). (This image was adapted from Weizman

2003).

Once integration happens, owing to the host cell regulatory machinery, the virus can

either remain dormant (viral gene expression is silent) or become activeFa consequence of

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stimulating infected host cells with mitogens (Karn 1999). The activation of transcription for

the proviral genome is regulated by transcription factors: NF-#$ and Sp1 and the Tat protein

(F igure 1.3). The HIV pre-mRNA that is transcribed from the proviral DNA contains several

splicing signals (Mishra 2008). In the nascent stages of the HIV replication cycle mostly 2 kb

mRNA transcripts to be produced (F igure 1.3). These mRNA transcripts are translated into

regulatory proteins: Tat, Nef and Rev (Mishra 2008; Romani 2010). The Tat protein is

imported in the nucleus where it binds to nascent RNA transcript (TAR RNA) and with the

help of Tat-associated kinases (TAK), dramatically stimulates transcription elongation and

increases the production of mRNA transcripts (Karn 1999; Stevenson 2003; Weizman 2003;

Mishra 2008). In order for the lifecycle to shift to the late phases, the production of unspliced

pre-mRNA transcripts are needed for assembly into the progeny virions. Moreover, in order

for HIV to produce its complete range of structural, accessory enzymatic proteins, unspliced

~9 kb and singly spliced ~4 kb transcripts are required (Mishra 2008). Once these unspliced

and singly spliced transcripts are generated they are translocated to the cytoplasm and

ribosomes by viral protein Rev with the help of host cell nuclear export machinery (F igure

1.3).

At the ribosomes, the unspliced RNA transcripts are translated into Gag and Gal-Pol

proteins, while the unspliced RNA is translated into Env, Vpu, Vif, and Vpr. Finally, new

progeny virions are packaged and released through the cell membrane surface of the host cell

by budding (F igure 1.3). Viral proteins Nef and Env mediate the budding mechanism;

degrading and down regulating cell surface CD4, thus avoiding immune response. This

stealthy release of new progeny into the interstitial of the body allows the virus to be

metastasized to other cells without detection and perpetuates the progression to AIDS and

decimated immune systems.

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T rans-activator of T ranscr iption: Tat is one of the six HIV-1 regulatory protein

products essential for transactivation of viral and cellular genes. It is expressed in both the

early and late stages of the viral replication cycle. Tat that is released in the nascent stages of

replication is found in both the nuclei and nucleolus of HIV infected cells; when it is

produced in the later stages, Tat is predominantly found in the extracellular environment. Tat

has a variable length of 86-104 amino acids and is encoded by two exons Fdepending on the

viral strain. The first-exon form encodes the first 72 amino acids, which are sufficient for Tat

transactivation. The second exon codes for amino acids 73-104. Moreover, the two-exon

form has an additional carboxyl terminal that, based upon the viral isolate varies in length

between 86 and 104 amino acids; the additional amino acids are appended at the carboxyl

terminal (Weissman et al. 1998; Jeang 1996; Aboul-ela et al. 1999). The generation of these

two forms of Tat is regulated during translation via splicing mechanisms: the 86 amino acid

version is produced from completely spliced mRNA and the 104 amino acid version from

partially spliced HIV mRNA transcripts (Weissman et al. 1999; Amendt et al. and Bilodeau

et al. 1999). Consequently, the one-exon form of Tat is expressed predominantly during the

nascent stages while the two-exon version of Tat materializes in the later stages (Amendt et

al. 1994; Romani 2010).

There are five structural regions of the Tat protein: the N-terminal domain, which

contains amino acids 1-20, the cysteine rich region that contains seven high conserved

cysteine residues (residues 22-37), the core region (amino acids 37-48), the basic region

(residues 48-72) and the carboxyl terminal domain (C-terminal; residues 72-86) (F igure 1.4).

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F igure 1.4: T rans-activator of T ranscr iption (Tat). L eft: Organization of Tat peptide. Right: Primary structure of HIV-1 Tat peptide with the Arginine-Rich Motif (ARM) residues

48-57 in red (Yang 2005). L eft: Primary structure of HIV-1 Tat peptide.

(The image on right was adapted and modified from Yang 2005).

Previous work has indicated that deletion and substitution experiments of residues in

the Cys-rich region have resulted in loss of trans-activation; suggesting that it is required for

Tat function (formation of intra-molecular disulphide bonds) but they are not directly

involved in TAR recognition (Aboul-ela 1999; Yang 2005). The basic region, which consists

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of Arginine Rich Motif (ARM), is conserved over several strains of the HIV-1 and regulates

the Transactivation activity of Tat. Furthermore, the basic region is an essential requisite for

the interactions between the protein and its nucleic acid conjugate, TAR RNA (Yang 2005).

In addition, some have discovered that the carboxyl-terminal domain (CTD) of Tat represses

the transcription of major histocompatibility class I genes (MCH I), which are the first line of

cell immune defense (Weissman 1998). Overall, Tat is a multifunctional protein that has

significant effects on both the virus and the host cell genes.

Extracellular Tat: In addition to intracellular Tat that activates HIV LTR, Tat is also

found in the extracellular matrix. Extracellular Tat along with helper gp120, are viral

products secreted by HIV-1 infected T-cells in the extracellular environment (Bugatti 2007;

Romani 2010). Cohesively, they act as immune-suppressors, activating quiescent T-cells and

targeting HIV-nonpermissive cells/non-HIV-infected cells for progression of the HIV-1

infection (Litovchick 2001; Bugatti 2007). A compilation of research studies elucidates the

entrance of extracellular Tat into cells via an endocytic pathway by binding to an invariable

amount of cell surface receptors, including vascular endothelial growth factor, heparan

sulfate proteoglycan chemokine receptors CCR2, CCR3 and CXCR4 (Xiao et al. 2000;

Bugatti 2007), and heparan sulfate proteoglycans (HSPGs) (Tyagi et al. 2001; Bugatti 2007).

The bindings of Tat by these receptors increase its local concentration in the extracellular

matrix (ECM) and mediate its internalization and trans-activating activity (Noonan 1996;

Vendeville 2004; Bugatti 2007; Miyauchi 2009). Studies of Tat-derived peptides have

demonstrated that residues 48-60 from the basic domain (protein transduction domain or

PTD) accounts for the functional internalization into cells (Buggati 2007; Romani 2010).

Furthermore, Tat contributes to the development of AIDS and other AIDS-associated

pathologies by concomitantly inducing oxidative stress in the blood-brain barrier cells (ECs)

(Price 2005) and causing apoptosis in cardiomyocytes (Fiala 2004), neurons (Singh 2004)

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and other immune cells. For example, Tat enters host macrophages and inhibits nitric oxide

synthase gene activity. This inhibitory effect of Tat on the production of nitric oxide renders

the host vulnerable to infections, since nitric oxide provides the first line of defense against

opportunistic pathogens (Romani 2010).

T rans-activation Response E lement (T A R) RN A : Replication of HIV-1 LTR

requires Tat to bind to trans-activation response element (TAR) RNA, a conserved 59-base

stem-2008%+3)*93*)1%209-31:%(/%361%20/E%31)@(/-2%)1E(0/%CGHIB%-3%361%J"%1/:%0A%-22%K=L-1

mRNA transcripts F igure 1.2 (Yang 2005). Several studies performed using mutant HIV-1

variants indicate that the Tat protein and the TAR RNA sequence are necessary for viral

replication and pathogenesis (Jeang et al. 1999; Karn 1999; Harrich et al. 1995). The

structural components of TAR RNA, spanning from nucleotides +1 to +57 (F igure 1.5)

includes: the stem-loop, upper arm, 3-base bulge, and the lower stem (Karn 1999; Yang

2005). The 3- base-bulge along with two base pairs above and below the bulge constitutes the

core elements for Tat binding (Yang 2005). Research has shown that the U-rich 3 base-bulge

residues (U 23, C24, U25 or UUU) near the apex of the TAR RNA stem are necessary for

specific binding and recognition of the Tat protein in vivo trans-activation. The mutations in

TAR RNA that affect the structure and base pairing in the U-rich bulge completely abolish

Tat association.

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F igure 1.5: T rans-activation Response E lement (T A R) RN A . (A) Secondary Structure and

Sequence of HIV-1 TAR RNA with critical trinucleotide residues essential for Tat binding

circled in red and the hexanucleotide loop elements squared in blue. This is the 29 base

residue used and presented in this study. Tat specifically binds and recognizes TAR RNA

through the 3-base-bulge (UCU). In the presence of cyclin T1, conformational rearrangements

in Tat permit interactions above in the apical loop and below in the lower stem (Karn 1999;

Aboul-ela 1996). (B) Molecular schematic of TAR RNA showing the bulge and stem-loop

regions (Ellis et al. 2011).

Binding of Tat to trinucleotide bulge of T A R RN A : It is generally understood that

electrostatic interactions modulate the RNA-protein complexation of TAR-RNA to Tat, a

finding elucidated by!Nuclear Overhauser Effect (NOE). NMR NOE experiments also

showed that upon association with basic residues in the Tat ARM, the configuration of TAR

RNA changes tremendously, allowing Tat to further interact with residues in the stem-region

and loop region (F igure 1.6) (Karn 1999; Anand 2008). Understanding of the dynamics of

Tat-TAR-RNA binding enables the design of drugs that would target the Tat peptide, or

alternatively, the TAR RNA. Furthermore, rational inhibitor designs that mimic the structural

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requirements and specificity for the recognition and binding of Tat to the 3 base-bulge are of

great interest for strategy aimed at controlling HIV-1 replication.

F igure 1.6: Interactions of T A R with the A R M of Tat. (A) The overall three-dimensional

view of Tat binding to the three-base bulge as well as parts of the stem-loop region of TAR.

Tat ARM residues Lys 50, Gln 54 and Arg 55 are highlighted as well as the stem-loop

residues of TAR (Anand 2008). (B) Highlights the Watson-Crick conformation of the

interactions of Lys51 and Arg55 with U10-G17. The guanidinium group of Arg55 is

coordinated to O2 and O4 of U13, O6 of G16, O6 of G17 and O4 of U10. In addition, Lys51

also coordinates to O6 of G17. These interactions are mediated by H-bonds. (C) Schematic

representation of the interactions between TAR nucleotides and residues of Tat (magenta).

(D) Provides a detailed view of Arg55 interactions with bases U13 and G16 in the TAR loop

region (Anand 2008). (This image was adapted from Anand 2008).

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Mutational studies have identified that in addition to acting as the binding site for

Tat, the TAR acts as the recognition signal for Tat cellular cofactor cyclin T1 (CycT1) a

component of the Tat-associated kinase (TAK)/Positive Elongation Factor (P-TEFb) CTD

kinase complex (Garber 1998b; Karn 1999; Raghunathan 2006). The CylcT1 once recruited

by Tat binds the apical stem loop sequence of TAR. It is important to note that, the binding of

the stem-loop sequence by the cofactor cyclin T1 (F igure 1.7) is required only for trans-

activation, but not for Tat binding (Karn 1999). Therefore, interfering with the interaction

between the Tat/CycT1 complexes can also be an attractive target for developing HIV-1

antiviral agents.

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F igure 1.7: The recognition of H IV-1 T A R RN A by Tat and Cyclin T1. The interaction of

HIV-1 Tat with CycT1 is critical for high-affinity, loop-specific binding to TAR RNA

(Garber et al. 1998a-b). The full length HIV-1 Tat protein binds very weakly to TAR RNA in vitro. The apical stem-loop and 3-base bulge sequence of TAR are critical for the highly

cooperative binding of Tat and CycT1 to TAR RNA. Additionally, high-affinity binding of

Tat to TAR RNA can also be achieved upon truncation of the trans-activation domain,

leaving the arginine-rich motif (ARM) of Tat to bind to the bulge of the RNA structure

CM-);1)%13%-2N%&OOP;BN%$(/:(/E%0A%H-3%30%Q49H&%099*)+%36)0*E6%(3+%64:)0860;(9%.90)15%@03(A%

and cysteine-rich region of the trans-activation domain. The cysteine rich domain of Tat

binds two zinc (Zn2+ ) coordinates to other cysteine re+(:*1+%0A%361%Q49H&,%9)1-3(/E%-%.+1'1/-

cysteine-Zn2+5%+4+31@%9)(3(9-2%A0)%36(+%-++09(-3(0/%CK*-/E%-/:%R-/E%&OOST%M-);1)%13%-2N%

1998a). Moreover, it has been proposed that binding of Tat to CycT1 induce a

conformational change in Tat, which promotes binding to TAR RNA as well as

concomitantly induces a conformational rearrangement in the apical loop of the TAR RNA

36)0*E6%-%@196-/(+@%0A%.(/:*91:%A(35%CM-);1)%13%-2N%&OOP;BN%(This figure was extracted from

Garber et al. 1998b).

Activation of H I V-L T R by Tat: Some concede that the host cellular transcription

machinery sustains basal levels of HIV-1 transcription (i.e. both short non-polyadenylated

and long polyadenylated mRNA transcripts). However, in the presence of Tat, increased

levels of long favorable HIV-1 mRNA transcripts predominate (Jeang 1996; Mischiati 2001).

The original conclusion to this observed phenomenon was that short transcripts resulted from

aborted transcripts and that TAR acts as a terminator sequence, forcing premature release of

the elongation polymerase in the absence of Tat. At the same time, there has been no

evidence to support this conclusion. Furthermore, in-depth studies have shown that this

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phenomenon occurs because TAR acts as a pause site that result in a brief kinetic block to

transcription (Muesing et al. 1987; Selby et al. 1989; Karn 1999). In the presence of Tat the

kinetic block is deactivated and transcription of viral LTR occurs.

In HIV-1 infected cells, the first step in activation of the HIV-1 LTR is the

recruitment of RNA polymerase II (RNA Pol II) (F igure 1.8). Once the RNA Pol II, along

with its mediators that regulate the carboxyl-terminal domain (CTD) of the enzyme is bound,

several downstream events must occur. The phosphorylation of the CTD by the Cylcin-

Dependent Kinase-7 (CDK-7) component of the Transcription factor II H (TFIIH) complex

allows the RNA POL II to clear the promoter and begin the transcription of TAR. Soon after

initiation and transcription of TAR, RNA Pol II is stalled by the repressive Negative

Elongation Factor (NELF), another component of basal transcription factor TFIIH. The

nascent RNA chain folds into the TAR RNA structure constituted of the 3-base bulge and

apical stem-loop. In order to reinitiate transcription, the HIV regulatory protein Tat is

recruited to the three-base bulge sequence of TAR and subsequently recruits the positive

transcription elongation factor complex b (P-TEFb)/Tat-associated kinase (TAK). The P-

TEFb complex consists of CDK9 and Cyclin T1. Tat interacts directly with the cyclin T1

subunit of P-TEFb through zinc (Zn2+) cation to induce the cooperative binding of Cylcin-

Dependent Kinase-9 (CDK-9) (F igure 1.8). This recruitment enables the phosphorylation of

the negative elongation factors as well as the CTD of RNA Pol II, which allows the RNA Pol

II to transcribe the remainder of the HIV-1 genome (Karn 1999). Furthermore, Tat binding

enhances the processivity of the RNA Polymerase II (RNA Pol II) elongation complex,

which induces transcription of HIV-1 long terminal region (LTR) (Karn 1999).

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F igure 1.8: Model for the activation of RN A polymerase I I by Tat and cellular co-factors. (a) Initiation. The RNA Pol II is recruited to the HIV LTR promoter through

interactions with TFIID and other basal transcription factors such as TFIIH, which contains

CDK7. The CTD of the RNA polymerase is phosphorylated by CDK9 kinase, allowing the

RNA Poll II to clear the promoter and begin transcription. (b) Promoter C learance. The

aborted transcript of RNA folds into stem-loop structure, TAR. In the absence of Tat RNA

pol II (grey) synthesized short non-polyadenylated RNAs (black squiggly line). (c) Tat binds T A R RN A and T A K . Association of Tat to the 3-base-bulge promotes the recruiting of P-

TEFb/TAK, forming a ternary complex by direct binding to Cyclin T1. The interface

between Tat and cyclin T1 is believed to involve cysteine residues from each protein that

participate in zinc binding (Wei et al. 1998 and Karn1999). After p-TEFb is bound, CDK-9

phosphorylates the two negative elongation factors as well as the carboxyl-terminal domain

of RNA Pol II. (d) Tat-activated elongation. The TAR is displaced from the polymerase and

transcription of the remainder of the HIV genome occurs (i.e. HIV LTR region). Tat-TAR

association increases the processivity of the RNA polymerase II. (This image was adapted

from Karn 1999).

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Summary: Altogether, the data available in the literature suggest that inhibition of

Tat/TAR RNA interactions and CyclinT1/TAR interaction could be of great interest for

controlling HIV-1 replication. Accordingly, this knowledge has catalyzed the search for

molecular compounds that specifically block Tat/TAR interactions. In this study we focus on

elucidating the binding of synthetic to the TAR-RNA to further develop a TAR-RNA drug

that may warrant pharmaceutical development.

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Section I I . Cur rent Drug T reatments:

There is currently no cure for HIV. Yet, the HIV pandemic remains one of the most

deadly threats to world health and presents a significant development challenge (Karn 1999;

UNAIDS 2012). There are approximately thirty-three million people living with HIV/AIDS

worldwide. However, in the thirty-two years since the discovery of HIV, only twenty-five

antiviral drugs are available, for mass use and production. These drugs have been able to

reduce HIV prevalence rates but they are by no means effective preventions or cures for the

disease.

Typically the regulation of HIV includes antiretroviral therapy (ART) and Highly

Active Antiretroviral Therapy (HAART). There are over twenty-three U.S. Food and Drug

Administration (FDA) approved antiretroviral drugs that are used to treat the disease. The

function of ARTs is to repress the growth and reproduction of HIV as well as allow people

infected to live longer, healthier lives. Using several of these drugs in combination also

allows for the rebuilding of the immune system. These drugs are classified by the phase of

the retrovirus life cycle that the drug inhibits; the seven categories are as follows: Entry

inhibitors, CCR5 receptors antagonist, Nucleoside Reverse Transcriptase Inhibitors (NRTIs),

Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs), Protease Inhibitors (PIs), Fusion

Inhibitors (FIs), and Intergrase Inhibitors (IIs). However, these drugs have a variety of

adverse side effects, which makes selecting a regiment complex and variable among

individuals. In addition, although HIV chemotherapy inhibits most viral replication, there is

still a remaining population of latently infected cells that remain unaffected.

Viral latency is one of the aspects of the virus that makes it difficult to cure. HIV

viral latency is the ability of the virus to integrate into resting T-Cells and other cellular

reservoirs (Stevenson, 2003). In the case that the cells are activated, viral production spirals

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off. A currently studied therapeutic approach involves activating resting cells and flushing

the virus out of hiding, making it vulnerable to antiretroviral drugs and the natural immune

response. The most recent drug that induces the expression of HIV RNA and genomes in

resting CD4+ cells is a histone deacetylase inhibitor, suberoylanilidehydroxamic acid SAHA

also known as vorinostat, VOR (F igure 1.9a). Histone deacetylases are recruited to the HIV

long terminal repeat (LTR) promoter, and are therefore one of the several restrictions that can

limit LTR expression and maintain viral latency (Archin et al. 2012).

F igure 1.9: Examples of Antiviral Drugs used to treat H I V . (A) Vorinostat is a histone

deacetylase inhibitor that acts on HIV-1 infected CD4+ cells inducing HIV RNA and genome

expression. (B) Maraviroc is an entry inhibitor as well as a chemokine receptor CCR5

antagonist, preventing HIV gp 160 proteins from associating with the cell. The chemokine

receptor CCR5 is an essential co-receptor is an essential co-receptor for a majority of HIV

strains and is necessary for the entry process of the virus into the host c

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Motivation and A ims:

Therapeutic E fforts: Inhibitors Targeting T A R-RN A: There are two bimolecular

strategies to inhibiting the Tat/TAR-RNA complex: antibiotic and small molecule analogues

that are able to selectively bind to the TAR-RNA and those that interact with the Tat ARM

(F igure 1.10). Pharmological compounds that have already been developed to bind to TAR-

RNA vary in structural components and fall into three subcategories: those that bind to the

UCU or UUU trinucleotide bulge tightly and consequently outcompete the endogenous

protein partner, Tat peptide; those that bind the 3-base-bulge together with either lower or

upper stem-loop region; and those that bind the stem-loop structure preventing trans-

activation, by impeding the TAR-RNA interaction with Cyclin T1 (Yang 2005).

F igure 1.10: C lassical Approaches to Tat-T A R inhibition. Class I inhibitors that bind

TAR-RNA and Class II that bind the Tat protein outcompeting endogenous is cognate RNA

partner. The blue circle and green Pac-Man structure represents the small molecules designed

to target Tat and TAR-RNA respectively.

Tat

TAR-RNA TAR-RNA

Tat

Tat

TAR-RNA

Tat

TAR-RNA

TAR-RNA

Tat

Tat

TAR-RNA

Uninhibited

Class I

Class II

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Small molecule Inhibitors targeting T A R RN A : =/%361%2-31%&OOU"+,%-/%(/'-)(-;24%

-@0*/3%0A%/0/8183(:(9%+@-22%@0219*21+%CVR%W%JUUU>-B%71)1%:(+90'1)1:%30%(/6(;(3%H-3XH<I%

complex formation. Molecules as small as argininamide, were of the first shown to inhibit the

association of TAR RNA to Tat protein. In fact it is considered one of the best-investigated

TAR RNA ligands (Krebs 2003; Thomas 2008). It also binds at the bulge region by via H-

bonding to the UCU residues as well as residue G26 in the stem (F igure 1.11a). These early

findings paved the way for the development of other molecular inhibitors of TAR that

possessed guanidine moieties. To enhance the affinity, or direct the specificity, argininamide

bifunctional ligands consisting of ethidium bromide (known intercalator of DNA) and

arginine were developed. An example of these arginine conjugates is shown in F igure 1.11b.

This ethidium-arginine conjugate inhibits the association of Tat/TAR by binding the

trinucleotide bulge of TAR RNA with high affinity. The basic guanidinium groups of both

arginine analogues are said to interact with the phosphate ion backbone of TAR RNA, while

361%90/Y*E-31:%Z-rings have been hypothesized to participate in ! " ! interactions. This

incorporation of the aromatic rings and therefore ! " ! interactions increases the strength

and specificity of the guanidinum compoundFserving as a pseudo anchor.

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F igure 1.11: Structures of small molecules that bind T A R . (A) Schematicof a modular

ligand based on the known specificities of ethidium for the C-G, C-G base pairs (shown in

blue) and Argininamide binds the 3-base bulge (shown in red) and arrests the motions of

HIV-1 TAR RNA (Stephen 2004, Thomas 2008). (B). Synthesized ethidium-arginine

conjugate. The guanidiums (positively charge at biological pH) interact electrostatically with

[Q[%;*2E1%C)1:B%76(21%361%;1/\1/1%)(/E+%Z-stack with G-C bases. (This image was adapted

and modified from Thomas 2008).

Inhibitors targeting both the 3-base bulge and the stem-loop region of TAR RNA are

designed with three attributes: the activator that is a functionalized arginine residue; the

anchor that is an aromatic conjugated pi system intercalator; and a linker that connects the

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activator and anchor (F igure 1.12). Artificial regulators with these three attributes not only

bind to TAR but also competitively block the interactions of Tat-TAR RNA.

F igure 1.12: Modular design for T A R stem-loop and 3-base-bulge inhibitors. The blue

circles (activator) represent the cationic residues of arginine or lysine responsible for the

electrostatic interactions between inhibitor and TAR-RNA. The linker connects the

intercalator (anchor) to the rest of the compound. (This image was extracted from Thomas

2008).

The aminoglycoside Neomycin-B-hexaarginine (NeoR) is a pivotal example of this type of

inhibitor (F igure 1.13). NeoR is a bi-functional inhibitor that effectively inhibits both Tat

trans-activation and Tat extracellular. NeoR accumulates in the cell nuclei and inhibits the

replication activities of HIV infected cells at concentration as low as 0.8 µM (Litovchick

2001). In addition, NeoR antagonizes Tat extracellular activities, such as increased viral

production, induction of CXCR4 expression; resulting in suppression of the HIV expression

and progression of AIDS (Litovchick 2001; Yang 2005). We believe that the potency of

NeoR is a consequence of its multivalent display of quanidiniums which increases the

flexibility of the compound. Furthermore, the fact that NeoR is a multitarget inhibitor, it is

therefore an attractive lead compound, capable of interfering with different stages of HIV

infection and AIDS pathogenesis (Litovchick 2001).

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F igure 1.13: Neomycin B-H exaarginine Conjugate. The molecule consists of sugar-like

backbone displaying 5 arginines, and one agmatine (decarboxylated arginine). These

guanidinium side chains are basic (positively charged in solution) under physiological

conditions and binds to the three-base bulge as well as the stem-loop of HIV-1 TAR RNA

preventing the association between TAR RNA and its cognate, Tat peptide (Litovchick 2001;

Karn 1999).

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Synthetic Polymers Targeting T A R RNA : Oligocarbamates and oligoureas were

the first synthetic peptidomimetics designed to interact with TAR RNA (F igure 1.14).

F igure 1.14: Structure of T A R RN A binding O ligocarbamate. The sequence of

oligocarbamate corresponds to Tat peptide ARM sequence 48-57

(48GlyArgLysLysArgArgGlnArgArgArg57) (Tamilarasu et al. 2001). The uses of unnatural

biopolymers containing carbamate backbone structures are attractive because they are

resistant to protease degradation in vitro. The use of the carbamate linkages (shown in red) in

this peptidomimetic increase the biological stability because they are protease resistant. (This

image was extracted from Tamilarasu et al. 2001).

Tamilarasu and co-workers found that peptidiomimetic oligomers, oligocarbamate and

oligourea are able to regulate HIV-1 gene expression both in vivo and in vitro. In HL3T1

cells, a HeLa (Human) cell line oligourea and oligocarbamate inhibit transcriptional

activation by outcompeting endogenous Tat protein with an IC50 of ~0.5 µM and 1.0 µM

respectively (Tamilarasu 2001). In addition to potency, it is important to highlight the fact

that these TAR-RNA binding oligomers are non-toxic to the human HeLa cells. The property

of non-cytotoxicity is important because molecules that are not selectively destructive to HIV

infected cells alone are potentially harmful causing adverse effects which are overall anti-

intuitive to our disease combating goals.

The most recent synthetic polymers targeting the nucleic acids of TAR RNA are

multivalent branched peptides. Branched peptides are generated from conventional peptide

synthesis using solid phase and resin techniques. However, the syntheses of the branched

forms have been shown to increases the biological stability of certain peptides in comparison

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to the monomeric peptides. In fact, it has been shown that branched peptides are resistant to

enzyme proteolysis (Bracci 2003; Falciani 2007). In 2012, Bryson and coworkers reported

that a branched peptide, FL4 increased binding, selectivity, affinity for the three-dimensional

structure of native TAR-RNA with an half maximal inhibitory concentration (IC50) of ~1.0

µM; a consequence of multivalency (F igure 1.15) (Bryson et al. 2012). The Branched

Peptide FL4, can traverse the cell membrane of HeLa cells, and exhibit no cytotoxicity.

F igure 1.15: Schematic of combination library of branched peptides. The peptides are

synthesized on a resin via photocleavable linkage, by an automative synthesis. (This image

was extracted from Bryson 2009).

However, in competition assays between FL4 and TAR-RNA in the presence of excess tRNA

resulted in an increase shift in binding suggesting that FL4 binds with low specificity to the

TAR and therefore only partially selective for the TAR-RNA interface (F igure 2.7) (Bryson

et al. 2012). That is the branched peptide FL4 does not bind directly to the UCU bulge. It can

be hypothesized that the flexibility of the backbone may be hindering the binding affinity of

the peptide. Such hurdle can be traverse by using a more rigid structural backbone.

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F igure 1.16: Bindings of F L4 to T A R in the presence of tRN A using E MSA . Peptide

concentrations increase from left to right: 0.001 µM, 0.01 µM, 0.03 µM, 0.1 µM, 0.3 µM, 1

µM, 3 µM, 10 µM, 30 µM, and 100 µM. (This image was extracted from Bryson 2012). The

band intensity increases largely in the presence of 1000x tRNA suggesting FL4 is partially

selective to TAR-RNA. (This image was adapted from Bryson et al. 2012).

Summary: So far, none of the previously identified inhibitors of HIV-1 transcription

described herein have been approved for clinical use as HIV-1 antiviral agents. The design

and use of multivalent polymers with essential structural requirements that mimic specificity

and recognition of HIV TAR-RNA would generate a novel anti-viral drug with a low

susceptibility to drug resistance as well as further insight into ceasing pathogenesis of HIV-

AIDS. Therefore, we wanted to specifically explore alternate scaffolds and structural

backbones that would display guanidinium functionality to mimic the arginine rich binding

domain of Tat. Most importantly, we were interested in using a scaffold/chemical backbone

that would present our ligands to the TAR-RNA under optimal conditions, be protease

resistant, biorthogonal to cellular environment, and non-toxic as well as have pharmological

properties such as cell permeability. Factors that affect cell ]permeability and potency of

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inhibitor are molecular weight, valency (spacing) and density (number) of arginine residues

(Bryson et al. 2012).

The design of drugs to inhibit RNA-protein interactions is not a trivial task. When

developing drugs that modulate these interactions poor selectivity and binding affinity is a

common hurdle to overcome. Issues with developing synthetic molecules fall into three

categories: cost, limitations, and difficulties in synthesis including the required steps of

reaction and purification. For example, synthetic peptides normally require a significant

amount of protecting groups chemistry, several sequential coupling steps (usually in order of

de-protection, then coupling of the next ligand) and rink amide resin (solid-phase and

expensive). It is important to note that though solid-phase peptide synthesis has become

routine, the procedure presents many disadvantages. They are: the method can be laborious

and tedious; non-compatibility of resin and growing peptide chain; and formation errors in

peptides causing truncated failure sequences. These disadvantages are unfavorable owing to

the fact that peptide synthesis has proven indispensable for the structural elucidation and

activity studies of many naturally isolated products.

Thorough examination and comparison of the aforementioned compounds found to

disrupt the Tat/TAR complexation share one thing in common; their cationic nature

resembles the ARM of the Tat. These mimics are functionalized to display lysine arginine

and other guanidiniums that emulate the highly basic structural region of Tat. Therefore

prototypic, molecule designs that target TAR-RNA must be functionalized with basic

guanidiniums and amines on a carbon scaffold. In addition, while the aforementioned

analogues and mimetics are cationic in nature, the potency, affinity and specificity of the

TAR binding compounds vary in backbone structure and scaffold. Moreover, it can be

concluded that the emerging picture from these pioneering findings indicates that new types

of RNA-specific chemical scaffolds must be developed (Wang 2009).

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A I MS: For this project, we strategized to create polymers displaying guanidinum

functionalities. Recently, an optimized synthetic route to creating biologically active

multivalent arrays displaying desired functionality was developed. This synthesis employs

ring-opening-metathesis-polymerization (ROMP) of norbornene succinimidyl (NHS) ester

monomers (F igure 1.17) (Strong 1999). The N-hydroxysuccinimide (NHS) functionality can

be modified with desired amine-containing epitotes; because the NHS ester group is

especially sensitive to amines in the presence of other less nucleophilic groups, which makes

it ideal for coupling amino acids to the polymer units (F igure 1.17). These ROMP-derived

polymers possess an attractive featureFthe ability to control density (mole fraction) by

altering stoichiometric ratios and the control over molecular weight, a property that can have

significant influence on cellular uptake (Tamilarasu 2001; Bryson 2009). Other inherent

advantages of using multivalent ROMP derived polymers is that multivalent displays

concomitantly increase avidity and selectivity as opposed to small molecules, which possess

low selectivity and weak binding. Therefore, there is a large possibility that through

developing these multivalent polymeric displays we cannot only increase the selectivity, but

also the affinity of our polymer to the RNA or protein.

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F igure 1.17: A General Scheme for Polymer Conjugation and Design. Synthetic

multivalent ligands (A) consist of a linear polymeric scaffold (black line) used to present

multiple copies of a ligand of interest (blue circle). We used a cylcopentane-based polymer

scaffold (B) and an amide bond connection to ligand resulting from the reaction between

polymer N-hydroxy succinimidyl-ester (NHS) group and ligand amine (C). The average

polymer length (n) was systematically varied. Synthesis of the NHS ester-substituted polymer

scaffold was accomplished by using the Ring-Opening Metathesis Polymerization (ROMP)

of NHS ester-substituted exo-norbornene monomer (D). This monomer was synthesized from

the exo-norbornene carboxylic acid isomer (E), which was recovered from a mixture of

endo/exo isomers (F) by iodolactonization reaction. The ligands of interest in these studies

abbreviated by the blue circle are the amino acid arginine and agmatine (a decarboxylated arginine).

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Synthetic Utility of R O MP-der ived Succinimidyl Ester Polymers: ROMP has been

used to develop scopes of biologically active polymers that have been used in several

structural elucidation and activity studies. In mouse B-cell activation ROMP derived

multivalent polymers were used to illicit immune responses by targeting B-cell receptors and

demonstrated no toxicity (Puffer et al. 2007), bacterial chemotaxis (Gestwicki et al. 2002)

and to develop cell permeable block copolymers (Kolonko et al. 2009). Gestwicki

demonstrated that multivalent polymers target methyl-accepting chemotaxis proteins (MCPs)

(Chemoreceptors) in bacteria subsequently influencing the signaling of cascade in bacteria.

As the valency of a galactose chemoattractant increased, a decrease in the average angular

velocity of E . coli strain AW05 (chemotaxically active E.coli) was observed, suggesting that

the bacteria were moving uniformly in response to the multivalent chemoattractant

(Gestwicki et al. 2002). Kolonko et al. also exploited this synthetic approach and length

control offered by ROMP to assemble cell permeable block copolymers (Kolonko et al.

2009). They found that block copolymers composed of half N-(3-amino propyl) guanidine

substituted and half alpha-chloroacetamide substituted were internalized into cell. The N-(3-

amino-propyl) guanidine functions as an artificial translocation domain (ATD) allowing the

polymer to traverse the cell membrane and the chloroacetamide group could be post modified

with intact proteins via reaction of cysteine side chain (Kolonko et al. 2009). Overall, they

used ROMP to generate polymeric ATD that can be used as delivery vehicles for

macromolecules as well as copolymer backbones that can promote intracellular protein

assemblies (Kolonko et al. 2009).

Determination of Protein-Nucleic Acid Interactions: Classical methods for the

detection of protein nucleic acid interactions include gel shift assays, filter-binding assays

and dot blots. However, Electrophoretic Mobility Shift Assay (EMSA) and dot blot assays

are the most popular and efficient of the three. Typical EMSA protocols, allows for solutions

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of combined proteins and nucleic acids to be incubated and the resulting oligiomers separated

by electrophoresis under native conditions through polyacrylamide or agarose gel. Native

conditions allow for the molecules to migrate complexes of protein- RNA to migrate;

therefore complexes travel more slowly than the corresponding free nucleic acid or protein. If

the starting nucleic acid was radioisotope labeled, or fluorescently labeled nucleic acid the

gel may be analyzed using autoradiography and a fluorescent scanner respectively.

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F igure 1.18: Approaches of Various L igand Displays: (A) Monovalent display cartoon.

This large volume of small molecule inhibitors (represented by circles) demonstrate weak

binding and low avidity. (B) Schematic of multivalent display. (C) It would potentially

require a large concentration of monovalent ligands to bind the TAR bulge sequence with

very low affinity and specificity. (D) We hypothesized that the multivalent polymeric dugs

may bind the TAR bulge at lower concentration because polymeric display provides greater

avidity and selectivity.

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Materials and methods:!

Section I . Chemical Synthesis Procedures, Purification and Character ization

General Procedures and Materials. All reactions were conducted under atmospheric

conditions unless otherwise noted. All other moisture and oxygen-sensitive reactions were

performed with syringe-septum cap techniques in flame-dried glassware under an inert

nitrogen (N2) atmosphere. Reactions were carried out at room temperature unless otherwise

specified. Unless otherwise noted, all materials (reagents and solvents) were used without

further purification as obtained from commercial suppliers. Dichloromethane (CH2Cl2) was

degassed and dried by sparging with ultra-high purity argon gas followed by passage through

an activated alumina column using a Glass Contour Seca Solvent Purification System and

water (H2O) was purified with a MilliQ purification system (Millipore). Reactions were

monitored and analyzed by thin-layer chromatography (TLC) using pre-coated Silica (SiO2)

plates available from Merck. Visualization of compounds was accomplished using two

methods: staining with a potassium permanganate stain (3 g KMnO4, 20 g K2CO3, 5 mL 5%

aqueous NaOH, 300 mL H2O) and/or ultraviolet (UV) light radiation at 254 nm. Flash

Column Chromatography and disposable PD-10 desalting columns (Sephadex G-25 Medium)

was used to purify all products unless otherwise stated. Silica (SiO2) gel flash

chromatography (SiliCycle, 40-63µM, pore size 60) was used as the stationary phase for

flash chromatography, while the mobile phase varied with each procedure. Proton and carbon

Nuclear Magnetic Resonance (1H NMR and 13C NMR respectively) were obtained on a

Varian 400MHz spectrometer. 1H NMR chemical shifts were calibrated in parts per million

',#

#

(ppm) and Hertz (Hz) using residual solvents CDCl3: 1H: 7.26, 13C: 77.23; CD3OD: 1H: 3.31,

13C: 49.15; DMSO-d6: 1H: 2.5, 13C: 39.51; and D2O: 1H: 4.8. The 1HNMR spectra are

tabulated as follows: chemical shifts, multiplicities (are described using the following

abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and resonances

that appear broad are designated (br) numbers of protons (integration) and coupling

constants. 13C NMR spectra are reported as values in parts per million relative to residual

CHCl3 (77 ppm) or CD3OD (49 ppm) as internal standards.

Exo 5-Bicyclo[2.2.1]hepta-2- (Norbornene)-2-Carboxylic Acid (2)

A mixture of commercially available exo/endo 5-norbornene-2-

carboxylic acid 1 (predominantly endo) (5.0 mL, 40.86 mmol, 1.0

eq) was dissolved in aqueous 0.75 M sodium bicarbonate

(NaHCO3) (3.75 g/60 mL H2O). Subsequently a solution of iodine

(I2) (9.3625 g. 36.89 mmol, 0.9 eq) and potassium iodide (KI) (17.6

g, 106.2 mmol, 2.6 eq) in H2O (50 mL) (brown/black in color) was added drop-wise and

completely with a pipette. A brown-yellow color persisted at the surface, while a dark

brown/black sludge (iodolactone) was suspended at the bottom of the flask. The mixture was

decanted separating the aqueous layer from the viscous sludge. The aqueous layer was

extracted with diethyl ether (Et2O) (3 x 20 mL) to remove the residual iodolactone sludge. A

10% sodium thiosulphate (Na2S2O3) solution (0.75 g/ 6.75 mL H2O) was added to the

aqueous layer to decolorize the solution. Using a 1.0M solution of sulfuric acid H2SO4 the pH

of the solution was adjusted to two as determined by litmus paper and a yellow precipitate

was observed. The mixture was extracted again with diethyl ether (4 x 40 mL). The

combined ether layers were dried using magnesium sulfate (MgSO4) and concentrated under

reduced pressure with rotary evaporator. The concentrated solution was placed under high

vacuum for 20 minsF24 hours. The final exo acid product was typically a white/yellow solid

'-#

#

color (1.217 g, 8.807 mmol, 21.6%). 1H NMR (400 MHz, CDCl3B^%_%&&NO`%C;)%+,%&KB,%SN&S%

(dd, J = 5.9 Hz, 1H), 5.97 (dd, J = 6.3 Hz, 4.2 Hz, 1H), 3.12 (br s, 1H), 2.95 (br s, 1H), 2.30-

2.25 (ddd, J = 10.5 Hz, 4.3 Hz, 2.2 Hz, 1H), 1.96 (dt, J = 12.7 Hz, 3.6 Hz, 1H), 1.55 (d, J =

8.5 Hz, 1H), 1.45-1.38 (m, 2H).

Succinimidyl ester (N HS) substituted exo-norbornene monomer (3)

The succinimidyl ester-substituted monomer was synthesized as

reported in Strong, L.E.; Keissling. L. L. J. Am. Chem. Soc.

1999, 121, 6193-6196. The exoFnorbornene acid (1.217 g,

8.807 mmol, 1.0 eq), N-(3-dimethylaminopropyl) N-

136429-);0:((@(:1%64:)09620)(:1%Ca>Q=%b%KQ2B%C`NJc%E,%&cN`%

mmol, 1.5 eq) and N-hydroxysuccinimide (NHS) (1.521 g, 13.2

mmol, 1.5 eq) were stirred in anhydrous reaction grade CH2Cl2

(50 mL) under N2 for 24 hours. The organic layer was extracted using 5% citric acid (3 x 65

mL), saturated sodium bicarbonate NaHCO3 (2 x 50 mL) and saturated NaCl (1 x 50 mL).

The dichloromethane CH2Cl2 layer was dried with magnesium sulfate (MgSO4) and

concentrated under reduced pressure on a rotary evaporator leaving a white powdery solid.

The residue was re-dissolved in minimal amount of dichloromethane (CH2Cl2) and purified

with flash column chromatography (2:1 hexanes/ethyl acetate). A total of 60 fractions were

collected and assessed by TLC. Fractions 8 ] 58 were combined and concentrated under

reduced pressure on rotary evaporator. A white solid powder (1.197 g, 5.087 mmol, 57.8%)

was isolated and was stored in the freezer. Silica gel Rf, 0.53 (1:1 hexane/ethyl acetate); 1H

NMR (400 MHz, DMSO-d6B^%_%SN&O%C::,%&KB,%SN&`%C::, 1H), 3.26 (br s, 1H), 2.99 (br s, 1H),

2.83 (s, 4H), 2.52 (ddd, 1H), 2.05 (dt, 1H), 1.54 (d, 1H), 1.51-1.45 (m, 2H).

(.#

#

Ruthenium Carbene Polymer ization Catalyst (6)

The Ruthenium Catalyst was synthesized as reported in

Love, J. A.; Morgan, J. P.; Trnka, T. M.; Grubbs, R. H.

.<%d)-93(9-2%-/:%K(E624%<93('1%I*361/(*@-Based

Catalyst that Effects the Cross Metathesis of

<9)420/(3)(21N5%Angew. Chem. Int. Ed. 2002, 41(21),

4035-4037. 3-bromopyridine 5 (238.3 µL, 2.47 mmol,

10eq) was added to 2nd generation Grubbs catalyst 4

(210 mg, 0.247 mmol, 1.0 eq) in a 24 mL Teflon-lined cap screw top vial. The solution

mixture was stirred for approximately 5-10 minutes at room temperature in air. Subsequently,

8.5 mL of room temperature pentanes was layered on top of the mixture. The vial was capped

under air and stored in the freezer at (~5o C) for 24 hours. A sintered glass funnel was then

used to filter the precipitate. Subsequently, the precipitate was washed with room temperature

pentane (4 x 4 mL). The ruthenium carbene catalyst 6 was dried on high vacuum to afford a

green powder (199.4 mg, 0.225 mmol, 91.3%) and stored in a freezer.

Succinimidyl (N HS) ester substituted Norbornene Polymer M :I = 10:1 (7a)

A solution of succinimidyl ester-substituted

norbornene monomer 3 (200.0 mg, 0.8502

mmol, 10 eq) dissolved in anhydrous

degassed dichloromethane was prepared in a

Teflon-capped screw-top vial. Subsequently,

75.2 mg (0.08502 mmol, 1.0 eq) of

ruthenium catalyst dissolved in degassed

CH2Cl2 was also added using a gastight

(%#

#

syringe. The succinimidyl ester and ruthenium solutions were made from stock

concentrations of 820 mg/ 8.2 mL and 202.6 mg/2.7 mL respectively. . The reaction was then

stirred under nitrogen (N2) for 30 minutes at -20o in a dry ice/isopropanol bath. Upon

observed color change from green to brown (indicating initiation and propagation of

polymerization) four drops of ethyl vinyl ether (in excess) were then added to terminate the

reaction and the solution was stirred overnight. The resulting polymer solution was divided

into 4 parts and precipitated into 25mL of 9:1 Et2O/Benzene while vortexing (polymer

solution added drop-wise). The solutions were then centrifuged at 1000-1500 rpm for

~30minutes, followed by decanting. The resulting solid material from each fraction was then

transferred and combined into one vial. The combined solid was placed on high vacuum

overnight, and the viscous product was triturated with ~1 mL of diethyl ether (Et2O). The

solution was concentrated on the rotary evaporator and again dried under high vacuum to

provide a greenish grey solid (M:I 10:1, 54.7 mg, 27.4 %) Selected 1H NMR (400 MHz,

DMSO-d6B^%_%eNcf-7.05 (m, 0.77H), 6.45-6.16 (m), 5.79-5.71 (m, 0.18H), 5.50-4.89 (m,

2.00H), 3.67-0.826 (m).

Succinimidyl (N HS) ester-substituted Norbornene Polymer M :I = 25:1(7b)


monomer 3 (200.0 mg, 0.8502 mmol, 10 eq)

dissolved in anhydrous degassed

dichloromethane (CH2Cl2) was prepared in a

Teflon-capped screw-top vial. Subsequently,

30.10 mg (0.03401 mmol, 1.0 eq) of

ruthenium catalyst dissolved in degassed



(&#

#

concentrations of 820 mg/ 8.2 mL and 202.6 mg/2.7 mL respectively. The reaction was then

stirred under nitrogen N2 for 30 minutes at -20 oC in a dry ice/isopropanol bath. Upon




into 4 parts and precipitated into 25 mL of 9:1 Et2O/Benzene while vortexing (polymer

solution added drop-wise). The solutions were then centrifuged at 1000-1500 rpm for ~30

minutes, followed by decanting. The resulting solid material from each fraction was then

transferred and combined into one vial. The combined solid was placed on high vacuum


solution was concentrated under diminished pressure on the rotary evaporator and again dried

under high vacuum to provide a grey solid (M:I 25:1, 64.0 mg, 32 % ). Selected 1H NMR

(400 MHz, DMSO-d6B^%_%eNcf-7.05 (m, 0.40H), 6.45-6.25 (m), 6.16-5.71 (dd), 5.50-4.91 (m,

2.00H), 4.39 (s), 3.99-0.82 (m).

Succinimidyl (N HS) ester substituted Norbornene Polymer M :I = 50:1 (7c)




degassed dichloromethane (CH2Cl2) was

prepared in a Teflon-capped screw-top vial.

Subsequently, 15.0 mg (0.0170 mmol, 1.0 eq)

of ruthenium catalyst dissolved in degassed




('#

#






solution added drop-wise). The solutions were then centrifuged at 1000-1500 rpm for ~30

minutes, followed by decanting. The resulting material from each fraction was then

transferred and combined into one vial. The combined solid was high placed on vacuum


solution was concentrated on the rotary evaporator and again dried under high vacuum to

provide a grey solid (M:I 50:1, 55.7 mg, 27.9 %). Selected 1H NMR (400 MHz, DMSO-d6):

_%eNcf-7.05 (m, 0.18H), 6.42 (m), 6.19-5.79 (dd), 5.50-4.91 (m, 2.00H), 3.98-0.468 (m).

Succinimidyl (N HS) ester substituted Norbornene Polymer M :I = 100:1 (7d)




degassed dichloromethane (CH2Cl2) was

prepared in a Teflon-capped screw-top vial.

Subsequently, 7.52 mg (0.0085 mmol, 1.0

eq) of ruthenium catalyst dissolved in

degassed CH2Cl2 was also added using a

gastight syringe. The succinimidyl ester and ruthenium solutions were made from stock




((#

#




solution added drop-wise). The solution mixtures were then centrifuged at 1000-1500 rpm for

~30 minutes, followed by decanting. The resulting material from each fraction was then

transferred and combined into one vial. The combined solid was high vacuumed overnight,

and the viscous product was triturated with ~1 mL of diethyl ether (Et2O). The solution was

concentrated on the rotary evaporator and again dried under high vacuum to provide a grey

solid (M:I 100:1, 72.6 mg, 36.3 %). Selected 1H NMR (400 MHz, DMSO-d6B^%_%eNcf-7.05

(m, 0.14H), 6.19-6.16 (dd), 5.72-4.97 (m, 2.00H), 4.39 (s), 3.30-2.46 (m), 2.01-1.79 (m),

1.49-1.44 (m), 1.33-1.19 (m).

N-(2-Amino)-5-guanidinopentanoic (A rginine) Conjugated Polymer 10mer

The succinimidyl ester-substituted polymer

7a (5.3 mg, 0.0225 mmol, 1.0 eq) was

dissolved in 225 µL anhydrous DMSO. To

the solution was added 7.6 mg (0.0437

mmol, 2.0 eq) of arginine and 12 µL (0.109

mmol, 5.0 eq) of N-Methylmorpholine was

added. The reaction was vortexed to dissolve

all reagents and then left to run for three days. The reaction was quenched with 3.5 µL (2.0

eq) of 3-amino-1, 2-propanediol. Using a PD-10 desalting size exclusion column (Sephadex

G-25 resin), the product was purified. The column was first washed five times with MilliQ

water. The crude product was then loaded onto the top of the column. The reaction tube was

washed twice with 100 µL of DMSO and vortexing. The resulting solutions were loaded

onto the column as well. After polymer loaded onto the column two portions of 400 µL of

()#

#

MilliQ water was added to the top of the column and allowed to load. The column was then

filled to the top with MilliQ water and the product was eluted into 6 fractions approximately

1 mL in volume. Due to the polymers limited water solubility, the eluents containing polymer

were observed as cloudy. All the fractions were concentrated using a speed vacuum for 24

hours and weighed to provide polymer 8a. Fractions with pure solid as determined by 1H

NMR were added to solvent a system containing 490 µG%>V?gX&U%hG%>2O and thereafter

vortexed and sonicated to dissolve. The pure solid weight for 8a was 4.8 mg (M:I 10: 96 %).

The same reaction sequence was used to provide polymer 8b (M:I 25:1, 3.6 mg, 61 %) as a

flocculent grey-white solid, polymer 7c (M:I 50:1, 4. mg, 94 %) as a flocculent colorless-

solid.

Compound 8a: Selected 1H NMR (400 MHz, DMSO-d6, D2gB^%_ 1.18 (br), 1.48, 1.82-2.01

(m), 2.24-2.35 (m), 2.46-2.55(m), 2.75- 2.89 (m), 3.19-3.37 (m), 4.38 (s), 4.902-4.96 (m),

5.25-5.50 (m), 7.04-7.50 (m, 5H).

Compound 8b: Selected 1H NMR (400 MHz, DMSO-d6, D2gB^%_ 1.09-1.19 (br), 1.488 (br),

1.81-2.01 (d), 2.24-2.35 (m), 2.46-2.56 (m), 2.75-2.89 (m), 3.2-3.37 (m), 4.39 (s), 5.18-5.27

(d), 6.95-7.21 (m, 5H), 7.48-7.789 (m).

Compound 8c: Selected 1H NMR (400 MHz, DMSO-d6, D2gB^%_ 1.02 (m), 1.19 (s), 1.47 (m),

1.82 (m), 2.29-2.35 (d), 2.44-2.59 (d), 2.62 (s), 2.89-3.00 (m), 3.21-3.28 (m), 3.39-3.46 (m),

4.43-4.66 (d), 5.13-5.27 (m), 7.6 (m, 5H)

(*#

#

N-(4-Aminobutyl) Guanidine [Agmatine] Conjugated Polymer 10 mer (9a)

The succinimidyl ester-substituted

polymer 7a (5.3 mg, 0.0225 mmol, 1.0

eq) was dissolved in 212 µL anhydrous

DMSO. To the solution 7.3 mg (0.0561

mmol, 2.5eq) of

N-(4-Aminobutyl) Guanidine

(agmatine) and11.7 µL (0.107 mmol,

5.0 eq) of N-Methylmorpholine was

added. The reaction was vortexed to dissolve all reagents and then left to run for three days.

The reaction was quenched with 3.5 µL (2.0 eq) of 3-amino-1, 2-propanediol. Using a PD-10

desalting size exclusion column (Sephadex G-25 resin), the product was purified. The

column was first washed five times with MilliQ water. The crude product was then loaded

onto the top of the column. The reaction tube was washed twice with 100 µL of DMSO and

vortexing. The resulting solutions were loaded onto the column as well. After polymer

loaded onto the column two portions of 400 µL of MilliQ water was added to the top of the

column and allowed to load. The column was then filled to the top with MilliQ water and the

product was eluted into 5 fractions approximately 1 mL in volume. Due to the polymers

limited water solubility, the eluents containing polymer were observed as cloudy. All the

fractions were concentrated using a speed vacuum for 24 hours to evaporate the water and

weighed (F1 0.0001 mg, F2 0.0035 mg, F3 0.0002 mg, F4 0.00 mg, F5 0.00 mg). Fractions

with pure solid as determined by 1H NMR were added to solvent a system containing 490 µL

>V?gX&U%hG%>2O and thereafter vortexed and sonicated to dissolve. The pure solid weight

for 9a was 3.8 mg (M:I 10:1,76% ). The same reaction sequence was used to provide

polymer 9b (M:I 25:1, 4.2 mg, 84% ) as a flocculent brown solid, polymer 9c (M:I 50:1, 4.1

(+#

#

mg, 82% ) as a flocculent brown solid.

Compound 9a: Selected 1H NMR (400 MHz, DMSO-d6, D2gB^%_ 1.17 (br), 1.49, 1.80-2.07

(m), 2.24-2.39 (m), 2.46-2.65(m), 2.75- 2.79 (m), 3.19-3.37 (m), 4.38 (s), 4.902-4.96 (m),

5.27-5.52 (m), 7.04-7.50 (m, 5H).

Compound 9b: Selected 1H NMR (400 MHz, DMSO-d6, D2gB^%_ 1.09-1.21 (br), 1.45 (br),

1.81-2.01 (d), 2.24-2.35 (m), 2.39-2.58 (m), 2.75-2.89 (m), 3.22-3.39 (m), 4.39 (s), 5.18-5.27

(d), 6.97-7.25 (m, 5H), 7.48-7.789 (m).

Compound 9c: Selected 1H NMR (400 MHz, DMSO-d6, D2gB^%_ 1.12 (m), 1.21 (s), 1.51 (m),

1.82 (m), 2.29-2.35 (d), 2.44-2.59 (d), 2.62 (s), 2.89-3.00 (m), 3.21-3.28 (m), 3.39-3.46 (m),

4.43-4.66 (d), 5.10-5.32 (m), 7.45 (m, 5H).

Norbornene-A rginine Monomer Conjugation (9)

The Norbornene- Arginine Monomer

Conjugation was synthesized as adapted

from a Choudhury protocol (Choudhury

2007). L (+)-Arginine 98+% (100 mg,

0.5740 mmol) was dissolved in 6 mL of

1:1 water/dioxane and treated with 72.4

mg (0.861 mmol) of sodium

bicarbonate (NaHCO3). The solution

was stirred for ~5 min until most of the arginine was dissolved. To the stirred solution was

added 147.8 mg (0.6297 mmol) of the N-hydroxysuccinimide (NHS) substituted norbornene

ester 3. The reaction mixture was stirred at 25 oC for 20 hr and then acidified to pH 4 by the

addition of 1 N sodium bisulfate (NaHSO4). The aqueous phase was extracted with 6 mL of

ethyl acetate and the organic layer was discarded. The aqueous phase was concentrated under

(,#

#

reduced pressure. The residue was suspended in 4 mL of methanol and the insoluble solid

was vacuum- filtered using a frit funnel. Subsequently, the solid was washed twice with 3mL

portions of methanol. The combined methanol extracts were concentrated under reduced

pressure. The vicious oil residue was further purified by flash chromatography on a silica gel

column. Elution with 15mL 1:1, 2:1, and then 3:1 methanol-ethyl acetate afforded ideal

separation. There were 25 fractions in total and the pure fractions 2-20 were qualitatively

assessed by thin-layer-chromatography and thereafter combined and concentrated on the

rotary evaporator. The viscous residual was dried under high vacuum to afford the Arginine-

substituted monomer 9 as a colorless foam: yield 188 mg (96 %); silica gel TLC Rf , 0.41 (4:1

methanol-ethyl acetate); 1K%!VI%CfUU%VK\,%>V?gB^%_%%&UNPJ%C;)%+,%&KB,%PNef%C;)%+,%`KB,%

7.67 (br d, 1H), 6.08 (dd, 1H), 6.04 (dd, 1H), 5.8 (d, 1H), 4.01 (dt, 1H), 2.78 (m, 2H), 2.29 (d,

1H), 1.95-1.15 (m, 10H).

(-#

#

Section I I : Biological Assays

Mater ials and Reagents:

O ligonucleotide. Non-labelled wild-type TAR RNA (MW = 9, 290.6 g/mole OD 260=3.1,

&&NJ%/@021+,%UN&&%@E,%+1i*1/91%J"-rGrCrC rArGrA rUrCrU rGrArG rCrCrU rGrGrG

rArGrC rUrCrU rCrUrG rGrC -c"B%7-+%-9i*()1:%A)0@%=/31E)-31:%H196/020E(1+%C=>H,%

Coralville, I). The extinction coefficient was 268, 900 L/(mol cm) at absorbance peak of 260

nm. RNase-Free HPLC by manufacturer purified TAR RNA and no further purification was

administered. 2.0 mg of Tat (47- 58) peptide (NH2-YGRKKRRQRRRP-COOH) (with

OONfUj%KdGQ%8*)(34T%VR%k%&SJeNOe%EX@02B%7-+%0;3-(/1:%A)0@%Z-Proteomics, LLC

(Huntsville, AL).

Molecular Biology Kits:

Electrophoretic Mobility-Shift Assay (EMSA) Kit was obtained from Invitrogen (Grand

Island, NY) and consisted of: SYBR® Green EMSA nucleic acid gel stain (Component A),

&UU%hG%0A%-%&U,UUUl%90/91/3)-31%(/%:(@13642+*2A0m(:1%C>V?gB,%?ndIgo%I*;4%aV?<%

protein gel stain (Component B), 650 mL of an aqueous 1X solution, Trichloroacetic acid

(TCA, Component C), 87.5 g, 6X EMSA gel-loading solution (Component D), 1 mL and 5X

;(/:(/E%;*AA1)%CQ0@80/1/3%aB,%`UU%hGN%

EMSA is a robust technique that provides a fast, easy and quantitative method to detect both

nucleic acid and protein in the same gel. This kit uses two fluorescent dyes for detection -

SYBR® Green EMSA nucleic acid gel stain for RNA or DNA detection and SYPRO® Ruby

EMSA protein gel stain for protein detection. The nucleic acids and proteins are stained

sequentially in the gel after electrophoresis, and labeling does not interfere with the protein

binding being assayed. All the reagents were used without further purification and diluted to

).#

#

achieve selective staining conditions.

Native PAGE Reagents:

Native polyacrylamide 16 % (w/v) PAGE gel cassettes (8 x 10) cm, Tris-Tricine based

RunBlue Native Run Buffer (20X), and RunBlue Native Sample Buffer (4X) were obtained

commercially from Expedeon. All the Native PAGE reagents were used as acquired and

diluted accordingly for each reaction condition.

Silver Staining Reagents:

Silver Nitrate ACS Grade and Formaldehyde, 37% Solution Proteomics Grade was obtained

from VWR (Amresco; Solon, OH, Cat# 97064-582 and Cat# 97064-886 respectively).

Sodium Thiosulfate, 0.0250 Normal (N/40) was obtained from VWR (Ricca; Cat#

RC790032). Absolute Ethanol (EtOH) and Acetic acid (HOAc)

was obtained from Sigma Aldrich (St. Louis, MO). Sodium bicarbonate was also obtained

from VWR (Amresco; Solon, OH).

Solutions:

DEPC-Treated Water:

Diethyl pyrocarbonate (DEPC)-treated water was prepared to free deionized water of

possible contaminating RNases. To every 1000 mL of deionized water approximately 1 mL

of 0.1% diethyl pyrocarbonate was added to the solution and mixed vigorously at ambient

temperature for 1 hr. The solution was then autoclaved from 45 minutes to inactivate DEPC

by hydrolyzing diethylpyrocarbonate to carbon dioxide and ethanol. These byproducts are

volatile so are released as vapors yielding RNase free water. The solution is then cooled to

room temperature prior to use.

)%#

#

TE (Tris-EDTA) Buffer Solution:

Stock buffer solutions of Tris/EDTA (Ethylenediamine Tetraacetic Acid) were prepared for

diluting TAR RNA samples. The TE solutions were made of

Tris(hydroxymethyl)aminoethane base (Tris-base), EDTA, HCl in DEPC treated water. In

each solution, final buffer concentrations were 10 mM (604.8 mg/0.5 L) Tris-base, and 0.5

mM (9.3 mg/0.5 L) EDTA at ~pH 8.0. The buffer was further sterilized by filtration through

a 0.22-µM filter.

Magnesium Binding Buffer Stock Solution:

A stock binding buffer solution of 100 mM Tris-HCl (606.8 mg/50 mL), 250 mM KCl (93.2

mg/50 mL), 5 mM MgCl2 (52.3 mg/50 mL), 5 mM dithiothreitol (DTT) (39.0 mg/ 50 mL)

and 25% Glycerin (12.5 mL/50 mL) was prepared using DEPC-treated water.

Silver Staining Solutions:

0.2% Silver nitrate:

0.2 g AgNO3

100 mL ddH2O

0.05% Formaldehyde:

50 µL of 37% formaldehyde

100mL of ddH2O

30% Ethanol:

300 mL of Absolute Ethanol

1 Liter of ddH2O

10% Acetic Acids:

50 mL of Glacial Acetic Acid

500 mL of ddH2O

)&#

#

0.08% Formaldehyde:

80 µL of 37% formaldehyde

100 mL of ddH2O

2.5 mM Sodium Thiosulfate:

0.31 g sodium thiosulfate (Na2S2O3)

500 mL ddH2O

3% Sodium Bicarbonate:

7.5 g Na2CO3

250 mL dd H2O

`U%hV%?0:(*@%H6(0+*2A-31^%

800 µL of 2.5 mM Na2S2O3

100mL ddH2O

Methods:

Preparation of Samples for Electrophoretic Mobility-Shift Assay (EMSA):

HIV-1 TAR RNA (0.11 mg, 11.5 nmole) was diluted with 115 µL of TE buffer to make a

final stock concent)-3(0/%0A%&UU%hV%C&UUUlBN%H1/-fold serial dilutions were made to generate

stock concentrations of 10 µM (100X), 1 µM (10X), and 100 nM (1X).

H70%@(22(E)-@+%0A%H-3%8183(:1%7-+%:(++02'1:%(/%&`U%hG%0A%Ha%;*AA1)%30%@-p1%-%A(/-2%+02*3(0/%

stock concentration of 10.05 mM. Additional ten-fold serial dilutions were performed to

E1/1)-31%+309p%90/91/3)-3(0/+%0A%&%@V,%&UU%hV,%&U%hV,%-/:%&%hVN

Preparations of dilutions for Electrophoretic mobility Shift Assay:

<%UN&%@Vk&UU%hV%+309p%+02*3(0/%0A%/*921(9%-9(:%7-+%8)1pared by dissolving (11.5nmoles,

UN&&@EB%0A%H<I%I!<%(/%&&J%hG%0A%A)1+624%8)18-)1:%Ha%;*AA1)%C&U@V%H)(+-HCL ~pH 8.0,

1mM EDTA) and gently vortexed to create and homogenous mixture. To decrease the

amount of freeze thaw cycles of the original stock of TAR RNA serial dilutions of 1:10,

1:100 and 1:1000 were prepared. A 1:10 dilution from this stock was by diluted by adding 10

)'#

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hG%0A%&UU%hV%H<I%+309p%-/:%:(++02'1:%(/%OU%hG%0A%Ha%;*AA1)%(/%-%207%-:61+(0/%1881/:0)A%30%

9)1-31%-%&U%hV%+309p%+02*3(0/%-/:%+7()21:%30%@ake solution homogenous. The 1:100 dilutions

7-+%@-:1%;4%-::(/E%&U%hG%0A%361%&U%hV%+02*3(0/+%30%-/0361)%OU%hG%0A%Ha%;*AA1)%30%E1/1)-31%

-%&%hV%+02*3(0/%76(96%7-+%A*)361)%:(2*31:%;4%+-@1%90/'1/3(0/%30%9)1-31%-%&^&UUU%

dilution/100 nM stock solution.

Concerns with TAR RNA sample quality, and the eventual failure of resolving bands

on the gel prompted spectrophotometric analysis using Nano Drop (ND-1000) UV-Vis

Spectrophotometer. The spectral analysis data of the HIV-1 stock and diluted samples

revealed ideal A260/280 ratios of 2.1.

General Procedure for TAR RNA Control Assays:

Five microliter aliquots of TAR RNA in TE buffer (10 mM Tris-HCl, 0.5 mM

EDTA) was then added to an empty low-adhesion eppendorf with RNase free low-adhesion

pipettes. In addition, 3 hG%0A%::%K2g%-/:%`%hG%0A%Jl%;(/:(/E%;*AA1)%C750 mM KCl, 0.5 mM

dithiothreitol, 0.5 mM EDTA, 50 mM Tris, pH 7.4). To make sure the control environments

of the TAR RNA resembled that of the forthcoming inhibition assay the TAR RNA was

incubated in 5 mL a 49:1 of DMSO/D2g%+02*3(0/%9)1-3(/E%-%A(/-2%)1-93(0/%'02*@1%0A%&J%hG%

and mixed gently but thoroughly. The reaction minutes was then incubated at 37 oC for

approximately 30 minutes.

General Procedure for Inhibition Assays without Magnesium Binding Buffer:

Initially, the binding of our ROMP-derived polymeric guanidiniums to the TAR RNA

interactions were studied by EMSA performed on 16% w/v Native polyacrylamide gels in

low ionic strength commercially available Native Run Buffer (Tricine, Tris-

hydromethylaminomethane, sodium bisulfite). The polymeric guanidiniums were diluted in

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fOU%hG%>V?gX%&U%hG%>2O to prepare each series at 2X concentrations. Seven and half

microliter aliquots of HIV-&%H<I%I!<%(/%Ha%;*AA1)%71)1%-::1:%30%eNJ%hG%-2(i*03+%0A%361%

conjugated multivalent polymer to give the desired final concentration of each component.

The binding of ROMP-derived multivalent guanidiniums to the TAR RNA was assayed by

incubating the commercially acquired wild type TAR RNA with increasing concentrations of

the specific-polymer lengths for approximately 30 minutes at 37 oQ%(/%361%8)1+1/91%0A%c%hG%

6X binding buffer (50 mM Tris-HCl (pH 7.4 at 20), 750 mM KCl, 0.5 mM EDTA, 0.5 mM

:(36(036)1(302BN%H61)1-A31),%J%hG%0A%-%fl%aV?<%E12-loading buffer solution was added to the

mixture for loading.

General Procedure for Inhibition Assays in the presence of Magnesium Binding Buffer:

Five microliter aliquots of HIV-1 TAR RNA in TE buffer were pre-(/9*;-31:%7(36%J%hG%

aliquots of magnesium binding buffer (20 mM Tris-HCL, 50 mM KCL, 1 mM MgCl2, 5 mM

DTT, 5% Gylcerin) for approximately 30 minutes at ambient temperature. Following pre-

(/9*;-3(0/,%361%H<I%I!<%7-+%3(3)-31:%7(36%J%hG%-2(i*03+%0A%361%-88)08)(-31%90/Y*E-31:%

multivalent polymer to give the desired final concentration of each component. The final

concentration of TAR RNA was 200nM while the concentration of specific polymer

(/9)1-+1:%-+%A02207+^%&%hV,%J%hV,%&U%hVN%%H61%303-2%'02*@1%@(m3*)1%0A%A(A311/%@(9)02(31)%7-+%

then incubated for 37 oQ%A0)%cU%@(/*31+N%H61)1-A31),%J%hG%0A%-%fl%aV?<%E12-loading buffer

solution was added to the mixture for loading.

Native-PAGE Gels Electrophoresis of TAR RNA control samples:

q02207(/E%361%-88)08)(-31%(/9*;-3(0/%0A%H<I%I!<%`NJU%hG%0A%Sl%aV?<%E12%20-:(/E%

solution was added and mixed gently. Before loading samples, the wells very rinse twice

thoroughly with ultrapure water. Thin RNase free, and low adhesion pipette tips were then

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used to load 12-&J%hG%0A%1-96%+-@821%/1-)%361%;0330@%0A%361%7122N%The RNA was then

electrophoresed on the 16 % non-denaturing polyacrylamide gels for 90 minutes at 130 V,

4oC.

TAR RNA SYBR® Green EMSA Nucleic Acid Staining:

The protocol used for the nucleic acid staining of TAR RNA was adapted from

Invitrogen product fact sheet. For a typical gel staining the SYBR® Green EMSA stain is

diluted into of TE (10 mM Tris-HCl, 1mM EDTA, pH~8.0). A total of 5 µL the 10 000X

stain concentrate is diluted into 50 mL of TE buffer generating a 1X final concentration. The

electrophoresed gel was then place in a clean Rubbermaid Servin Saver plastic staining

container followed by addition of sufficient 1X SYBR Green stain. The gel was then

incubated in the 1X stain covered with aluminum foil to protect from light with continuous

agitation on an orbital shaker at 50 rpm from ~40 minutes at ambient temperature (25 oC).

Note that each 50 mL staining solution was kept in the cold room at 4 oC in the dark and

reused for a total of three-four times. Following this, the gel was washed twice with 150 mL

of RNase free dH2O for ~10 seconds to remove excess stain.

Silver Staining:

The protocol used for staining the Protein was adapted from a Chevallet protocol

(Chevallet 2006) and in the laboratory of Melissa Kosinski-Collins, under the supervision of

Deborah Bordne.

Following electrophoresis the gel was incubated in a fixer solution of 30% ethanol

(EtOH) and 10% acetic acid (HOAc) for 2 hrs. Subsequently, the gel was washed with 30%

EtOH twice every 15 minutes for 30 minutes and thereafter rehydrated by washing with dd

H2O for approximately 25 minutes to remove all the acetic acid, reduce background staining

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and increase the gel sensitivity. The gel was then sensitized by soaking in 0.02%, (2.5 mM)

sodium thiosulfate (Na2S2O3) for 2 minutes and washed with ddH2O two times every 5

minutes for 10 minutes. Afterwards, the gel was impregnated with silver nitrate by incubating

for 30 minutes in at 4o C cold. Following this, the gel was then washed with ddH2O twice in 1

min. The gel was then placed in a new staining tray and developed in 3% sodium bicarbonate

(Na2CO3B,%UNUJj%A0)@-2:164:1%CJU%hG%cej%QK2gB,%`U%hV%+0:(*@%36(0+*2A-31%C!-2S2O3).

The developer solution was changed immediately when a yellow color change was observed.

The process was terminated when staining was sufficient. The gel staining was terminated by

washed with ddH2O for 30 seconds and incubation in 10 mL of acetic acid (HOAc) for five

minutes. Lastly, the gel was then left at 4 ºC in 1.0 % HOAc for storage. Prior to analysis the

gel was washed in ddH2O for 3 x 10 min to ensure complete removal of acetic acid.

Staining with SYPRO® Ruby EMSA protein gel stain:

The protocol for staining Tat protein with SYPRO® Ruby stain was adapted from

Invitrogen fact sheet and in the laboratory of Melissa-Kosinski Collins.

To a solution a bottle containing 87.5 g of Trichloroacetic (TCA) a 100 mL of

SYPRO® Ruby EMSA protein gel stain was added completely. The solution was agitated

and shaken to dissolve the TCA. The gel was then placed in a clean Rubbermaid Servin

Saver plastic staining container followed by addition of 50 mL SYPRO® Ruby EMSA

protein gel stain in TCA. The gel was incubated with continuous gentle agitation on an

orbital shaker at 50 rpm for approximately 3 hours in the dark. Subsequently, the gel was

washed twice with 150 mL of dd H2O for about 10 seconds. The gel was further de-stained

with 10 % methanol and 7% acetic acid for 60 minutes. The gel was again washed in twice

with 150 mL of dd H2O to remove all the acetic acid.

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Data Acquisition, Processing and Analysis:

Electrophoresis data were collected on Molecular Imager Gel Doc XR acquired from

Bio-Rad Laboratories Inc., a Typhoon Scanner (Amersham Biosciences) and Image

Quantification Software (Armersham Biosciences). Xcel graphs were further generated to

tabulate the quantities of binding.

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Results: Section I : Synthesis of Guanidinylated-Substituted Polymers The first major goal of our research project was to synthesized multivalent

peptidomimetics with enhanced inhibitor potency, specificity and affinity for interaction with

the 3-base-bulge interface of tertiary folding TAR-RNA. Through synthetic rationale

garnered from researching previously reported TAR-RNA inhibitors, we aim to generate

multivalent oligomers of defined lengths and low polydispersities using post-polymerization

modification strategy to conjugate ligands onto a polymeric scaffold (Strong 1999). Post-

synthetic ligand modification would allow us to attain the structural requirements for

inhibiting TAR RNA/Tat association. Our efforts will begin by synthesizing monomers

containing amine-reactive succinimidyl ester groups. Subsequently, the polymeric scaffolds

of well-defined lengths will be generated via ring-opening-metathesis polymerization

(ROMP) of the amine-reactive monomers. ROMP is an attractive method for polymerization

because it utilizes a reactive ruthenium carbine catalyst, which can tailor polymers of well-

defined lengths by varying the monomer to catalyst initiator ratios (Strong 1999; Puffer 2007;

Gestwicki 2002).

Norbornene Monomers: Synthesis of the isomerically pure exo- norbornene

monomer (2) for ROMP was generated in one step (Scheme 1). An iodolactonization reaction

of a commercially available mixture of exo and endo stereoisomers of 5-norbornene-2-

carboxylic acid (1) was performed.

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Scheme 1: Isolation of Exo-Norbornene. Iodolactonization of a commercially

available mixture of endo/exonorbornene carboxylic acid (1) afforded the exo isomer

(2). Reagents and conditions: Predominantly endo/exo-norbornene acid (1) (5 mL,

40 mmol, 1.0 eq), I2 (9.37 g, 36.9 mmol, 0.87 eq), KI (17.6 g, 106.2 mmol, 2.6 eq),

and NaHCO3 (3.75 g/60 mL), H2O, 25o C, 4 h.

The general mechanism of the iodolactonization is detailed in F igure 3.1. The predominantly

endo mixture was dissolved in aqueous sodium bicarbonate (NaHCO3) and treated with an

amalgamation of iodine (I2) and potassium iodide (KI). The endo isomer (1 endo), which

reacts very quickly with iodine, is precipitated as iodolactone, a brown sludge, while the exo

isomer reacts slowly to form a di-iodide compound (F igure 3.1). Because the diiodide is

unstable at room temperature it decomposes readily back to the alkene and the iodine.

Subsequently, the exo product was isolated via extraction with diethyl ether; the iodolactone

dissolved in the ether layer was discarded, while the exo-isomer remained in aqueous layer.

Acidification of the aqueous solution with sodium thiosulfate removed excess iodine and

afforded the 2 exo product. It was necessary to isolate the 2 because mechanistic studies have

shown that the exo iosomer reacts faster with the ROMP catalyst. Furthermore, the endo

conformation will sterically and electronically inhibit polymerization in downstream

reactions as a result of: (1) unfavorable steric interactions between the growing polymer

chains and the incoming isomer and (2) the orientation and electronics of the carbonyl group

additionally plays a critical role in retarding the rate of propagation. Overall, the exo isomer

is necessary because, this configuration allows for greater reactivity under ring-opening-

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metathesis conditions. A yield of 21.6% of the exo isomer was obtained. Since we started

with a predominantly endo mixture this yield was acceptable.

F igure 3.1: Iodolactonization M echanism for Isolation of Exo-norbornene. In a basic

aqueous solution of NaHCO3 all the norbornene is deprotonated by the base to generate

carbonic acid and the carboxylate ion. The carbonic acid decomposes to carbon dioxide and

water. The alkene in the endo-isomer reacts more readily with iodine to form a cyclic iodide

intermediate. The stereochemistry of the endo-isomer allows for fast intramolecular

nucleophillic attack of the carboxylate ion, which relieves the ring strain by forming the

iodolactone intermediate. The di-iodide species is unstable at ambient temperature and

decomposes back to the iodine (I2) and the exo-alkene. Extraction with diethyl ether removes

the iodolactone in the organic layer.

In order to produce monomers that are selective to amines in the presence of other

nucleophiles facilitating ligand conjugation, the isomerically pure exo product was

completely converted to the amine-reactive ester monomers (3) by the general mechanism

shown in F igure 3.2. Use of the coupling agent N-(3-dimethylaminopropyl)-!"-

ethylcarbodiimide (EDCI) in combination with N-hydroxysuccinimide (NHS) in methylene

chloride under inert N2 atmosphere transformed the exo-carboxylic acid to the N-

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succinimidyl ester substituted exo-norbornene monomer (3). EDCI activated the carboxylic

acid group of 2 forming an unstable O-acylisourea 2b that is more labile for the attack of the

NHS hydroxyl nucleophile. Acidification and extraction of the reaction mixture using citric

acid, allowed for the removal of the urea by-product 2c. The activated carboxylic acid ester

(3), was purified by flash column chromatography, to afforded an 85% yield and

subsequently analyzed by Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy.

Scheme 2: Subsequent Conversion of Exo-Norbornene to Succinimidyl Ester . The exo

isomer (2), is subsequently converted to the activated carboxylic acid ester, amine-reactive N-succinimidyl ester substituted exo-norbornene (3). Reagents and conditions: Isomerically

pure exo- norbornene acid (2) (1.22 g, 8.81 mmol, 1.0 eq), EDCI (2.53g, 13.2 mmol, 1.5 eq),

NHS (1.52 g, 13.2 mmol, 1.5 eq), CH2Cl2, 25o C, 24 h, 57.8%.

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F igure 3.2: Mechanism for the formation of the Amine-reactive Esters. The carbodiimide

(EDCI) reacts with the exo-acid (2) to form the unstable activated carboxylic acid, O-

acylisourea (2b). The O-acylisourea futher reacts with the NHS to form a more stable NHS-

activated ester product, which is selectively susceptible to amines in the presence of other

nucleophiles.

Ruthenium Carbene Polymer ization Catalyst: To generate the ruthenium carbine

catalyst for ROMP, ligand substitutions of commercially available second generation

M)*;;"+%9-3-24+t (4) using 3-bromopyridine (5) was conducted to yield the bispyridine

carbene complex (6). The 2nd generation Grubbs was reacted overnight in air at room

temperature and filtered through a sintered glass funnel (Scheme 3). The formation of the

ruthenium carbene catalyst (6) for Ring-Opening Metathesis Polymerization (ROMP) was

necessary to obtain the desired polymer lengths. The second generation catalyst could

potentially terminate polymer formation before reaching the appropriate length due to its high

reactivity forming varying lengths of polymers with high polydispersities. This reaction

replaces the tricyclohexylphosphine ligand on 4 with two more labile and electron-deficient

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3-bromopyridine ligands increasing the catalyst initiation rate > 1, 000,000 fold. The higher

ratio of initiation to rate of propagation makes catalyst 6 more useful in polymerization.

Importantly, the dissociation rates of ligands are directly related to catalytic efficiency. This

suggests that in comparison to the tricyclohexylphosphine ligands, the dissociation of 3-

bromopyridine is extremely rapid owing to faster initiation and even faster chain propagation

due to slower rebinding. Overall, the strong affinity and binding of the phosphine ligands

makes the catalyst less reactive and less likely to prematurely terminate the polymerization.

The ruthenium carbene catalyst 6 would react until all the norbornene NHS ester monomers

are consumed. Therefore by exploiting monomer-initiator-stoichiometries we can tailor

polymers with varying average lengths with low polydispersities. A green powdery product

(6), in 91% yield was isolated by precipitation in pentanes.

Scheme 3: Synthesis of Ruthenium Carbene Polymer ization Catalyst. Coupling of 3-

bromopyridine (5) with 2nd Generation Grubbs Catalyst (4) at room temperature results in the

generation of Ruthenium Carbene-Initiator Polymerization Catalyst (6). Reagents and conditions: M)*;;"+%`nd Generation Catalyst (4) (210.0 mg, 0.247 mmol, 1.0 eq), 3-

bromopyridine (5) C`cPNc%hG,%`Nfe%@@02,%&UNU%1iB,%pentanes (8.5 mL), -20o C, overnight,

91%.

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G eneral Succinimidyl Ester Substituted Polymeric Scaffold: The succinimidyl substituted

polymers was synthesized under inert nitrogen (N2) atmosphere by ring-opening-metathesis

polymerization using bispyridine-carbene catalyst (6) (Scheme 4). Control over average

polymer length was accomplished by variation of the monomer-to-initiator (M/I) catalyst

stoichiometric ratio. Ratios of 10:1 monomer: catalyst, 25:1, 50:1, and 100:1 were used to

obtain polymers of average lengths n~10, n~25, n~50 and n~100 (7a-d). A solution of NHS

ester monomer (3) dissolved in anhydrous (degassed) dichloromethane was cooled in a dry

ice/isopropanol bath to -`U%rQN%H61%8024@1)(\-3(0/%)1-93(0/%7-+%(/(3(-31:%*80/%-::(3(0/%0A%

solution of ruthenium catalyst (6) in degassed dichloromethane and termination after

complete consumption of the substituted norbornene (3) with ethyl vinyl ether, an electron

rich olefin. The excess ethyl vinyl ether undergoes metathesis with the living polymer chain

end carrying Grubbs catalyst to generate a metathesis-inactive Fischer carbene. After 12

hours, the polymer was precipitated into falcon tube from vortexing diethyl ether (Et2O).

After centrifuging and decanting the tubes, the precipitate was dried under high vacuum,

leaving a greenish solid for the 10-mer and grey solid for the 25-mer, 50-mer and 100-mer.

Polymerization yields ranged between 27 and 37%. The low percent yields were presumably

due to too much dicholoromethaneFa solvent which our polymers was soluble inFin the

vortexing tubes. Note that using more diethyl ether and/or more falcon tubes can optimize the

yields. The resulting polymers were characterized by 1H NMR spectroscopy to afford the

average length (Mn) values. The Mn values were determined by comparing the 1H-NMR

integration signals of the polymer alkene protons to that of the terminal phenyl protons

(Puffer 2007). The calculated Mn values more or less corresponded to the expected values

from the monomer initiator ratios, M:I =10:1, 25:1, 50:1, 100: 1.

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Scheme 4: Synthesis of Succinimidyl Ester Substituted Polymer Scaffolds. Using

Ruthenium Carbene-Initiator Catalyst (6), succinimidyl ester polymers (7a-d) with the

average lengths n~10, n~25, n~50 and n~100 were derived from monomer units of N-succinimidyl ester substituted exo-norbornene (3). Reagents and conditions: i) 10-mer: Amine reactive ester (3) (200.0 mg, 0.8502 mmol 10 eq), Ruthenium Carbene-Initiator

Catalyst (6) (75.2 mg, 0.08502 mmol, 1.0 eq) -20oC, 30 mins, ethyl vinyl ether (3-5 drops),

24 hr, 27.4%. ii) 25-mer: Amine reactive ester (3) (200.0 mg, 0.8502 mmol 25 eq),

Ruthenium Carbene-Initiator Catalyst (6) (30.1 mg, 0.03401 mmol, 1.0 eq) -20oC, 30 mins,

ethyl vinyl ether (3-5 drops), 24 hr, 32 %. iii) 50-mer: Amine reactive ester (3) (200.0 mg,

0.8502 mmol 50 eq), Ruthenium Carbene-Initiator Catalyst (6) (15.0 mg, 0.0170 mmol, 1.0

eq), -20oC, 30 mins, ethyl vinyl ether (3-5 drops), 24 hr, 27.9%. iv) 100-mer: Amine reactive

ester (3) (200.0 mg, 0.8502 mmol 100 eq), Ruthenium Carbene-Initiator Catalyst (6) (7.52

mg, 0.008502 mmol, 1.0 eq), -20oC, 30 mins, ethyl vinyl ether (3-5 drops), 24 hr, 36.3%.

These polymers will be used in our EMSA assays.

Our next objective was to modify the succinimidyl substituted polymeric scaffolds

with our amine-bearing ligands of choice. To generate polymeric drugs to target the TAR

RNA, the amino acids arginine and agmatine were coupled to the polymers (Scheme 5). In

our experimental protocol, a 1.0-eq solution of NHS-polymer (7a-c) dissolved in dimethyl

sulfoxide (DMSO), 2.0 eq of Homo-S-Arginine was added, followed by 5.0 eq of N-

Methylmorpholine (NMM) at room temperature. N-Methylmorpholine is an organic base that

acts a coupling reagent to facilitate the formation of amide bond between the polymeric acyl

esters and the most reactive primary amine of the amino acids arginine and agmatine.

Overall, the synthesis of the amides in 8a-c and 9a-c proceeds via zwitterionic intermediate

that forms as a result of lone pairs on the amine attacking the carbonyl carbon, resolving its

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charge separation by collapse of the tetrahedral intermediate allowing elimination of the NHS

ester from the carbon center. The reaction mixture was then vortexed to facilitate dissolution

of all reagents. The reaction was then allowed to run for three days. Excess 3-amino-1, 2-

propanediol was used to convert any remaining N-hydroxysuccinimide (NHS) ester groups

into neutral functionality. The products were then purified and isolated using a PD-10

desalting size-exclusion column, eluted with Milli-Q water. PD-desalting columns separate

compounds by large differences in molecular weight. Globular or large molecular weight

compounds pass through column quickly, while compounds such as salts and small

molecules pass through slowly. Therefore, this technique provides an efficient way to

separate our large molecular weight polymeric products. Yields of the multivalent arginine

displays 8a-c ranged from 61-96%, while the yields from the agmatine displays ranged from

76-91% yield. As the polymer length increased the recovered yield decreased significantly.

The approximate weights of 8a-c polymers assuming 100% conjugation are 3000 Da, 8000

Da, and 16 000; while the weights of polymers 9a-c are 2800 Da, 7000 Da, and 14 000 Da.

These weights will be important when assessing the RNA-polymer complexes.

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Scheme 5: Conjugation of Amines to Synthetic Polymer Scaffolds. Coupling of amine-

bearing guanidinums with the polymeric NHS ester. Quenching with excess 3-amino-1, 2

propanediol, terminates the reaction. Reagents and conditions: i) 8a-c: Respective

succinimidyl ester-substituted polymer 7a-d (5.3 mg, 0.0225 mmol, 1.0 eq), arginine (7.6 mg,

0.0437 mmol, 2.0 eq), N-Methylmorpholine (11.7 µL, 0.107 mmol, 5.0 eq) of, DMSO, 25 oC,

c:-4+,%cNJ%hG%c-amino propanediol, 61-96%. ii) 9a-c Respective succinimidyl ester-

substituted polymer 7a-d (5.3 mg, 0.0225 mmol, 1.0 eq), N-(4-Aminobutyl) Guanidine

(agmatine) (7.3 mg, 0.0561 mmol, 2.5eq) and N-Methylmorpholine (11.7 µL, 0.107 mmol,

5.0 eq),%>V?g,%c:-4+,%cNJ%hG%c-amino propanediol, 76-91%.

N-Norbornene-S-A rginine Monomer : In order, to make general comparisons

between the multivalent polymers and small molecules, conjugated monomers of arginine

were successfully synthesized (Scheme 6). The procedure for this synthesis was adapted and

modified from Choudhury. N-succinimidyl-ester substituted Norbornene (3) was added to a

solution of Homo-S-Arginine, dissolved in a 1:1 mixture of water-dioxane treated with

sodium bicarbonate and stirred at room temperature for 16 hr. Acidification, followed by

extraction with ethyl acetate isolated the crude product. The crude product was purified by

flash chromatography, which afforded a 96% yield of N-Norbornene-S-Arginine Monomer

(9). 1H NMR Spectroscopy was used to further assess the conversion as well as purity of the

product. All peaks corresponding to the alkene, amide and methylene protons were observed.

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Scheme 6: Synthesis of A rginine Norbornene Control Monomers. Activated NHS-

Norbornene was coupled with arginine in 1:1 water-dioxane solution. Sodium bicarbonate

served two roles (1) as catalytic base for resolving the charge separation that occurs as a

result of the primary amine attacking the carbonyl center (2) increasing the solubility of the

reactive species. Reagents and conditions: Amine reactive ester (3) (148 mg, 0.630 mmol,

1.01 eq), Arginine (100 mg, 0.574 mmol, 1.0 eq), NaHCO3 (72.3 mg, 0.861 mmol, 1.5 eq), 4

mL 1:1 water-dioxane, 25 oC, 16 hr, 96%.

N-(4-Aminobutyl) Guanidine (Agmatine) Monomer : The synthesis of agmatine

was unsuccessful. The hypothesized route to generating these monomers is described in

Scheme 7. Though the reaction procedure was orthogonal to the conjugation of the

succinimidyl-esters we experienced isolation and purification issues. We exhausted several

extraction and flash chromatography procedures with no luck. Due to limited time and

resources this synthesis was postponed.

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Scheme 7: G eneral route for the synthesis of Agmatine Monomers. Attempts to generate

the Agmatine monomers. Reagents and Conditions: Amine reactive ester (3) (20.6 mg,

0.0876 mmol, 1.097 1iB,%<E@-3(/1%C&PN`%@E,%UNUeOe%@@02,%&NU%1iB,%!VV%C`J%hG,%UN`&`%

@@02,%JNU%1iB,%fJU%hG%>V?g,%`f%6)- 3 days, 0%.

Summary: Altogether, we hypothesized that we would be able to compare the

arginine derivatives of arginine and agmatine to assess the role of the carboxylic acid in the

inhibition of Tat/TAR-RNA interactions. In addition, the variability in polymer length will

also allow us to identify whether the valency increases or decreases the affinity of the

potential RNA-binding drugs. To test this hypothesis, we will assay the RNA-binding

activities of our guanidinylated polymers using an EMSA-based approach. We expect that

since the polymers display guanidiniums an essential requisite for the electrostatic interaction

between the RNA and its endogenous cognate, we should observed some level alteration in

the electrophoretic mobility shift of TAR RNA especially at high concentrations of the

polymer.

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Section I I : E lectrophoretic Mobility Shift Assays The identification of multivalent oligomers that bind TAR RNA with great affinity

and specificity could potentially serve as a potent therapeutic antiviral agent in the fight

against HIV/AIDS. To this end, our laboratory collaborated with Dr. Melissa Kosiniski-

Collins to develop a quantitative assay that allows us to analyze the effects of our ROMP-

derived synthetic polymers on TAR RNA. To investigate whether our synthetic polymeric

guanidinium compounds possess desired reactivity, we performed binding studies using the

Electrophoretic Mobility Shift Assay (EMSA) technique. To implicate TAR RNA folding

and activity, we first dissolved the lyophilized RNA in Tris/EDTA buffer, pH 7.5. In our

protocol, we incubated the wild type HIV-1 TAR RNA at biological cesQ, followed by

subsequent gel electrophoresis (4oC, 130V, 90 mins) to determine the optimal concentration

required for visualization with commercially acquired SYBR® Green Nucleic acid EMSA

stain. By titrating varying concentrations of RNA we observed the gel mobility shifts as

illustrated in F igure 3.3. Using the Typhoon 6410 Variable Mode Scanner, we concluded

that the SYBR® GREEN stain was sensitive to 43 ng of TAR RNA or more. We also

concluded that for future binding/inhibition experiments, TAR RNA concentrations between

200 ]300nM would be ideal.

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F igure 3.3: Native Gel E lectrophoresis of control T A R RN A samples with increasing concentrations. Increasing concentrations of TAR RNA was incubated at 37oC in a total

volume mixture of 15 µL including 32.7% DMSO and 0.67% D2O (final concentration v/v)

to control against polymer solvents. Subsequently, the reaction mixture was electrophoresed

into a 16% native PAGE gel. The gel was visualized using a Typhoon Variable Mode

Scanner at ?n$I%E)11/"+%excitation wavelength of 520 nm. The dark concentrated bands

correspond to the labeled TAR RNA (red arrow). We suspect the band underneath free RNA

is an RNA degradation product (black bracket). TAR RNA concentrations of 200 and 300nM

gave the cleanest band of the group.

Binding of Guanidinylated R O MP-derived Polymers to the H I V-1 T A R RN A in

the absence of Magnesium: In our experimental protocol for monitoring binding between

the arginine 10-mer and TAR RNA, we incubated TAR RNA (300nM) with an increasing

concentration of polymer ranging from &%hV,%30%fUU%hV%CF igure 3.4). After incubation, the

RNA-polymer complexes were resolved by electrophoresis along with control solutions of

strictly polymer and strictly TAR RNA. The various bands (F igure 3.4) suggest that the

+&#

#

arginine conjugated 10-mer binds the TAR RNA, decreasing its mobility through the gel.

Furthermore, the juxtaposition of the RNA-polymer complex band with the free RNA band

provided convincing evidence that arginine 10-mer is an RNA-binding molecule active in the

1-fUUhV%)-/E1N%The number of bands and their respective intensities are not sensitive to

small changes in polymer concentration, but large changes show an increase in the number of

visualized RNA-polymer complexes and a decrease in free RNA (Table 3.1). In the

concentration range of 1F10 µM, all the retarded band shifts were similar. In contrast, in the

range 200F400 µM we observed three or more high density oligomeric precipitates (F igure

3.4). The fluorescence in the retarded bands was quantified using a fluorescent Image Quant

software.

Table 3.1: Quantified A rginine 10-mer binding exper iment. This table corresponds to the

gel shifts in F igure 3.4. The gel shifts seen in the figure were categorized into free RNA and

complexed/bound RNA. The fluorescence is strictly a measurement of the SYBR stain on the

RNA, which was quantified using Imager Quant software. The values of Fluorescence are

reported in generic Absorbance Units (AU) as obtained from the Typhoon scanner. % bound

was calculated as complexed RNA AU divided by total absorbance times 100%.

10-mer Arginine:

d024@1)%ChVB

Free RNA

Fluorescence

Complex

Fluorescence % Bound

1 3449106 1704197 33.1

5 3510325 1685399 32.4

10 2761667 1260555 31.3

200 2423354 2090077 46.3

400 1708616 3653452 68.1

0 2793172 0 0

After confirming that the developed polymers bind TAR RNA, we inquired about the

binding mechanism of TAR RNA to the polymers. Initially, we hypothesized that since the

10-mer polymer is approximately the same length as the arginine rich domain in Tat, then the

multiple gel shift bands suggest that RNA-polymer interactions were non-specific. In

addition, such bindings could compel the RNA to fold into various configurations that could

+'#

#

be accounted for in the different gel bands. This hypothesis would further suggest that the

increase in the polymer length and molecular weight may decrease the number of gel shift

bands. To make convincing conclusions about the binding mechanism as well as the effect of

polymer length in the RNA-binding assays, we moved forward with the arginine-conjugated

compounds.

F igure 3.4: T itration of T A R-RN A with R O MP-der ived A rginine Peptidomimetic (10-mer). Increasing amounts of arginine 10-mer polymer were mixed with 25 ng of TAR RNA,

incubated for 30 minutes at 37oC and then separated into a 16 % non-denaturing

polyacrylamide gel for 130 V, for 90 minutes, at 4oC. The gel was stained with SYBR®

Green EMS stain components of the Electrophoretic Mobility-Shift Assay Kit. After staining,

the image was scanned using a Typhoon 9410 Variable Mode Scanner at an excitation

?n$IoE)11/"+%1m9(3-3(0/%wavelength of 520 nm. This native electrophoresis gel shows the

TAR RNA-polymer complexes. The leading band is free TAR (red arrow) while the trailing

bands are hetero-complexes of the arginine 10-mer and TAR (black arrow). In addition, the

absence of the polymer showed no inhibition of the TAR RNA, as was expected.

+(#

#

In contrast to the arginine 10-mer, the bindings of arginine conjugated 25-mer and

50-mer to the wild type RNA in the 1F&U%hV%)-/ge resulted in only one shifted RNA band

(F igure 3.5-6). The results suggested that the 25-mer and 50-mer specifically recognize the

TAR RNA and retards the migration of the RNA. However, at considerably higher

concentrations (approximately 200!M), we begin to observe multiple retarded band shifts

suggesting less specific binding exist between the TAR RNA and the polymers. Given the

fact that the 25-mer and 50-mer are longer in length than the 10-mer, it is possible that

alternative polymer-RNA stoichiometries exist. The length would attribute multiple RNA

binding sites for a single polymer unit.







25-mer Arginine:

d024@1)%ChVB

Free RNA

Fluorescence

Complex


1 3448799 1202956 25.9

5 3705031 1002198 21.3

10 4131736 993494 19.4

200 2210197 1521484 40.8

0 4112013 0 0

+)#

#

F igure 3.5: T itration of T A R RN A with R O MP-der ived A rginine Peptidomimetic (25-mer). Increasing amounts of arginine 25-mer polymer were mixed with 25 ng of TAR RNA,

incubated for 30 minutes at 37 oC and then separated into 16 % non-denaturing

polyacrylamide gel at 130 V, for 90 minutes, at 4oC. The gel was stained with the SYBR®

Green EMSA stain component of the Electrophoretic Mobility-Shift Assay Kit. After

staining, the gel was analyzed using a Typhoon 9410 variable mode imager at SYBR®

E)11/"+%excitation wavelength of 520 nm. This native electrophoresis gel shows the TAR

RNA-polymer complexes. The leading band is free RNA (red arrow) while the trailing bands

are hetero-complexes of the arginine 25-mer and RNA (black arrows).

+*#

#







50-mer Arginine:

Polymer (hVB

Free RNA

Fluorescence

Complex


1 1393080 50890 3.52

5 2865762 82811 2.80

10 2099457 56872 2.64

200 1513969 406472 21.17

0 2815080 0 0

++#

#

F igure 3.6: T itration of T A R-RN A with R O MP-der ived A rginine Peptidomimetic (50-mer). Increasing amounts of arginine 50-mer polymer were mixed with 25 ng of TAR RNA,

incubated for 30 minutes at 37 oC and then separated into 16 % non-denaturing

polyacrylamide gel at 130 V, for 90 minutes, at 4oC. The gel was stained with the SYBR®

Green EMSA stain component of the Electrophoretic Mobility-Shift Assay Kit. After

staining, the gel was analyzed using a Typhoon 9410 variable mode imager at SYBR®

E)11/"+%excitation wavelength of 520 nm. This native electrophoresis gel shows the TAR

RNA-polymer complexes. The leading band is free RNA (red arrow) while the trailing bands

are hetero-complexes of the arginine 25-mer and RNA (black arrows).

Due to the success of the preliminary Native-PAGE experiments, our group was

convinced that the equilibrium-binding constant of the arginine polymers to the HIV-1 TAR

RNA could be increased. That is, we believed the binding was being perturbed by the

presence or absence of an important binding cofactor. Initial concerns led to the discussion of

whether the binding environment was optimal. There was a general agreement that the

presence of salt ions and pH were essential for any reputable binding reaction. In fact, the

addition of too much salt may have destabilized the binding interactions between the polymer

+,#

#

and RNA, decreasing the interactions actually observed on the gel. We concluded that the

initial 750 mM KCl binding buffer was concentrated enough to destabilize the binding

interactions. By lowering the salt concentration, we observed increased binding slightly, but

this enhancement was not significant enough to warrant the end to development of a better

binding reaction buffer. In fact, this lack of significant increase in binding led us to question

whether binding system promotes and maintains the native folding of the TAR RNA.

Incorrect folding may also have influenced the presence other bands in the gel.

To alleviate our concerns about the TAR RNA folding, as well as the hypothesized

multiple binding states of the various polymers, we decided to explore binding buffer systems

that contained both biological KCl and magnesium ion (Mg2+) concentrations. We then

studied the effects of introducing 50 mM KCl and 1 mM MgCl2 in the binding reaction and

same no evidence of misfolded or degraded RNA (Figure 3.7a). The effects of the Mg2+ ion

on the electrophoretic mobility of TAR RNA are shown in F igure 3.7b. Evidently, the

presence of the divalent Mg2+ cation in solution allowed for more sensitive binding between

TAR RNA and the polymers, with interactions being observed within polymer concentration

ranges of 1-10!M.

+-#

#

Table 3.4: Quantified polymer binding experiment. This table corresponds to the gel shifts

in F igure 3.7b. The gel shifts seen in the figure were categorized into free RNA and





ARG 10-mer:

d024@1)%ChVB Free RNA Complex % Bound

1 1463685 266890 15.4

5 1811174 358795 16.5

10 1675748 274198 14.1

ARG 25-mer

1 1600827 306245 16.1

5 1698572 312540 15.5

10 1698572 312540 15.5

ARG 50-mer

1 1657244 191358 10.4

5 1644738 347365 17.4

10 1550906 253107 14.0

,.#

#

F igure 3.7: T itration of A rginine-Conjugated polymers with H I V-T A R after pre-incubation in Mg2+

binding buffer . (A) A sample

Native-PAGE gel of TAR RNA

control and Arginine 10-mer

reaction. (B) The titration of RNA

with increasing concentration of

arginine-10-, 25-, 50-mer in the

presence of Mg2+. The titration of

RNA with increasing concentration

of arginine-The large streaking may

be attributed to RNA degradation as

a result a result of increased RNase

activity due the presence of Mg2+ in

solution. The presence of both

degraded and misfolded RNA may

explain the lack of multiple shifts in

RNA mobility since we assumed

that the polymers should bind 100%

of the folded/active RNA.

,%#

#

To make further conclusions regarding whether our ROMP-derived polymers

possessed unique binding to the TAR RNA, we decided to evaluate the RNA-binding effects

of the free arginine and the arginine-conjugated norbornene monomer (9). The results of

these experiments under the optimized Mg2+ buffer conditions revealed multiple band shifts

for both the arginine-conjugated monomer and the free arginine (F igure 3.8). These results

suggest that the monomers may be affecting the mobility shift of the RNA in two ways: (1)

varying stoichiometric ratios of polymer and RNA, or (2) binding of the monomers forces the

TAR RNA to form a variety of tertiary structures. Both possibilities of large stoichiometries

or multiple configurations could cause the alteration of electrophoretic migration of TAR

RNA. In comparison, the separation of both the free arginine and the conjugated-arginine

norbornene monomer from free TAR RNA is orthogonal to those observed for the arginine

10-mer without the Mg2+. These results allow us to conclude that the arginine 10-mer

polymer binds more specifically to the HIV-1 TAR RNA than the monomers.

,&#

#

F igure 3.8: T itration of T A R RN A with arginine conjugated 10-mer (A), norbornene-arginine monomer (B) and free arginine (C). At all concentrations we observed only one

retarded band in lanes labeled A , while in the monomer and free arginine lanes we observed

multiple bands. These results may suggest that the polymeric guanidinium binds the TAR

RNA specifically. The higher shifted bands (labeled 2) clearly displayed larger stoichiometric

ratios of monomer to RNA or an alternative non-specific binding mode to RNAFowing to

the fact that the bands migrate relative to 3D configuration.

,'#

#

Discussion: Section I : Chemical Synthesis of Multivalent Guanidinium Inhibitors

Selective inhibition of the Tat/TAR interaction has long been considered an attractive

target for the development of potent antiviral agents. However, several caveats temper this

enthusiasm. First of all, it is difficult to inhibit viral protein-RNA interfaces with small

molecules. Secondly, scientists find it difficult to design RNA-binding molecules with well-

defined molecular recognition properties. Lastly, though numerous compounds have been

shown to bind TAR RNA none of these molecules warrant pharmaceutical development due

to non-specific binding, toxicity, poor biological activity (due to poor cellular uptake), or a

combination of these problems (Karn 1999; Yang 2005; Davidson 2009; Wang 2009). To

overcome some of these difficulties, we focused on a series of guanidinylated polymeric

peptide mimics of the Arginine Rich Motif (ARM) of the Tat protein, rationally developed

through structural considerations (Strong 1999; Karn 1999; Yang 2005; Davidson 2009). We

reasoned that the multivalency, and specificity of these polymers would show increased

selectivity for the TAR RNA compared to other reported polymeric peptidomimetics. In

addition, the large molecular weight of the polymer might allow for more potent and specific

interactions compared to small organic structures (Karn 1999; Yang 1999; Anthanassiou

2007; Davidson 2010; Tamilarasu 2001). Based on this rationale, we successfully

synthesized ROMP-derived polymeric guanidinium compounds that were characterized by

1H NMR Spectroscopy.

The synthesis of the reactive activated carboxylic ester monomer proceeded

efficiently (Scheme 1). Isomerically pure exo norbornene (2) was isolated from a

,(#

#

predominantly endo mixture of exo and endo 5-norbornene-2-carboxylic acid (1) using a

selective iodolactonization brew of I2 and KI in water to remove the excess endo isomer.

There was a 21.5% yield recovery, which is consistent with the amount of exo-stereoisomer

present in the starting reaction. The exo product in its entirety was converted to the amine-

reactive ester (3). The exo isomer was coupled with EDCI and NHS, followed by isolation

and purification using extraction and flash chromatography sequentially. A white solid was

recovered in 85% yield. It is important to note that the final yield and purity of the

Norbornene NHS ester 3 were optimized by selectively increasing the packing of the

stationary phase and/or varying the polarity of the eluent solvent during flash

chromatography. The high purity was reflected by the 1H NMR spectrum. All the expected

peaks were observed and distinguishable in the spectrum.

The synthesis of the bispyridine ruthenium carbene polymerization catalyst (6) from

the 2nd generation Grubbs catalyst (4) proceeded with great efficacy affording a colossal

yield of 91%. This yield was orthogonal to the published literature value of 89% (Love

2000). Owing to the fact that ring-opening metathesis polymerization can provide oligomers

of varying average lengths, the catalyst was employed in ROMP to yield the amine reactive

polymers 7a-d. By varying the ruthenium-carbene catalyst to monomer initiators ratios,

polymers 7a-d of varying lengths n~10, n~25, n~50 and n~100 were generated. All

polymerization reactions proceeded efficiently, consuming all the activated NHS ester

monomers. The acquired yields were 27%, 32%, 28% and 36% respectively. The resulting

solids were all grey in color except the 10-mer, which appeared greenish-grey. Though the

percent yields were low, there was more than enough grannular product acquired for the post-

polymerization ligand conjugations of the polymers with the guanidinium derivatives of

arginine and agmatine.

,)#

#

Post-synthetic modification of the amine-selective polymer scaffolds (7a-c) with

amine bearing guanidinums afforded products 8a-c and 9a-c. The polymers 7a-c were

dissolved in DMSO and coupled with arginine and agmatine to yield 8a-c and 9a-c

respectively. Initial issues with post modification occurred as a result of arginine and

agmatine insolubility in reaction solvent, DMSO. The literature reaction time for these post

polymerization modifications are on average 8 hrs (Kolonto 2009) but, due to the solubility

issue, the reaction time for our experiments was optimized to three days with sporadic

agitation by vortexing to increase collisions between the reactive species.

The yields of the conjugated polymers ranged from 61-96%. There was significant difference

in product yield based on length of polymer. In fact, recovery of the guanidinylated 100mer

was repeatedly unsuccessful. Successful conjugation was determined by 1H NMR

spectroscopy. Since the guanidinium signals are not distinguishable from the cyclopentane

polymer backbone, the percentile conversion was determined by integration of the methylene

signals of 3-amino-1-propanediol against the alkene signal of the polymer backbone (Kolonto

2009). We hypothesized that we would be able to compare the quandinium derivatives of

arginine and agmatine to assess the role of the carboxylic acid in the inhibition of Tat/TAR-

RNA interactions.

,*#

#

Section I I : Biological Assay

After successful synthesis, a biological assay that would give us both a qualitative

and quantitative assessment of the potency, selectivity and efficacy of our polymers in

binding HIV-1 TAR RNA needed to be developed. We hypothesized that the conjugated

ROMP-derived oligomers of arginine varying lengths can illuminate different biological

recognitions of the TAR RNA. Our understanding of the RNA-ligand systems suggested that

EMSA was the best method to afford us the preliminary and practical results we sought. In

this discussion, we will provide a chronological assessment and development of all the data

acquired from EMSA technique.

Preliminary Control Assays: Previous TAR-peptidomimetic binding assays

conducted by Mischiati et al., Davidson et al., and Bryson et al. revealed that the

concentration of TAR RNA required for observation of bound RNA complexes was

approximate range of 10F20 ng/nl (Mischiati 2001; Davidson 2009, Bryson 2012).

However, owing to the different preparatory schemes of the TAR RNA as well as labeling

dye, we decided to first conduct preliminary control assays with the TAR RNA. These

studies revealed that the minimum sensitivity of the commercially available SYBR ® Green

stain used in this study was approximately 43 ng of TAR RNA or more. Consequently, in

order to effectively assess inhibition we had to optimize the TAR RNA signal in the gel.

Optimization of the assay revealed that the optimal amount of TAR RNA was between 260

and 520ng for accurate quantitative analysis of the binding associations of TAR-

peptidomimetic polymer (F igure 3.3).

Binding of Arginine Conjugated Polymers to HIV-1 TAR RNA without Mg2+ : In the

first set of binding experiments, we analyzed our polymeric arginine compounds differing in

,+#

#

length. A single concentration of HIV-1 TAR RNA (300 nM) was incubated with these

compounds at concentrations ranging from &%hV to `UU%hV without Mg2+. After incubation,

RNA-polymer complexes were resolved by polyacrylamide gel electrophoresis. The initial

results of the argininge conjugated 10-mer polymers were promising (F igure 3.4). In both the

arginine conjugated 10-mer binding experiments, we saw that the mobility of a fraction of

TAR RNA migrated more slowly through the gel in comparison to the control. The altered

EMSA patterns revealed three retarded band shifts, which suggest that our compounds were

able to recognize and prevent the free migration of TAR RNA. From these initial binding

experiments of TAR RNA with arginine 10-mer, we envisaged that the bands represented

different oligomerization states, implying the possibility of three binding modes: (") multiple

RNA folds while bound, (ß) multiple molecules of TAR RNA per one polymerFa

consequence of zipping which may be dependent on polymer length, and (#) multiple

polymers per one RNA molecule. Please refer to the binding modes previously described for

the remainder of the discussion; they will be referred to as their letters ", ß, and #. All three

binding modes are possible given that in the microenvironment the binding mechanism of

RNA-polymer is unknown. Refer to F igure 4.1 for a detailed comparison of the proposed

binding modes.

,,#

#

F igure 4.1: Binding-modes of RN A-polymer . The possible modular binding mechanisms of

the ROMP-derived polymers to HIV-1 RNA as observed in the initial binding experiments of

arginine-10mer to TAR RNA. (A) Mechanism of binding mode ", indicating binding of

polymer to different RNA folding states. (B) Describes only binding mode ß, indicating

multiple polymers bound per RNA. (C) Description of binding mode #, indicating multiple

RNA per polymer. (D) Description the combination of binding modes ß and #. Kinetic

equations may be derived from these possible-binding mechanisms occurring between the

TAR RNA and the guanidinylated polymers. We hypothesized that the on and off rates Kon

and Koff of ligand binding may be severely dependent on the salt concentration, pH, and co-

factors such as Mg2+.

Given binding system ", we hypothesized that the binding between the ROMP-

derived 10-mer would not be specific or selective for biologically active TAR 3-nucleotide

bulge, a tertiary fold which is conserved over all strains of the HIV-1 virus (Aboul-ela 1996;

Karn 1999). This would suggest that the polymer did not simply attach to the TAR RNA

bulge but also to the negatively charged phosphates in the backbone of the misfolded TAR

RNA molecules. However, owing to these speculations and observations we reasoned that

with longer polymers (25-mer and 50-mer), we should observe multiple or fewer gel shift

bands, consequences of either binding mode " or # respectively. These two binding modes

can be modeled using the kinetic equations in F igure 4.1. These kinetic equations will

,-#

#

subsequently be used as a framework for our discussion regarding the bindings of 25-mer and

50-mer under conditions similar to the 10-mer.

Continued binding reactions with arginine-conjugated 25-mer and 50-mer provided

evidence suggesting that the polymer bindings are a result of at least binding mechanism".

We were unable to discern whether ß and # were also occurring. Furthermore, the polymers

have no tertiary structure and as a result we cannot determine whether the bindings of a 10-,

25-, or 50-mer travel differently through the gel. As we can see from comparing the gels in

F igures 3.4-6 (using the 200nM lane as the standard for comparison), all the gel shift bands

(labeled b) are all in the same position, reflecting that the bindings of either 10-, 25- or 50-

mer are the same and that the predominating factor in gel mobility is the folding state of the

RNA molecule while bound. To justify this reasoning, ongoing work seeks to stain Native-

PAGE gels containing the varying polymer lengths using Coomassie stain. This will

delineate further if the polymers have a tertiary conformation and whether they travel through

361%E12%-3%:(AA1)1/3%)-31+,%76(96%3614%+60*2:/"3

Major concerns about the binding modes compelled us to contemplate ways

to further parse out binding-modes", ß, and #. At first we strategized a test for binding mode

". This experiment would involve some form of crystallographic or Cryo Electron

Microscopy technique that we did not have the time or resources to test. In addition, in

absence of Mg2+, a few configuration of TAR RNA may exist. On the other hand, we were

very confident we could differentiate between the presence of binding mechanisms ß and #

using the following tests: (1) titration of a low concentration of RNA against a single

concentration of polymer and (2) simple mobility shift assay comparing properly folded TAR

RNA in the presence of excess 10-, 25-, and 50-mer polymer and all necessary biological

cofactors.

-.#

#

Binding of Arginine Conjugated Polymers in the presence of Mg2+ : Given our

previous results, we realized that a crucial factor in our experiments may have been

misfolding of the TAR RNA due to the lack of Magnesium ion in solution. In the biological

microenvironment, charged groups are essential for the binding interactions of the hetero-

complexes of protein-nucleic acids and vary due to the binding modes and charge

complementarities between the molecules. It is important to note that these interactions are

often electrostatically guided: molecules that are highly charged like the macromolecules

RNA and DNA are steered towards their interacting partners via the electric force that brings

the molecules together. That is, the interactions between native proteins and their

macromolecular cognate are specific and they can recognize each other among hundreds of

thousands of candidates (Wang 1996). Upon binding, there are induced ionization changes

due to the proton donating and accepting in ligand-macromolecule associations. However,

these proton donor/-991830)%@196-/(+@+%0)(E(/-31%A)0@%361%(/:('(:*-2%8D-"+%0A%3(3)-3-;21%

groupsF a consequence of pH and salt ion concentration in the microenvironment.

Therefore, different binding interactions can occur at different pH values. For example,

bovine-t-lactoglobulin complexation forms a dimer at low pH but forms a tetramer at high

pH. In this case Sakurai and coworkers showed that the addition of salts such as NaCl, KCl,

and NaClO4 stabilized the varying interaction states (Sakuri 2001). Consequently, changing

the salt concentration can cause the binding constant to change by several orders of

magnitude.

Acknowledging this phenomenon, we concluded that a crucial factor for the binding

reaction is the solution in which the binding reaction was performed. We theorized that

optimizing the ligand-protein electrostatic interactions required the use of an appropriate

-%#

#

buffer, pH, and salt concentration and to include any co-factors required for the interactions

involved. Due to the oligiomerization of the complexes observed from our assays, we

hypothesized that the presence of salts and other additives may reduce non-specific binding

of polymerFTAR RNA associations.

Initially, the addition of salts to the binding reaction provided inconclusive evidence

that buffer/salt crucially increased the on and off bindings of the polymeric guanidiniums to

TAR RNA. In fact, the presence of salt in our binding reaction resulted in little or no retarded

gel shift bands. This suggested that the salt destabilized the binding of the polymer to the

TAR by solvating it. Furthermore, cofactors crucial for the structural integrity and biological

activity and folding of RNA needed to be used. We discovered that we neglected to

incorporate the divalent cation magnesium (Mg2+) (Misra 1998, Misra 2002). Put simply,

rigorous studies with RNA showed that the stability of RNA tertiary structure is crucially

dependent on the concentration and presence of Mg2+ (Leipply 2010).

After pre-incubation of TAR RNA in 1 mM Mg2+ binding buffer and titration with

10-mer, 25-mer, and 50-mer arginine-conjugated polymers there was only one gel shift band

besides the free RNA observed on the gel. The absence of the multiple retarded bands

provides convincing evidence that the occupied RNA sustained multiple folding patterns

without the Mg2+ present in the binding reaction.

In contrast to the gel patterns generated in absence of Mg2+, we observed only one

retarded band for each of the polymer lengths (F igure 3.8). Despite the varying

concentrations of polymer and polymer length there was no difference in the percent RNAF

polymer bound (Table 3.4). At face value, these results suggest that the essential requisite for

binding of the three conjugated polymers is the presence of the guanidine moieties. This

observation reaffirms previous studies that the arginine rich motif of Tat recognizes HIV-1

-&#

#

TAR RNA. However, the single shifted band indicates one of two things: that the binding of

RNA to polymer is independent of the concentration and the valency of the polymer, or the

concentrations used in this experiment is significantly above the dissociation constant (KD).

It is expected that an increase in the concentration of the polymer with respect to

RNA should push the reaction forward, but this does not happen (F igure 4.2). As shown in

Table 3.4, all the tied up RNA-polymer complexes resulted in similarly resolved bands

despite the concentration of polymer. Theoretically, each polymer may have multiple binding

sites per RNA. At the concentrations worked with in this study, all the RNA should have

been bound due to polymer saturation. However, after three repetitions we saw that only

about 15-30% of the RNA was complexed with the polymers.

F igure 4.2: The equilibrium constant for the binding of ligand to single site. The KA is

also known as the association or affinity constant. [RNA*P] is the concentration of RNA-

polymer complex, [RNA] is the free concentration of TAR RNA and [P] is the free

concentration of polymer.

There is one other plausible conclusion that satisfies to this apparent independence of

concentration on the binding between the polymersFTAR RNA. That is, the retarded band

shift is indicative of only the correctly folded or active RNA such that we have already

saturated the RNA at concentrations 1µM of polymer. The fact that only a few percentage of

the RNA is in the viable active form is quite disappointing. Altogether, these results suggest

that the introduction of the magnesium into the binding buffer strongly enhances the

specificity and RNA-binding activities of the guanidinylated conjugates.

Comparing Monomers of Arginine to ROMP-derived 10-mer in presence of Mg2+: In

F igure 3.8 we saw observed only one retarded band shift for the Arginine-10mer, but for

-'#

#

arginine-conjugated norbornene and free arginine we observed two to three band shifts

suggesting that the monomers sequester RNA. Since the Arginine 10-mer does not sequester

the RNA as indicated by the one gel shift band, it can be further concluded that the polymer

binds to the RNA in a more specific manner as opposed to the monomers which binds the

RNA in multiple ways causing it fold differently. The different configurations of the bound

up RNA travel differently on the gel and results in the distinguished gel shift bands.

-(#

#

Conclusion:

In conclusion, the major finding in this work is the identification of RNA-binding

ROMP-derived guanidinylated polymers exhibiting structural affinity to the HIV-1 TAR

RNA. However, in other to improve the binding ratios of polymer to RNA we must conduct

further tests to optimize the binding reaction buffer. A reputable binding buffer solution will

provide us with the means to calculate binding constants such as KA. In addition, we may be

able to conduct subsequent tests to evaluate the selectivity and specificity of the

guanidinylated polymers. In addition, it is expected that TAR RNA binding molecules

potentially inhibits the association of Tat/TAR. To verify this expectation, ongoing work

seeks to study the effects of the ROMP-derived polymers on the Tat/TAR interactions using

EMSA.

-)#

#

Future Directions:

Targeting the Tat-peptide represents a parallel strategy to inhibit the association of

Tat/TAR-RNA. We plan to functionalize our various length polymer scaffolds to display

sulfates or carboxylates. We hypothesize that these anions will electrostatically interact with

the basic ARM of Tat thus preventing the association of Tat to TAR-RNA 3 base bulge.

Sulfate amines such as sulfanilic acid and 5-amino-1-pentane sulfonic acid are ideal because

they have both anionic and amine functionality optimal for exploration. The difference in

carbon lengths will allow for the exploration of proximity between the polymer scaffold and

sulfonic groups. A similar assessment may also be conducted between the carboxylic acid

amines of 4-aminomethyl benzoic acid and caproic acid.

-*#

#

Bibliography:

Anand, K.; Schulte, A.; Vogel-$-96@-4),%DNT%?961AA\1p,%DNT%M141),%VN%.?3)*93*)-2%(/+(E63+%

into the Cyclin T1]Tat]H<I%I!<%3)-/+9)(83(0/%-93('-3(0/%90@821m%A)0@%a=<LN5%

Nat. Struct. Mol. Biol. 2008, 15(12), 1287-1292.

Aboul-ela, F.; Karn, J.; Va)-/(,%MN%.?3)*93*)1%0A%K=L-1 TAR RNA in the absence of ligands

)1'1-2+%-%/0'12%90/A(E*)-3(0/%0A%361%3)(/*92103(:1%;*2E1N5%Nucl. Acids Res. 1996, 24,

3974]3981.

Aboul-12-,%qN%.?3)-31E(1+%A0)%361%:1+(E/%0A%I!<-;(/:(/E%+@-22%@0219*21+N5%Future Med. Chem. 2010, 2(1), 93-119.

Archin, N. M.; Liberty, A. L.; Kashuba, A. D.; Choudhary, S. K.; Kuruc, J. D.; Crooks, A.

M.; Parker, D. C.; Anderson, E. M.; Kearney, M. F.; Strain, M. C.; Richman, D. D.;

Hudgens, M. G.; Bosch, R. J.; Coffin, J. M.; Eron, J. J.; Hazuda, D. J.; Margolis, D.

VN%.<:@(/(+3)-3(0/%0A%'0)(/0+3-3%:(+)*83+%K=L-1 latency in patients on antiretroviral

361)-84N5%Nature 2012, 487, 482-489.

Amendt, B. A.; Hesslein, D.; Chang, L. J.; Stoltzfus, C. M. .d)1+1/91 of negative and

positive cis-acting RNA splicing elements within and flanking the first Tat coding

exon of the Human Immunodeficiency Virus Type-&N5 Mol Cell Biol. 1994, 14,

3960]3970.

$1)3-E/02(0,%<NT%u0):-/,%VN%INT%$-)9-)20,%uNT%d-)p(/,%!N%.R0)2:%K1-236%g)E-/(\-3(0/5%H61%

HIV Drug Resistance Report -`U&`N5%WHO, 2012; pp 1-84.

Bilodeau, P. S.; Domsic, J. K.; Stoltzfus,%VN%QN%.Splicing Regulatory Elements

within tat Exon 2 of Human Immunodeficiency Virus Type 1 (HIV-1) are

characteristic of Group M but not Group O HIV-&%?3)-(/+N5 J. Virol. 1999, 73(12), 9764]9772.

$)-99(,%GNT%q-29(-/(,%QNT%G122(,%$N%.?4/3613(9%8183(:1+%(/%361%A0)@%0A%:1/:)(@1)+%;190@1%

)1+(+3-/3%30%8)031-+1%-93('(34N5%J. Biol. Chem. 2003, 278, 46590]46595.

Bryson, D. I.; Zhang, W.; Ray, W. K.; Santos, W. L. .Screening of a branched peptide library

with HIV-1 TAR RNAN5%Mol. BioSyst. 2009, 5, 1070-1073.

$)4+0/,%>N%=NT%v6-/E,%RNT%V9G1/:0/,%dN%VNT%I1(/1p1,%HN%VNT%?-/30+,%RN%GN%.H07-):%

Targeting RNA Structure: Branched Peptides as Cell-Permeable Ligands to TAR

I!<N5%ACS. Chem. Biol. 2012, 7(1), 210-217.

-+#

#

Bugatti, A.; U)(/-3(,%QNT%I-'122(,%QNT%>1%Q21)9i,%aNT%G(1p1/+,%?NT%I*+/-3(,%VN%.K18-)(/-

Mimicking Sulfonic Acid Polymers as Multitarget Inhibitors of Human

=@@*/0:1A(9(1/94%L()*+%H481%&%H-3%-/:%E8&`U%d)031(/+N5%Antimicrobial Agents and Chemotherapy 2007, 51(7), 2337-2345.

Q-0,%KNT%H-@(2-)-+*,%!NT%I-/-,%HN%VN%.g)(1/3-3(0/%-/:%<AA(/(34%0A%K=L-1 Tat Fragments in

H-3wH<I%Q0@821m%>131)@(/1:%;4%q2*0)1+91/91%I1+0/-/91%a/1)E4%H)-/+A1)N5%

Bioconjugate Chem. 2006, 17, 352-58.

Chevallet, M.; Luche, S.; Rabilloud, HN%.Silver staining of proteins in polyacrylamide gels.5%

Nat. Protoc. 2006, 1(4), 1852]1858.

Choudhury, A. K.; Golovine, S. YNT%>1:p0'-,%GN%VNT%K1963,%?N%VN%.?4/361+(+%0A%d)031(/+%

Q0/3-(/(/E%V0:(A(1:%<)E(/(/1%I1+(:*1+N5%Biochemistry 2007, 46, 4066-4076

Q)071,%!N%nNT%M0:A)14,%>N%=NT%$-m31),%<N%MN%.!-3*)1%p(221)%H%9122+%-)1%3-)E13+%A0)%K*@-/%

=@@*/0:1A(9(1/94%'()*+%(/A193(0/N5%Immunology 2003, 108, 1-2.

Cullen, B. R. Transactivation of human immunodeficiency virus occurs via a bimodal

mechanism. Cell 1986, 46, 973-982.

Davidson, A.; Patora-Komisarska, K.; Robinson, J. A; Varani, GN%.a++1/3(-2%+3)*93*)-2%

requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat

8)031(/N5 Nucleic Acid Res. 2011, 39(1), 248-256.

>176*)+3,%?NT%M12;-):,%KN%<NT%q(/1,%?N%VN%.!1*)08-360E1/1+(+%0A%<=>?N5%Mol. Med. 1996, 2,

16-23.

Dingwall, C.; Ernberg, I.; Gait, M. J.; Green, S. M.; Heaphy, S.; Karn, J.; Lowe, A. D.;

Singh, M.; Skinner, M. A.; Valerio, R. (Human immunodeficiency virus 1 Tat protein

binds transactivation-responsive region (TAR) RNA in vitro. Proc.Natl Acad. Sci. USA 1989, 86, 6925-6929.

Ellis, J. S.; Thompson, M. .Q0/A0)@-3(0/-2%+3-31+%0A%/*921(9%-9(:]peptide complexes

@0/(30)1:%;4%-90*+3(9%7-'1%8)08-E-3(0/%-/:%@0219*2-)%:4/-@(9+%+(@*2-3(0/N5%Chem. Sci. 2011, 2, 237-255.

q-29(-/(,%QNT%G0\\(,%GNT%d(/(,%<N%.V0219*2-)%;-+(+%0A%;)-/961:%8183(:1+%)1+(+3-/91%30%1/\4@e

8)031024+(+N5%Chem. Biol. Drug. Des. 2007, 69, 216]221.

Fiala, M.; Popik, W.; Qiao, J. H.; Lossinsky, A. S.; Alce, T.; Tran, K.; Yang, W.; Roos, K.

dNT%%<)360+,%uN%.K=L-1 induces cardiomyopathy by cardiomyocyte invasion and gp120, Tat, and cytokine apoptoti9%+(E/-2(/EN5%Cardiovasc. Toxicol. 2004, 4, 97-107.

Fleuridor, R.; Wilson, B.; Hou, R.; Landay, A.; Kessler, H.; Al-Harth(,%GN%.CD1d-restricted

natural killer T cells are potent targets for human immunodeficiency virus infection.5 Immunology 2003, 108 (1), 3-9.

-,#

#

Fricker, S.P.; Anastassov, V.; Cox, J.; Darkes, M.C.; Grujic, O.; Idzan, S.R.; Labrecque, J.;

Lau, G.; Mosi, R.M.; Nelson, K.L.; Qin, L.; Santucci, Z.; Wong, R.S.Y.

.Q6-)-931)(\-3(0/%0A%361%@0219*2-)%86-)@-9020E4%0A%<V>c&UU^%<%+819(A(9%

antagonist of the G-8)031(/%90*821:%Q61@0p(/1%)191830),%QlQIfN5%Biochemical Pharmacology 2006, 72(5), 588-596.

Garber, M. E.; Wei, P.; Kewal-Ramani, V.; Mayall, T. P.; Herrmann, C. H.; Rice, A. P.;

Littman, D. R.; Jones, K. A. .The interaction between HIV-1 Tat and human cyclin

T1 requires zinc and a critical cysteine residue that is not conserved in the murine

CycT1 proteinN5%Genes Dev. 1998a, 12, 3512-3527

M-);1),%VN%aNT%R1(,%dNT%u0/1+,%DN%<N%.HIV-1 Tat Interacts with Cyclin T1 to Direct the P-

Haq;%QH>%D(/-+1%Q0@821m%30%H<I%I!<N5 Cold Spring Harb. Symp. Quant. Biol. 1998b, 63, 371-380.

M1+37(9p(,%uN%aN,%G-*)-%GN%DN%.=/31)-receptor Communication through Arrays of Bacterial

Q61@0)191830)+N5%Nature 2002, 415, 81-84.

Harrich, D.; Mavankal, G.; Mette-Snider, A.; Gaynor, R. B. .Human immunodeficiency virus

type 1 TAR element revertant viruses define RNA structures required for efficient

viral gene expression and replication.5 J. Virol. 1995, 69(8), 4906]4913.

Harrich, D.; Ulich, C.; Gaynor, R. B. .A critical role for the TAR element in promoting

efficient human immunodeficiency virus type 1 reverse transcription.5 J. Virol. 1996,

70, 4017-4127.

K*-/E,%KN%RNT%R-/E,%DN%HN%.?3)*93*)-2%96-)-931)(\-3(0/%0A%361%@13-2%;(/:(/E%+(31%(/%361%

cysteine-rich region of HIV-&H-3%8)031(/N5%Biochem. Biophys. Res. Commun. 1996,

227, 615-621.

Jeang, K. T.; Chun, R.; Lin, N. H.; Gatignol, A.; Glabe, C. G.; Fan, H. .=/%'(3)0%-/:%(/%'('0%

binding of human immunod1x9(1/94%'()*+%3481%&%H-3%8)031(/%-/:%?8&%3)-/+9)(83(0/%

A-930)N5%J. Virol. 1993, 67, 6224]6233.

u1-/E,%DN%HN%.K=L%-/:%Q122*2-)%q-930)+N5%%Molecular Virology Section 1996, 0, 1-13.

Jeang, K. T.; Xiao, H.; and Rich, E.A. (1999). .Multifaceted activities of the HIV-1

transactivator of transcription Tat.5 J. Biol. Chem. 1999, 274, 28837]28840.

Karn, J. "Tackling Tat." J. Mol. Biol. 1999, 293(2), 235-254.

Khalil,%<N%VNT%I(@,%uN%GN%.RNA-Protein Interactions (/%K*@-/%K1-236%-/:%>(+1-+1N5 Semin. Cell. Dev. Biol. 2011, 22(4), 359]365.

Kolonto, E. M.; Pontrello, J. K.; Mangold, S. L.; -/:%D(1++2(/E,%GNGN%.%M1/1)-2%?4/3613(9%

I0*31%30%Q122%d1)@1-;21%$209p%Q08024@1)+N5%J. Am. Chem. Soc. 2009, 131, 7327-

7333.

--#

#

D)1;+,%<NT%G*:7(E,%LNT%$0:1/,%gNT%V(96-12%RN%MN%.Targeting the HIV Trans-Activation

Responsive Region]Approaches Towards RNA-Binding Drugs.5Chem. Bio. Chem

2003, 4, 972-978.

Leipply, D. Draper, D. E. .The dependence of RNA tertiary structure stability on Mg2+

concentration: interpretation of the Hill equation and 901AA(9(1/3N5%Biochemistry 2010,

49(9), 1843]1853.

G(30'96(9p,%<NT%G-8(:03,%<NT%a(+1/+31(/,%VNT%D-2(/p0'(96,%<NT%$0)p0',%MN%.!10@49(/%$-

Arginine Conjugate, a Novel HIV-1 Tat Antagonist: Synthesis and Anti- HIV

<93('(3(1+N5 Biochemistry 2001, 40, 15612-15623.

Love, uN%<NT%V0)E-/%uNdNT%H)/p-,HNVNT%M)*;;+,%INKN%%.<%d)-93(9-2%-/:%K(E624%<93('1%

Ruthenium-$-+1:%Q-3-24+3%36-3%aAA193+%361%Q)0++%V13-361+(+%0A%<9)420/(3)(21N5%

Angew. Chem. Int. Ed. 2002, 41(21), 4035-4037.

!00/-/,%>N%-/:%<2;(/(,%<N%.q)0@%361%0*3+(:1%(/^%am3)-9122*2-)%-93('(3(1+%0A%K=L%H-3N5Adv. Pharmocol. 2000, 48, 229-250.

Mishra, Subrata H. "Structure and Energetics of RNA - Protein Interactions for HIV RREIIB

Targeting Zinc Finger Proteins." (2008). Chemistry Dissertations. Paper 22.

http://digitalarchive.gsu.edu/chemistry_diss/22/

V(+)-,%LN%DNT%>)-81),%>N%aN%.g/%361%I021%0A%V-E/1+(*@%=0/+%(/%I!<%?3-;(2(34N5%Biopolymers (Nucleic Acid Sciences) 1998, 48, 113]135.

Misra, V. K.; Draper, D. E. .The linkage between magnesium binding and RNA folding.5%J. Mol. Biol. 2002, 317(4), 507-521.

V(4-*96(,%DNT%D(@,%nNT%G-3(/0'(9,%gNT%V0)0\0',%LNT%V12(p4-/,%MN%$N%.K=L%1/31)+%9122+%'(-%

endocytosis and dynamin-:181/:1/3%A*+(0/%7(36%1/:0+0@1+N5%Cell 2009, 137(3), 433-444.

V*1+(/E,%VN%<NT%?@(36,%>N%KNT%Q-80/,%>N%uN%.I1E*2-3(0/%0A%@I!<%-99*@*2-3(0/%;4%-%K*@-/%

Immunodeficiency Virus trans-93('-30)%8)031(/N5%Cell 1987, 48, 691-701.

Pitt, S. W.; Majumdar, A.; Serganov, A.; Patel, D. J.; Al-Hashimi, H. M. "Argininamide

binding arrests global motions in HIV-1 TAR RNA: comparison with Mg2+-induced

conformational stabilization." J. Mol. Biol. 2004, 338(1), 7-16.

d)(91,%HN%gNT%a)9-2,%!NT%!-p-0p1,%INT%$-/p+,%RN%<N%.K=L-1 viral proteins gp120 and Tat

induce oxidative +3)1++%(/%;)-(/%1/:03612(-2%9122+N5%Brain Res. 2005, 104, 557-63.

d*AA1),%aN%$N,%d0/3)1220,%uN%DNT%%K0221/;19p,%uN%uNT%D(/p,%uN%<NT%D(1++2(/E,%GN%GN%.<93('-3(/E%$%

Q122%?(E/-2(/E%7(36%>1A(/1:%V*23('-21/3%G(E-/:+N5%ACS Chem. Biol. 2007, 2(4) 252-

262.

%..#

#

R-/Y-/%>-+,%?NT%u-@112,%?N%.$(020E4%0A%361%K=L%!1A%8)031(/5%Indian. J. Med. Res. 2005, 121,

315-332.

Rusnati, M.; Urbinati, C.; Caputo, A.; Possati, L.; Lortat-Jacob, H.; Giacca, M.; Ribatti, D.;

d)1+3-,%VN%.d1/30+-/%d024+*2A-31%-+%-/%=/6(;(3(0)%0A%am3)-cellular HIV-&%H-3N5%J. of Biol. Chem. 2001, 276(25), 22420-22425.

?12;4,%VN%uNT%$-(/,%aN%?NT%G*9(7,%dNT%y%d131)2(/,%$N%VN%.?3)*93*)1,%+1i*1/91%-/:%80+(3(0/%0A%

the stem-loop in TAR determine transcriptional elongation by Tat through the HIV-1

long termin-2%)181-3N5%Genes Dev. 1989, 3, 547-558.

Singh, I. N.; Goody, R. J.; Dean, C.; Ahmad, N. M.; Lutz, S. E.; Knapp, P. E.; Nath,

<NT%K-*+1),%DN%qN%.<808303(9%:1-36%0A%+3)(-3-2%/1*)0/+%(/:*91:%;4%6*@-/%%%

immunodeficiency virus-1 Tat and gp120: Differential involvement of caspase-3

-/:%1/:0/*921-+1N5%%G . J. Neurovirol. 2004, 10, 141-151.

Sodroski, J.; Patarca, R.; Rosen, C.; Wong-?3--2,%qNT%K-+123(/1,%RN%<N%.G09-3(0/%0A%361%3)-/+-

acting region on the genome of human T-9122%24@8603)08(9%'()*+%3481%===N5%Science

1995a, 229, 74-77.

Sodroski, J. G.; Rosen, C. A.; Wong-Staal, F.; Salahuddin, S. Z.; Popovic, M.; Arya, S.;

M-220,%IN%QNT%K-+123(/1,%RN%<N%.H)-/+-acting transcriptional regulation of human T-

cell leukemia virus 3481%===%20/E%31)@(/-2%)181-3N5%Science 1995b, 227, 171-173.

?3)0/E,%GN%aNT%-/:%D(1++2(/E,%GNGN%.<%M1/1)-2%?4/3613(9%I0*31%30%>1A(/1:,%Biologically

<93('1%V*23('-21/3%<))-4+N5%J. Am. Chem. Soc. 1999, 121(26), 6193-6196.

Tamilarasu, N.; Huq, I.; Rana,%HNVN%.H-)E13(/E%I!<%7(36%d183(:0@(@13(9%g2(E0@1)+%(/%

K*@-/%Q122+N5%Bioorg. Med. Chem. Lett. 2001, 11, 505-507.

H4-E(,%VNT%I*+/-3(,%VNT%d)1+3-,%VNT%M(-99-,%VN%.=/31)/-2(\-3(0/%0A%K=L-1 Tat requires cell

+*)A-91%618-)-/%+*2A-31%8)0310E249-/+N5%J. Biol. Chem. 2001, 276, 3254-3261.

UNAIDS .M20;-2%I180)3^%UNAIDS )180)3%0/%361%E20;-2%-(:+%18(:1@(9%`U&`N5%WHO, 2012;

pp 1-110.

[);(/-3(,%QNT%Q6(0:122(,%dNT%I*+/-3(,%VN%.d024-/(0/(9%>)*E+%-/:%L()-2%g/90E1/1+(+^%-%!0'12%

Approach to Control Infection, Tumor-assoc(-31:%=/A2-@@-3(0/%-/:%</E(0E1/1+(+N5%

Molecules 2008, 13, 2758-2785.

Vendeville, A.; Rayne, F.; Bonhoure, A.; Bettache, N.; Montcourrier, P.; Beaumelle, B.

.K=L-1 Tat enters T cells using coated pits before translocating from acidified

endosomes and elic(3(/E%;(020E(9-2%)1+80/+1+N5%Mol. Biol. Cell. 2004, 15, 2347-2360.

Wang, D.; Iera, J.; Baker, H.; Hogan, P.; Ptak, R.; Yang, L.; Hartman, T.; Buckheit, Jr., R.

W.; Desjardins, A.; Yang, A.; Legault, P.; Yedavalli, V.; Jeang, K. T.; Apella, D. H.

.V*23('-21/3%$(/:(/E%g2(E0@1)+%=/6(;(3%K=L%H-3-TAR Interaction Critical for Viral

I182(9-3(0/N5 Bioorg. Med. Chem. Lett. 2009, 19(24), 6893]6897.

doi: 10.1016/j.bmcl.2009.10.078

%.%#

#

R-/E,%vNT%R-/E,%lNT%I-/-,%HN%VNT%.d)031(/%0)(1/3-3(0/%(/%361%H-3-TAR complex determined

;4%8+0)-21/%860309)0++2(/p(/EN5J. Biol. Chem. 1996, 271, 16995-

Weeks, K. M.; Ampe, C.; Scultz, S.QNT%?31)3\,%HN%<NT%Q)0361+,%>N%VN%.q)-E@1/3+%0A%361%K=L-1

3-3%8)031(/%+819(A(9-224%;(/:%H<I%I!<N5%Science 1990, 249, 1281-1285.

Weissman, J. D.; Brown, J. A.; Howcroft, K. T.; Hwang, J.; Chawla, A.; Roche, P. A.;

Schiltz, L.; Nakatani, Y.; Singer, D. S. .K=L-1 Tat binds TAFII250 and represses

TAFII250-:181/:1/3%3)-/+9)(83(/%0A%@-Y0)%6(+3090@8-3(;(2(34%92-++%=%E1/1+N5%Pro. Natl. Acad. Sci. 1998, 95, 11601-11606.

R1(\@-/,%KNT%H0),%nN%.H-)E13(/E%I!<%7(36%?@-22%V0219*21+N5%ChemBiochem 2003, 4, 998-

1007.

Ya/E,%VN%.>(+90'1)(1+%0A%H-3-TAR Interaction Inhibitors for HIV-&N5%Curr. Drug Targets Infect Disord. 2005, 5(4), 433-444.

v6-0,%KNT%G(,%uNT%u(-/E,%GN%.=/6(;(3(0/%0A%K=L-1 TAR RNA ] Tat peptide complexation using

8024C-9)42(9%-9(:BN5%Biochem. Biophys. Res. Commun. 2004, 320 (1), 95-99.

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