HIV-1 Protease as a Target for Antiretroviral Therapy

by

Ian William Windsor

B.S. BiochemistryUniversity of Minnesota Twin Cities, 2011

M.S. BiochemistryUniversity of Wisconsin-Madison, 2017

Submitted to the Department of Chemistryin Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHYIN CHEMISTRY

at the

Massachusetts Institute of Technology

February 2019

2019 Massachusetts Institute of Technology. All rights reserved.

Signature of Author:Signature redacted

Department of ChemistryDecember 17, 2018

-Signature redactedCertified by:

Ronald RainesFirmenich Professor of Chemistry

Thesis Supervisor

Accepted by:

MASSACHUSETS INSTOF TECHNOLOGY

MAR 212019

LIBRARIESARCHIVFS

Signature redactedRobert W. Field

ITUTE Haslam and Dewey Professor of ChemistryChairman, Departmental Committee on Graduate Students

1II

This doctoral thesis has been examined by a committee of professorsfrom the Department of Chemistry as follows:

Signature redactedNovartis Professor of

Alexander M. KlibanovChemistry and Biological Engineering

Thesis Committee Chair

-Signature redacted

Ronald T. RainesFirmenich Professor of Chemistry

Thesis Supervisor

Signature redacted

Elizabeth M. NolanAssociate Professor of Chemistry

Thesis Committee Member

2

HIV-1 Protease as a Target for Antiretroviral Therapy

By

Ian William Windsor

Submitted to the Department of Chemistry onJanuary 7, 2019 in Partial Fulfillment of the Requirements

for the Degree of Doctor of Philosophy in Chemistry

Abstract

Human immunodeficiency virus (HIV) is the causative agent of acquiredimmunodeficiency syndrome (AIDS). HIV employs three enzymes in its lifecycle, including aprotease that enables maturation of polyprotein precursors. Despite decades of progress studyingthe lifecycle of HIV and elaboration of therapeutics targeting nearly every aspect of the viral lifecycle, a cure remains elusive. Breakthroughs in HIV research have occurred alongsidefoundational advances of molecular biology, biotechnology, and medicinal chemistry, highlightingthe importance revisiting old questions with new approaches. The goal of this thesis is to advanceour biochemical knowledge of HIV-I protease and develop novel therapeutics targeting this keyviral enzyme.

In Chapter 1, I introduce HIV and the role that HIV-1 protease plays in life cycle andcurrent treatment strategies. In Chapter 2, I describe an assay that enables the determination ofsub-picomolar inhibition constants for competitive inhibitors of HIV-1 protease. This advance wasmade possible by a peptide substrate selected by phage display. I report in Chapter 3 the enhancedhydrogen bonding in the recognition of this peptide by HIV-1 protease as revealed by X-raycrystallography. The mechanism of aspartic proteases, including HIV-1 protease, has been thesubject of numerous enzymology studies spanning over half a century. In Chapter 4, I revealunappreciated non-covalent interactions within substrates of aspartic proteases that assist incatalysis. In addition to biochemical studies, this thesis includes chapters that account thedevelopment of novel antivirals. In Chapter 5, I describe the rational drug design of a boronic acidanalog of the clinical inhibitor darunavir with improved potency. A limitation of boronic acids ismetabolic instability; in Chapter 6, I reveal an intramolecular protecting group that can conferoxidative stability to boronic acids. Finally, in Chapter 7, I describe an engineering approach toinactivate human RNase 1. The inactivation relies on installing a substrate for HIV- I protease, the

cleavage of which unmasks cytotoxic activity. Together these chapters describe new ways forward

and novel therapeutics targeting HIV-1 protease. My thesis also includes an Appendix, whichdescribes the elaboration of boronic acid-based covalent pharmacological chaperones of humantransthyretin.

3

Thesis Supervisor: Ronald T. Raines

Title: Firmenich Professor of Chemistry

4

Acknowledgments

The Raines Laboratory has been an incredible place to receive training at the interface betweenchemistry and biology. I thank Prof. Ronald Raines for the opportunity to work on a variety ofexciting projects with relevance to human health. His approach to mentoring has made me anindependent researcher and instilled in me an attention to detail that I will carry on in my futureresearch endeavors.

The work presented here in my thesis would not be possible without the support of mycoworkers in the Raines Group. I thank Dr. Chelcie Eller, Dr. Kevin Desai, Dr. Trish Hoang, Dr.Joelle Lomax, and Dr. James Vasta, for helping me get started in the lab and being a constantsource of advice and support. I value the short time I overlapped with Dr. Michael Palte and hismany project ideas. My chemical training was made possible through numerous collaborationswithin the Raines Group. I thank Dr. John Lukesh, III, Dr. Thomas Smith, and Brian Graham fortheir hands-on training in organic synthesis. I also thank Dr. Brian Gold for his mentoring incomputational chemistry. Certainly, each member of the Raines group has contributed to my workthrough group meetings and everyday conversations.

I have been privileged to receive substantial training outside of the Raines Group. I amgrateful for Prof. Katrina Forest at the University of Wisconsin-Madison. Early in my studies, Iwas able to learn alongside her students, and she provided me with the resources necessary toprepare structures by X-ray crystallography. I must also thank Dr. Kittikhun Wangkanont who alsotrained with Prof. Forest for sharing his expertise and friendship during numerous trips to ArgonneNational Lab. I am also grateful for the training I received from Prof. David O'Connor at the AIDSVaccine Research Lab at UW-Madison. His staff scientist Dr. Dawn Dudley has helped mecharacterize live HIV in experiments that are ongoing and did not make it into my thesis. Thatentire group is friendly and helpful and made me feel safe working with a deadly virus. I have alsobeen fortunate to attend workshops including the NAMD molecular dynamics workshop hostedby the Theoretical and Computational Biophysics Group at the University of Illinois Urbana-Champaign, the NMRFAM workshop at UW-Madison, and a Rosetta Workshop hosted by Prof.Jens Meiler at Vanderbilt University.

I originally began my graduate studies at UW-Madison. In addition to my collaboratorsthere, I must thank my previous thesis committee members Prof. Julie Mitchell and Prof. AnjonAudhya for their support over several years. Transferring to MIT in 2017 was an easy decisionthat I made with a heavy heart. My arrival to MIT was delayed as I conducted viral studies duringthe summer of 2017 and I thank Val Ressler and Dr. Emily Garnett for setting up the biochemicallaboratory at MIT. I am grateful for the support from my new thesis committee members Prof.Elizabeth Nolan and Prof. Alexander Klibanov. I am also grateful for my ongoing collaborations

5

at MIT with Christine Isabella and Dr. Caitlin McMahon of the Kiessling Group, Dr. Ivan Buslovof the Buchwald and Pentelute Groups, and Scott Sheppard of the Cummins Group. The staff inthe Chemistry Office has been instrumental in navigating the process of transferring to MIT and

graduation.

It has been remarkable to be part of the Raines Group through two cycles of grant renewals

and see the process that enabled Ron to fund many projects over many years. My work has beenfunded by ROl GM044783 and ROl CA073808 from the NIH. I was privileged to receive an NIHBiotechnology Training Program Fellowship (T32 GM008349) and a pre-doctoral fellowship fromGenentech while at UW-Madison.

I must also thank Prof. Claudia Schmidt-Dannert and Prof. Jeffrey Gralnick at theUniversity of Minnesota Twin Cities for providing me with my first research experiences andhelping me enroll in graduate school. I thank Dr. Swati Choudary and Dr. Maureen Quin for theirhands-on training in biochemistry and helping me find my passion for science. I also thank Dr.Raghunath Padiyath at 3M for his mentoring and advice.

I have had tremendous support from friends and family during my graduate studies. I valuemy teammates from the Discertators ultimate frisbee team and the Warthogs intramural hockeyteams for their companionship and helping me stay active. I thank my parents William Windsorand Starla Guckenberg and my sister Autumn Windsor for their love and encouragement. By far,the most pivotal event I experienced during graduate school was meeting my loving partner SusanBright. She has been a constant source of support and inspiration and I cannot imagine my lifewithout her.

6

Table of Contents

PREFACE

Abstract ................................................................................................................................... 3

A cknowledgm ents................................................................................................................... 5

Table of Contents .................................................................................................................... 7

List of Tables ....................................................................................... 15

List of Figures ....................................................................................................................... 18

List of Charts......................................................................................................................... 22

List of Schem es..................................................................................................................... 22

Abbreviations........................................................................................................................ 23

CHAPTER 1

Introduction: Current and Future Prospects for the Treatment of HIV ..................................... 26

1. 1 Significance..................................................................................................................... 27

1.2 Hum an Im m unodeficiency Virus................................................................................. 28

1.2.1 The Lifecycle of HIV ............................................................................................ 28

1.2.2 Treatm ent of HIV ................................................................................................. 30

1.2.3 H IV M utations and Setbacks for a Cure .............................................................. 31

1.2.4 H IV Latency............................................................................................................. 32

1.3 Biochem istry of HIV -1 Protease................................................................................. 33

1.3.1 The Structure of HIV-1 Protease ......................................................................... 33

1.3.2 Catalysis by HIV Protease ..................................................................................... 34

1.3.3 Substrate Recognition by HIV -1 Protease............................................................ 36

7

1.4 Inhibition of HIV- 1 Protease ....................................................................................... 38

1.4.1 Natural Product-inspired Inhibitors of Aspartic Proteases ................................... 38

1.4.2 The First Generation of Clinical Inhibitors.......................................................... 39

1.4.3 The Second Generation of Protease Inhibitors ..................................................... 40

1.4.4 Drug Resistance and the Future of Protease Inhibitors........................................ 41

1.5 Versatile Recognition by Boronic Acids ..................................................................... 43

1.5.1 Chemistry of Boronic Acids ................................................................................ 43

1.5.2 Covalent Recognition of Proteins by Boronic Acids............................................ 45

1.5.3 Non-covalent Recognition of Proteins by Boronic Acids.................................... 46

1.6 Ribonucleases as Antiviral Therapeutics..................................................................... 48

1.6.1 Structure of RNases .............................................................................................. 48

1.6.2 Catalysis by RNases.............................................................................................. 49

1.6.4 Evasion of Ribonuclease Inhibitor........................................................................ 50

1.6.5 Antiviral Activity of RNases ................................................................................ 51

CHAPTER 2

Fluorogenic Assay for Inhibitors of HIV- 1 Protease with Sub-picomolar Affinity .................. 62

2.1. Introduction.................................................................................................................... 64

2 .2 . R esu lts............................................................................................................................ 6 5

2.2.1. Substrate Design. ................................................................................................. 65

2.2.2. Assay Design. ......................................................................................................... 66

2.2.3. M ichaelis-M enten Kinetics. ............................................................................... 67

2.2.4. Determination of Ki Values with M orrison's Equation. ...................................... 67

8

23 , Di scun..................................................................................................................... 60

2.4. M aterials and M ethods................................................................................................. 69

2.4.1. M aterials. ................................................................................................................ 69

2.4.2. Plasm id Preparation ............................................................................................ 69

2.4.3. Protein Purification .............................................................................................. 70

2.4.4. Enzym atic Activity Assays................................................................................... 71

2.4.5. Data Analysis.......................................................................................................... 72

2.5. A cknow ledgm ents.......................................................................................................... 73

CHAPTER 3

Substrate Selected by Phage Display Exhibits Enhanced Side-chain Hydrogen Bonding with HIV-

1 Protease ...................................................................................................................................... 82

3.1. Introduction.................................................................................................................... 84

3.2 Results and D iscussion ................................................................................................ 84

3.2.1 SGIFLETS Binds in Alternative Orientations ..................................................... 84

3.2.2 SerlA and Gly2A at the P4 and P3 Positions Occupy Alternative Conformations. 85

3.2.3 Glu6 and Ser8 Form a N etwork of Hydrogen Bonds .............................................. 86

3.2.4 Thr7 Plays a Lim ited Role..................................................................................... 87

3.3 M aterials and M ethods................................................................................................ 88

3.3.1. Protein..................................................................................................................... 88

3.3.2. Peptide..................................................................................................................... 89

3.3.3. Crystallization......................................................................................................... 89

3.3.4. Data Collection and Processing ........................................................................... 89

9

3.3.5 Structure Solution and Refinement..................................................................... 89

3 .4 C on clu sion ...................................................................................................................... 9 1

3.5 Acknowledgements..................................................................................................... 91

CHAPTER 4

An n--+7* Interaction in the Bound Substrate of Aspartic Proteases Replicates the Oxyanion Hole

112

104

106

106

108

109

112

112

113

113

114

114

.....................................................................................................................................................

4.1 Introduction...................................................................................................................

4.2 Results and Discussion .................................................................................................

4.2.1 Conform ation of the P1 Residue............................................................................

4.2.2 Conformation of the P 1' Residue ...........................................................................

4.2.3 M echanistic Insights ..............................................................................................

4.3 M aterials and M ethods..................................................................................................

4.3.1 Structural Analyses ................................................................................................

4.3.2 DFT Optim ization..................................................................................................

4.3.3 NBO Analysis ........................................................................................................

4.4 Conclusions...................................................................................................................

4.5 Acknowledgem ents.......................................................................................................

CHAPTER 5

Sub-Picom olar Inhibition of HIV- 1 Protease with a Boronic Acid ............................................

5.1 Introduction...................................................................................................................

5.2 Results and Discussion .................................................................................................

5.3.1 General...................................................................................................................

10

134

136

137

141

5. . C Syn arthe ................................................................................................. 142

5.3.3 Protein Preparation ................................................................................................. 145

5.3.4 Enzym e K inetics and. M ate........ ..... ...................... ............................ ..... 146

5.3.4.1 I ha enis nten K in e is .......................................................................... 146

5.3.4.2 M ichaelis-M enten Kinetics ......................................... ................................... 146

5.3.4.3 Inhibition K inetics.......................................................................................... 147

5.3.5 Cytotoxicity.............................................................................. 147

5.3.6 X -Ray Crystallography .......................................................................................... 148

5.3.6.1 Protein Crystallization .................................................................................... 148

5.3.6.2 X -Ray D iffraction ........................................................................................... 148

5.3.6.3 Structure Solution and Refinem ent................................................................. 149

5.3.7 Com putational A nalysis......................................................................................... 150

5.3.7.1 D FT Optim ization w ith G aussian ................................................................... 150

5.3.7.2 N atural Bonding Orbital A nalysis .................................................................. 150

5.3.7.3 A tom s in M olecules (A IM ) A nalysis.............................................................. 151

5.4 A cknow ledgm ents......................................................................................................... 152

5.5 H ydrogen Optim ization Coordinates ............................................................................ 153

5.6 N M R Spectra ................................................................................................................ 172

CHAPTER 6

Benzoxaborolone A Boronic Acid with Remarkable Oxidative Stability in Aqueous Solution

..................................................................................................................................................... 1 7 6

6.1 Introduction................................................................................................................... 178

11

6.2 Results and D iscussion ................................................................................................. 180

6.2.1 pKa V alues..............................................................................................................180

6.2.2 Susceptibility to Oxidation. ................................................................................... 181

6.2.3 A ffinity for Saccharides.........................................................................................182

6.2.4 Com putational A nalyses. ....................................................................................... 182

6.3 M aterials and M ethods.................................................................................................. 185

6.3.1 G eneral................................................................................................................... 185

6.3.2 pKa Determ ination...............................................................................................185

6.3.3 X -Ray Crystallography.......................................................................................... 186

6.3.4 Chem ical K inetics.................................................................................................. 186

6.3.4.1 Instrum entation and M aterials ........................................................................ 186

6.3.4.2 W avelength Optim ization and Calibration ..................................................... 187

6.3.4.3 Initial V elocity K inetics.................................................................................. 187

6.3.4.4 Evaluation of pH -D ependence........................................................................ 188

6.3.5 Saccharide Binding................................................................................................ 188

6.3.6 Com putational A nalyses........................................................................................ 189

6.3.6.1 D FT Optim ization w ith Gaussian................................................................... 189

6.3.6.2 N atural Bonding Orbital A nalysis .................................................................. 189

6.4 Conclusions................................................................................................................... 190

6.5 A cknow ledgm ents......................................................................................................... 190

6.6 A tom ic Coordinates of Optim ized Structures............................................................... 232

12

CHAPTER 7

Strain Inactivation in a Circular RNase 1 Zym ogen................................................................... 242

7.1 Introduction................................................................................................................... 244

7.2 Results........................................................................................................................... 246

7.2.1 Design of a Circular Zym ogen Construct .............................................................. 246

7.2.2 Design and Assessment of Glycine (G) Series of Zymogens ................................ 246

7.2.3 Design and Assessment of Strained (Str) Series of Zymogens.............................. 247

7.2.4 A ssessing Zym ogen Activation by HIV -1 Protease .............................................. 247

7.2.5 Modelling the Structural Basis of Str2 Zymogen Inactivation .............................. 248

7.2.6 Modelling the Structural Basis of Proteolytic Activation of Str2 by HIV-1 Protease

......................................................................................................................................... 2 4 8

7.3 D iscussion..................................................................................................................... 249

7.4 M aterials and M ethods.................................................................................................. 251

7.4.1 Protein Expression and Purification....................................................................... 251

7.4.2 Enzym e Activity A ssays........................................................................................ 252

7.4.3 Therm al Stability A ssays ....................................................................................... 253

7.4.4 M odelling with Rosetta.......................................................................................... 254

APPENDIX A

Stilbene Boronic Acids Form a Covalent Bond with Human Transthyretin and Inhibit its

Aggregation.................................................................................................................................272

A . 1 Introduction..................................................................................................................274

A .2 Results..........................................................................................................................277

13

A .3 D iscussion .................................................................................................................... 28 1

A .4 M aterials and M ethods................................................................................................. 285

A .4.2 M aterials .. ynthe............................................................................................... 285

A .4.2 Chem ical Synthesis.............................................................................................. 285

A .4.2.1 M aterials.m.. a.... .............................................. .......................................... 285

A .4.2.2 Conditions p ...... ........................................... ........................................ 285

A .4.2.3 Solvent Rem oval ......................................................................................... 286

A .4.2.4 N M R Spectroscopy................................................ ........................................ 286

A .4.2.5 M ass Spectrom etry......................................................................................... 286

A .4.2.6 M elting Points..................................................... ........................................... 286

A.4.2.7 Compound Purity .................................................................. 287

A.4.2.8 Synthesis............................................................................ 287

A .4.3 Protein Expression and Purification...................................................................... 305

A .4.4 Com petitive Fluorescence A ssay.......................................................................... 306

A .4.5 Fibril-Form ation A ssay ......................................................................................... 307

A.4.6 Protein Crystallization and X-ray Structure Determination.................................. 308

A .5 Conclusion ................................................................................................................... 3 10

A .5 H PLC Traces................................................................................................................ 365

A .6 N M R Spectra................................................................................................................ 366

R E FE R E N C E S .............................................................................................................. 395

14

List of Tables

Table 2.1. Inhibition Constants Reported for Amprenavir, Darunavir, and Tipranavir............ 74

Table 2.2. Kinetic Parameters of Popular HIV- 1 Protease Substrates and Substrate 1 ........... 75

Table 3.1. Endogenous and Optimized HIV-1 Protease Substrate Sequences.......................... 92

Table 3.2. Crystallographic Data Collection and Refinement Statistics ................................... 93

Table 4.S1. Measured Parameters from PDB Entries ................................................................ 115

Table 4.S2. Measured Parameters from CSD Entry SOWJUL .................................................. 117

Table 4.S3. Coordinates Extracted from PDB Entry 6bra and Optimized Hydrogen Atoms ... 118

Table 4.S4. Coordinates Extracted from PDB Entry 3b80 and Optimized Hydrogen Atoms ... 120

Table 5.1. Values of Ki for Inhibition of HIV-1 Protease .......................................................... 158

Table 5.S1. Crystallographic Data Collection and Refinement Statistics.................................. 159

Table 6.1. Rate Constants for the Oxidation of Boronic Acids and Biological Thiols at pH ~7.4

..................................................................................................................................................... 1 9 2

Table 6.2. Experimental and Computational Kinetic Parameters for the Oxidation of Boronic

A cid s ........................................................................................................................................... 19 3

Table 6.3. Values of Ka (M-1) for Boronic Acids and Saccharides............................................ 194

Table 6.S1. Crystal Data and Structure Refinement .................................................................. 195

Table 6.S2. Atomic Coordinates (x 104 ) and Equivalent Isotropic Displacement Parameters (A2 x

103) for P 180 6 5 ........................................................................................................................... 19 6

Table 6.S3. Anisotropic Displacement Parameters (A 2 x 103) for P 18065................................ 197

T able 6.S4. B ond Lengths for P 18065 ....................................................................................... 198

T able 6.S5. B ond A ngles for P 18065......................................................................................... 199

15

Table 6.S6. Hydrogen Coordinates (x 104 ) and Isotropic Displacement Parameters (A 2 x 103 ) for

P 18 0 6 5 ........................................................................................................................................ 2 0 1

Table 6.S7. Hydrogen Bonds for P18065 .................................................................................. 202

Table 6.S8. Values of Ac During Oxidation Reactions (M-1cm ) ............................................ 203

Table 6.S9. Second-Order Rate Constants for the Oxidation of Boronic Acids (M-'s-').......... 204

Table 6.S10. Calculated Energies of Starting Materials, Transition States, and Products (Hartrees)

..................................................................................................................................................... 2 0 5

Table 6.S11. Calculated Free Energies of Activation and Reaction (kcal/mol) ........................ 206

Table 6.S12. Boron-Oxygen and Competing Donor-Acceptor Interaction Energies ............... 207

Table 7.1. G and Str Zym ogen D esigns ..................................................................................... 255

Table 7.2. Properties of Str Series Zym ogens ............................................................................ 256

Table 7.S1. Activity and Stability of RNase 1 and Zymogens .................................................. 257

Table 7.S2. Str Zymogen Kinetic Parameters at pH 5.0 ............................................................ 258

Table 7.S3. Total Scores of Models Calculated by Rosetta....................................................... 259

Table A.1. Interaction of Diphenols 1-4 with Wild-type TTR and its V30M Variant...... 312

Table A.2. Interactions of Carboxylic Acids 5-8 with Wild-type TTR and its V30M Variant. 313

Table A.3. Interactions of Compounds 9-12 with Wild-type TTR and its V30M Variant ....... 314

Table A.S1. Bond Angles and Bond Lengths of Planar Boronic Esters in Small-molecule Crystal

S tru ctu res .................................................................................................................................... 3 15

Table A.S2. Crystallographic Data Collection and Refinement Statistics for the TTR-2 Complex.

..................................................................................................................................................... 3 16

Table A.S3. Data Collection and Refinement Statistics for the TTR-3 Complex. ................... 317

16

Table A.S4. Crystallographic a and Refine et Statistics for the TTr 4 Complex

Table A.S. Crystallographic Data Collection and Refinement Statistics for the TTR5 Complex.

..................................................................................................................................................... 3 19

Table A.S6. Crystallographic Data Collection and Refinement Statistics for the TTR-6 Complex.

..................................................................................................................................................... 32 0

Table A.S7. Crystallographic Data Collection and Refinement Statistics for the TTR 7 Complex.

..................................................................................................................................................... 3 2 1

Table A.S8. Crystallographic Data Collection and Refinement Statistics for the TTR-8 Complex.

..................................................................................................................................................... 3 2 2

Table A.S9. Crystallographic Data Collection and Refinement Statistics for the TTR 10 Complex.

..................................................................................................................................................... 3 2 3

Table A.S10. Crystallographic Data Collection and Refinement Statistics for the TTR 11

C o m p lex ...................................................................................................................................... 3 2 4

Table A.S11. Non-covalent Interactions and Distances in TTR Ligand Complexes ................ 325

Table A.S12. Observed c--hole Bond Lengths and Bond Angles in TTR Ligand Complexes .. 327

17

List of Figures

Figure 1.3. Structures of Aspartic Proteases Bound to Ligands. ............................................. 53

Figure 1.4.1. Structure of Pepstatin, Statine, and a Tetrahedral Intermediate .......................... 55

Figure 1.4.2. FDA Approved Inhibitors of HIV Protease........................................................ 57

Figure 1.5. Equilibria of Boronic Acids in Aqueous Solution................................................. 59

Figure 1.6. Structures of RNases Bound to Ligands................................................................. 61

Figure 2.1. Structure of Substrate 1 ......................................................................................... 77

Figure 2.2. Catalysis of the Hydrolysis of Substrate 1 by HIV-1 Protease............................... 79

Figure 2.3. Inhibition of HIV- 1 Protease by Amprenavir, Darunavir and Tipranavir .............. 81

Figure 3.1. Structure of the D25N HIV-1 Protease CA/p2 Complex...................................... 95

Figure 3.2. Electron Density and Interactions of SGIFLETS Bound in the Active Site of D25N

H IV -1 P rotease.............................................................................................................................. 97

Figure 3.3. Alternative Conformations of P3 and P4 Residues .............................................. 99

F igure 3.4. R ole of A sp30 .......................................................................................................... 101

Figure 4.1. Structural Features of Aspartic Proteases................................................................ 123

Figure 4.2. Ramachandran Plot of P1 and P1' Residues in Substrates Bound to The Active Site of

Inactivated A spartic Proteases .................................................................................................... 125

Figure 4.3. Main-chain Conformation of the P1 Residue .......................................................... 127

Figure 4.4. Main-chain Conformation of the P1' Residue ......................................................... 129

Figure 4.5. Putative Mechanism of Catalysis by HIV- 1 Protease ............................................. 131

Figure 4.6. Orbital Interactions of the Scissile Peptide Bond During Catalysis by HIV-1 Protease

..................................................................................................................................................... 13 3

18

Figure 5.1 Interaions witb a bstraP, Drunavir, or its Analg '-aN d the 2' Susie of HIV-1

P ro tea se ....................................................................................................................................... 16 3

Figure 5.2. Orbital Interactions in a Model of Boronic Acid 1 and Residue 30 of HIV- 1 Protease

..................................................................................................................................................... 1 6 5

Figure 5.S1. Raw and Processed Kinetic Data from Assays of HIV- 1 Protease Activity ......... 167

Figure 5.S2. Depiction of Electron Density of HIV- 1 Proteases with Bound Boronic Acid 1.. 169

Figure 5.S3. Toxicity of Darunavir and Boronic Acid 1 for Human Cells................................ 171

Figure 6.1. Structure of Phenylboronic Acid, Benzoxaborole, Benzoxaborolone, and Hydration

and Protonation States that are Relevant in Aqueous Solution................................................... 209

Figure 6.2. pH-Dependence of the Rate Constant for the Oxidation of Boronic Acids by Hydrogen

P ero x id e ...................................................................................................................................... 2 1 1

Figure 6.3. Calculated Structure of the Transition State for the 1,2-aryl Shift During the Oxidation

of PBA, 2-HMPBA, and 2-CPBA by Hydrogen Peroxide ......................................................... 213

Figure 6.4. Images of Key Orbitals in the Transition State for the 1,2-aryl Shift During the

Oxidation of PBA , 2-HM PA, and 2-CPBA ................................................................................ 215

Figure 6.S1. Representative "B-NMR Spectra Used to Determine the pKa Values of 2-CPBA

..................................................................................................................................................... 2 1 7

Figure 6.S2. UV-Spectroscopic Basis for Assays of the Oxidation of Boronic Acids .............. 219

Figure 6.S3. Kinetic Traces of the Oxidation of Boronic Acids by Hydrogen Peroxide at pH 7.4

..................................................................................................................................................... 2 2 1

Figure 6.S4. 1 H-NMR Spectra Acquired During the Oxidation of Boronic Acids ................... 227

19

Figure 6.S5. Optimized Structure of Each Hydrogen Peroxide Complex and Product During the

O xidation of B oronic A cids ........................................................................................................ 229

Figure 6.S6. Graph of the Extent of Protodeboronation of Boronic Acids at Three pHs.......... 231

Figure 7.1. Activity and Stability of Circular Zymogens of RNase 1 ...................................... 261

Figure 7.2. Modelling of Str2 Zymogen and Complex with HIV-1 Protease............................ 263

Figure 7.S1. Expression of 3G Zym ogen................................................................................... 265

Figure 7.S2. Proteolytic Treatment of RNase 1 with HIV- 1 Protease....................................... 267

Figure 7.S3. A ctivation K inetics of Str2.................................................................................... 269

Figure 7.S4. Top 10 Scoring Rosetta Models of Str2 and Complex with HIV- 1 Protease........ 271

Figure A.1. Three-dimensional Structure of the TTR Resveratrol Complex ............................ 329

Figure A.2. Three-dimensional Structures of TTR-Ligand Complexes that Contain a Boronic Acid

G ro u p .......................................................................................................................................... 3 3 1

Figure A.3. Three-dimensional Structures of TTR Ligand Complexes that do not Contain a

B oronic A cid G roup .................................................................................................................... 333

Figure A.4. Halogen-bonding Interactions in the TTR 10 and TTR 11 Complexes................. 335

Figure A.S1. Graphs Showing the Results of ANS Competition Assays .................................. 337

Figure A.S2. Graphs Showing the Results of 96-h Fibril-formation Assays............................. 339

Figure A.S3. MALDI-TOF Mass Spectra to Probe the Reversibility of TTR Inhibition ......... 341

Figure A.S4. Electron Density in the TTR-2 Complex.............................................................. 343

Figure A.S5. Electron Density in the TTR 3 Complex.............................................................. 345

Figure A.S6. Electron Density in the TTR-4 Complex.............................................................. 347

Figure A.S7. Electron Density in the TTR-5 Complex.............................................................. 349

20

Figure A.S8. Flectron nntv in the TTR 6 Coomppe x 31

Figure A.S9. Electron Density in the TTR7 Complex.............................................................. 353

Figure A.S1O. Electron Density in the TTR8 Complex........................................................ 355

Figure A.S11. Electron Density in the TTR-1 Complex...................................................... 357

Figure A.S12. Electron Density in the TTR 11 Complex.......................................................... 359

21

List of Charts

C hart A .1. D iphenol Ligands..................................................................................................... 360

Chart A.2. Carboxylic Acid Ligands......................................................................................... 361

Chart A.3. Diboronic Acid and Related Ligands....................................................................... 362

List of Schemes

Scheme 5.S1. Route for the Synthesis of Boronic Acid 1. Overall yield: 54% (unoptimized).. 157

Scheme 6.1. Boronic Acid Oxidation Pathway .......................................................................... 191

Scheme A.1. Routes for the Synthesis of Stilbenes 2-6............................................................. 363

Scheme A.2. Routes for the Synthesis of Stilbenes 7-11........................................................... 364

22

bbre-viationsT .

2-CPBA ............................................................................................. 2-carboxyphenylboronic acid2-HM PBA ............................................................................. 2-hydroxym ethylphenylboronic acidA ...................................................................................................................... Angstr6m (10-10 m )ACE ................................................................................................ angiotensin converting enzym eAID S .................................................................................. acquired im m unodeficiency syndrom eAIM .................................................................................................................. Atom s in M oleculesAM I ........................................................................................................................ Austin m odel IAN S ....................................................................................... 8-anilinonaphthalene-1-sulfonic acidART ................................................................................................................ antiretroviral therapyAZT ......................................................................................................................... azidothym idineBCA .................................................................................................................... bicinchoninic acidBCP ..................................................................................................................... bond critical pointCCR ............................................................................................................ CC chem okine receptorCD ............................................................................................................. cluster of differentiationCXCR ..................................................................................................... CXC chem okine receptorsDABCYL .............................................................. 4-((4-(dim ethylam ino)phenyl)azo)benzoic acidDCM ..................................................................................................................... dichlorom ethaneDEPC ............................................................................................................ diethyl pyrocarbonateDFT .......................................................................................................... density functional theoryDM F ................................................................................................................. dim ethylform am ideDM SO ................................................................................................................. dim ethylsulfoxideDNA .............................................................................................................. deoxyribonucleic acidDTT ............................................................................................................................. dithiothreitolEDAN S .......................................................... 5-(2-am inoethylam ino)- I -naphthalenesulfonic acidEDTA ............................................................................................. ethylenediam inetetraacetic acidE .................................................................................................................... extinction coefficientEPR .............................................................................................. electron param agnetic resonanceER ............................................................................................................... endoplasm ic reticulumESCRT .......................................................... endosom al sorting com plexes required for transportESI ............................................................................................................... electrospray ionizationFAM ............................................................................................................................... fluoresceinFDA ............................................. Food and Drug Adm inistration of the United States of Am ericaFRET ........................................................................................... Fbrster resonance energy transferG .......................................................................................................................................... glycine

gp .................................................................................................................................. glycoproteinHAART .................................................................................... highly active antiretroviral. therapyHIV .................................................................................................. hum an im m unodefiency virusHPLC .................................................................................... high pressure liquid chrom atographyHRM S ....................................................................................... high-resolution m ass spectrom etryIEFPCM ........................ integral equation formalism variant of the polarizable continuum model

23

IN ....................................................................................................................................... integraseIPTG ................................................................................... isopropyl p-D-1-thiogalactopyranosideITC ................................................................................................. isotherm al titration calorim etryKa.................................................................................................. equilibrium association constantkcat.......................................................................................................................... turnover num ber

kcat/KM .................................................................................................................. catalytic effiencyKd................................................................................................. equilibrium dissociation constantK ........................................................................................................................ inhibition constantKm ...................................................................................................................... M ichaelis constantkobs ................................................................................................................ observed rate constantLBHB .................................................................................................... low-barrier hydrogen bondlogP ........................................................................................... octanol-water partition coefficientLS-CAT......................................................................... Life Sciences Collaborative Access TeamM ............................................................................................................................................. m olarM ALDI ........................................................................ m atrix-assisted laser desorption/ionizationM D ................................................................................................................... m olecular dynam icsM DR .............................................................................................................. m ultidrug-resistantM R ............................................................................................................... m olecular replacem entm RNA .................................................................................................................... m essenger RNAMTS .............. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-

tetrazoliumM W CO ....................................................................................................... m olecular weight cutoffN2(g).............................................................................................................................nitrogen gasN2(l).......................................................................................................................... iquid nitrogenNBO ............................................................................................................ natural bonding orbitalNIAID .......................................................... National Institute of Allergy and Infectious DiseasesNIH ..................................................................................................... National Institutes of HealthNM R .................................................................................................... nuclear m agnetic resonanceNM RFAM ........................................................ National M agnetic Resonance Facility at M adisonNN RTI .................................................................... non-nucleoside reverse transcriptase inhibitorNpu....................................................................................................................Nostoc punctiformeNRTI .............................................................................. nucleoside reverse transcriptase inhibitorN S ................................................................................................................. non-structural protein

C ............................................................................................................................ degrees CelsiusONC ............................................................................................................................... Onconase*ORTEP........................................................................................ Oak Ridge therm al-ellipsoid plotPAGE ....................................................................................... polyacrylam ide gel electrophoresisPBA ................................................................................................................... phenylboronic acidPBS ......................................................................................................... phosphate-buffered salinePCR ........................................................................................................ polym erase chain reactionPDB .................................................................................................................... Protein Data BankPDE ..................................................................................................................... phosphodiesterasepH ............................................................... negative of the logarithm of the hydrogen ion activityPI..........................................................................................................................protease inhibitor

24

V_ I Tfl nf t1l 0- 1 I-N " :l r;tl'% "-I rVF fk -11--.1 A 'SOCi.1+;f_%" CO"Starita ....................................................... nia..", Ai V % %-,P-L L,11%w 1%-15L41 ILI" I I kJ.L L,11% LL%. I $.I d ica $ IGLLIVII %., 113L

PPII ................................................................................................................ poly-l-protein type 11P R ........................................................................................................................................ p ro tea sePSI ............................................................................................................... pounds per square inchQM /M M ........................................................................ quantum m echanic s/m olecular m echanicsRFU ........................................................................................................ relative fluorescence unitsRI ................................................................................................................... ribonuclease inhibitorRM SD .................................................................................................. root-m ean- square deviationRNA ....................................................................................................................... ribonucleic acidRNase ........................................................................................................................... ribonucleaseRT .................................................................................................................... reverse transcriptaseS ....................................................................................................................................... S v ed b ergSDS ............................................................................................................. sodium dodecyl sulfateSiv ................................................................................................ sim ian imm unodeficiency virusSPPS .................................................................................................. solid-phase peptide synthesisSSHB ............................................................................................................................ short-strongS tr ........................................................................................................................................ strain edT .......................................................................................................................................... th y m u sT 4 ....................................................................................................................................... th yro x in eTAM RA ........................................................................................... carboxytetram ethy1rhodam ineTEA ............................................................................................................................. triethylam ineTFA .................................................................................................................... trifluoroacetic acidTHF ......................................................................................................................... tetrahydrofuranTLC ....................................................................................................... thin-layer chrom atography

TM ................................................................................................ m idpoint of therm al denaturationTOF ............................................................................................................................. tim e of flightTris ........................................................................... 2-am ino-2-(hydroxym ethyl)propane- 1,3-diolTTR .............................................................................................................................. transthyretinUV .................................................................................................................................... ultravioletv is .......................................................................................................................................... v isib le

VMax ...................................................................................................................... maxim al velocityVO ............................................................................................................................... initial velocity

25

Chapter 1

Introduction: Current and Future Prospects for the

Treatment of HIV

26

1.1 Significance

An estimated 37 million individuals are infected with HIV worldwide in 2017 and AIDS associated

complications have resulted in 35 million deaths.' Since the AIDS epidemic began in 1981,

academia and industry, sometimes in collaboration, have sought to find a cure for HIV.2 The war

against AIDS began when a trio of labs independently discovered HIV.4 6 Identification of this

new class retrovirus, the lentiviruses, responsible for AIDS unlocked the possibility of testing and

the development of targeted therapeutics. Though HIV was first clinically observed in the United

States, the virus is thought to have originated in Africa through human adaption of SIV transmitted

through contact with an infected animal.7 The majority of infected individuals reside in

economically disadvantaged regions of Africa and India, making accessibility to antivirals a

prominent goal of public health organizations. Antivirals with greater potency that require smaller

dosage and compounds with enhanced shelf lives conferred by greater chemical stability are highly

desired. Virological differences amplify shortcoming in the treatment of HIV in the developing

world as therapeutic have been optimized to treat HIV-1, type M, subtype B, which is common in

the US and Europe, and not subtype C that infects the majority of people and is prominent in

Africa.8 Finally, the emergence of drug resistance has transformed HIV treatment from a general

strategy to personalized medicine that requires tailored therapeutics. 9

27

1.2 Human Immunodeficiency Virus

HIV can be transmitted via several mechanisms. Sexual intercourse is the prominent route,

although infection can arise from contact with contaminated blood, from mother to child via birth,

and intravenous drug use. One quarter of infected individuals are not aware they are infected and

contribute to nearly half of new infections, making testing and prevention a prominent feature of

eradication efforts.10 Differential presentation of acute infection and the symptoms being similar

to common pathogens lead to challenges in diagnosis. Following the acute stage, viral replication

is repressed by the immune system during the chronic infection stage." Treatment outcomes are

more favorable with earlier commencement of antiretroviral therapy (ART). The progression of

HIV to AIDS occurs within a few years without treatment to well over a decade with treatment.

AIDS is the result of depletion of the immune system, which makes the patient susceptible to

pathogens that are not problematic to an otherwise healthy individual.

1.2.1 The Lifecycle of HIV

HIV is unlike any other pathogen facing humanity due to the cell types that are permissive hosts

to infection. The lifecycle of HIV begins by a recognition event called attachment where the viral

protein gp120 binds the CD4 receptor of a CD4+ T-cell.1 2 Activated T-cells express greater

quantities of receptors utilized by HIV upon antigenic stimulation as part of the adaptive immune

response; thus, HIV infects and kills a subset of cells tasked with fighting the infection." Binding

of the co-receptor, either CXCR4 or CCR5, leads the other viral membrane protein gp4l extending

an a-helical domain into the host-cell membrane.' 4 This process takes roughly an hour and leads

28

to fusion of the viral and host cell membrane.15 Fusion deposits the viral RNA genome and viral

enzymes into the cytosol.

Historically, the first targets for therapeutic intervention were the viral enzymes. The

enzyme reverse transcriptase (RT) synthesizes a DNA copy of the viral genome in a template-

dependent manor using the viral RNA.1 6 Cyclization of the DNA through the action of integrase

in conjunction with accessory proteins forms the preinitiation complex which transduces into the

nucleus where the proviral genome becomes covalently integrated into the host cell genome. 7

Association with nuclear proteins and the accessibility of transcriptionally active regions promote

integration.1 8 Evolution of retrovirus has achieved compact, approximately 10 kilobase viral

genomes.

Instead of independently regulating transcription and translation of viral RNA and proteins,

the viral proteins are expressed as long poly-proteins known as gag and gag-pol. The process of

maturation separates the individually folded domains of the poly-proteins, enabling the assembly

of the virus particle. The final viral enzyme in the life cycle, HIV protease hydrolyzes a main-

chain amide in the unfolded, extended regions between viral proteins allowing them to fold and

assemble into their quaternary structures. 19-20 Additionally, frame shifting enables differential

abundances of viral mRNA to properly titer the amount of viral protein expressed.21

Prior to maturation, the viral poly-proteins assemble on the inner leaflet of the host cell

plasma membrane. The gp4l and gpl20 proteins are expressed as a tandem precursor protein

called env that is translated into the ER. Endogenous protease separate the two before transport to

the plasma membrane.22 Here, the membrane proteins and the cytosolic polyproteins coordinate

29

with host cell proteins from the ESCRT pathway to begin the process of budding.23 Much of the

maturation occurs post-budding, though studies with a photo-cleavable HIV- 1 protease inhibitor

reveal budding can occur without HIV-1 protease activity and upon degradation of the inhibitor,

budded, immature particles can mature to infection particles. 24 Nonetheless, HIV protease is

reported to target endogenous proteins in the cytosol that contributes to pathogenesis.25

1.2.2 Treatment of HIV

Numerous aspects of the viral life cycle have been successfully targeted in the clinic. The first HIV

inhibitor, of any class, was azidothymdine (AZT), an RNA analog that indirectly inhibits reverse

transcription. This class of drugs is called nucleotide RT inhibitors (NRTIs) and works by serving

as a substrate for RT to act as chain terminators by preventing further polymerization after

incorporation into nascent DNA.26 Another class of RT inhibitors target a hydrophobic cavity that

is required for proper enzyme dynamics. These non-nucleotide RT inhibitors (NNRTIs) fill this

hole and trap the enzyme preventing additional turnover.2 7 Protease inhibitors (PIs) are another

major class, which will be extensively reviewed in Section 1.4. Historically, the last enzyme

targeted for antiviral therapy was integrase (IN). Inhibitors of IN target the enzyme bound to DNA

and interact with active site magnesium ions.28 The fourth and most recent IN inhibitor received

FDA approval in 2018.29

Fusion and entry of HIV is also a prominent clinical target. HIV must first bind the primary

receptor, CD4. An antibody selected to bind CD4 was found to inhibit post-attachment co-receptor

engagement and provides the basis for the entry inhibitor ibalizumab, which achieved FDA

approval early in 2018 .30 Additionally, small molecules were elaborated to antagonize the CCR5

30

co-receptor leading to the development of maraviroc. 3 1 Finally, entry requires a conformational

chain of the env trimer in which the pair of 3-helix bundles of gp4l form a six-helix bundle.32

Peptides based on the C-terminal helix traps the intermediate and acts as inhibitor of viral

replication by preventing fusion.33

HIV inhibitors are used in combinations. This approach is referred to as ART but also

highly active ART (HAART). Typically, ART starts with a NRTI, a NNRTI, and an IN inhibitor,

and later includes other classes, like protease inhibitors. As the duration of infection progresses,

the drug combination is optimized to maintain low viremia.34 These molecule must be taken daily

and in large quantities. A prominent issue with HIV treatment is accessibility as well as

adherence. 35 Halting therapy can be detrimental and allow the infection more rapidly become drug-

resistant.

1.2.3 HIV Mutations and Setbacks for a Cure

Mutations in the HIV genome accumulate in patients over the course of treatment. A selection

occurs that enables viral proteins to continue the lifecycle without binding the inhibitors. The low

fidelity of RT is responsible for the mutagenesis. Unlike the robust proofreading of DNA

polymerases, which makes a mistake one in every 109-10 bases replicated, HIV RT has an error

rate of one in 104 bases.36 The adaptability of HIV can lead to drug resistance and treatment failure,

but also enables HIV to evade the adaptive immune response. The only viral proteins assessable

for recognition by the immune system are found on the surface of the viral membrane: gp120 and

gp41. gp 120 has can dramatically change to avoid neutralization by antibodies yet still maintain

affinity to CD4.37 gp120 is additionally masked by glycans when it is produced by the host cell,

31

limiting recognition by the immune system.38 An encouraging target for HIV vaccines is the helix

used by gp4l to insert into the membrane. This feature of the protein is highly conserved but only

solvent accessible for a short time. There are new frontiers in understanding immune recognition

that are promising in developing HIV antibodies that are broadly neutralizing. 39

1.2.4 HIV Latency

A confounding factor of HIV treatment is the lack of viral clearance. Antivirals can suppress HIV

infection to below detectable limits; however, halting therapy will lead to rebounding viremia.

This is due to latency. Latency is the result of cells that become infected but harbor the provirus in

a transcriptionally inactive state. A prominent example is quiescent CD4+ T-cells.40 Latent cells

inefficiently produce viral RNA and are reported to make low levels of virus in patients receiving

treatment.41 Latency is also responsible for reestablishing infection after suspension of antiviral

treatment. Emerging approaches seek to treat HIV by eradication of latent viral reservoirs. 42 These

approaches employ epigenetic modification to increase transcription of the virus.4 3 Viral infection

results in cytopathic effects that can kill the once latent cell.44 Current setbacks of this approach

are global transcriptional activation and cytopathic effects appear to be insufficient to kill all

infected cells.45

32

1.3 Biochemistry of HIV-1 Protease

Understanding the manner in which enzymes operate is crucial in the development of targeted

therapeutics. Kinetic-based assays and X-ray crystallography have been workhorse methodologies

in illuminating the mechanism of HIV-1 protease. The study of proteases has relied heavily on

solid-phase peptide synthesis (SPPS). 4 6 Prior to SPPS, the scope of accessible substrates for

proteolytic studies were limited.47 Additionally, incorporation of chromophores and FRET pairs

into substrates enabled continuous quantitation by absorbance and fluorescence spectroscopy.48-49

Kent and coworkers synthesized HIV-1 protease by SPPS that yielded diffraction quality crystals,

providing among the first publicly available structures, after heterologous expression approaches

failed to provide sufficient materials.5 0 5 1 The clinical relevance of HIV-1 protease and the tools

developed for its study have made it a model protein.

1.3.1 The Structure of HIV-1 Protease

HIV-1 protease, like all retroviral proteases, are homodimeric aspartic proteases (Figure 1.3.A).

Recently, a retroviral protease homolog was discovered in yeast.53 Eukaryotes utilize numerous

monomeric aspartic proteases with varied specificity suited for their biological functions. 54 For

example, pepsin is a non-specific protease that degrades dietary protein (Figure 1.3.B). Meanwhile,

renin specifically cleaves angiotensinogen to liberate the angiotensin precursor peptide, which in

turn, regulates blood pressure (Figure 1.3 .C). 55-56 These two classes, the monomeric eukaryotic

and the homodimeric retroviral protease, have limited sequence and structural similarity, but they

share a conserved active site. That active site consists of a pair of aspartic acid residues for which

this family is named. These residues are found at the beginning of a "DTG" sequence that is

33

arranged by a 2-fold axis as the result of inter-domain hydrogen bonding. This motif is called the

fireman's grip and is responsible for much of the affinity between the monomers of retroviral

proteases.57

Aspartic proteases employ flaps to assist in binding substrates. Monomeric proteases use a

single flap to form hydrogen bonds directly with the carbonyl oxygens of the amides flanking the

scissile amide. Retroviral proteases instead donate hydrogen bonds to a conserved water that

donates hydrogen bonds to these groups. EPR and MD studies have revealed these flaps are

dynamic but favor the closed state when a ligand is bound. 58-59 The flaps contribute to the potency

of HIV protease inhibitors and will be discussed in Section 1.4.3.

1.3.2 Catalysis by HIV Protease

Enzymes catalyze reactions by lowering the kinetic barrier to chemical reactions. HIV protease

shares a common mechanism with all aspartic proteases. Aspartic proteases provide a roughly

101 '-fold rate enhancement compared to the non-catalyzed reaction. 0 This family facilitates amide

hydrolysis by activating water for nucleophilic attack using acid-base catalysis. The mechanism

of aspartic proteases has been studied for several decades. 61 Though a rather complete picture of

catalysis has emerged, there are still unanswered questions. The reaction begins by the formation

of a Michaelis complex where a mono-protonated active site binds a water molecule and the

peptide substrate. The aspartate deprotonates water leading to nucleophilic while the other aspartic

acid protonates the scissile carbonyl oxygen to form the geminal-diol intermediate. The proton

abstracted from water is then transferred to the scissile amide nitrogen leading to a cationic gem-

diol. Scission of the bond leads to formation of the products. The second proton transfer and

34

sciss1IIo Stepa are the least unDetood and the irdeay bei i.Lery to Lthe..' tranition1L1V.J

states and may constitute a single step.62 A proton transfer occurs between the nascent termini prior

to product dissociation.

Crystallography has provided substantial insights into the mechanism. Flap dynamics are

important for substrate binding and the protease has been observed with both open and closed

flaps. A structure with open flaps reveals how the active site is solvated prior to the peptide

substrate binding. 63 A catalytically inactivate variant of HIV protease can be prepared by installing

the D25N mutation. This single atom change alters the active-site residue but still enables the

protein to attain the proper fold.64 Many structures of inactivated HIV protease bound to substrates

have be solved to reveal the Michaelis complex.65 Several structure of inefficiently cleaved

substrates have trapped the gem-diol intermediate. 66-67 Finally, a structure has been solved with

the product bound. 68 The only missing structure is the protonated gem-diol, however, this species

is likely too unstable to capture.

Kinetic-based assays have also revealed signification mechanistic details. Specificity

constants for the cleavage of peptides are dependent on substrate sequence and range from 101 to

105 M-s-1 for endogenous substrates. 69 An activated substrate containing non-canonical amino

acids exhibits the highest observed rate constant of kcat/Km = 2.1 x 107 M-s'-1. 70 Another substrate,

selected by phage display, is the best substrate containing only canonical amino acids, with kcat/KM

= 5.0 x 105 M's-1. Kinetic isotope effects have provided unique insights into the mechanism.

Hydrolysis studies with [ 180]H20 have revealed that HIV-1 protease can incorporate 180 into

substrates. 71 This suggests the first step of forming the gem-diol intermediate is reversible.

Additional incorporation of 13C and 180 into the carbonyl, 5N into the scissile amide, and 2H into

35

C" of the P1 residue have revealed that wild-type and drug-resistant mutants have the same rate-

determining step. 72 Modelling and computational prediction of the kinetic isotope effects suggests

that step is the N protonation.

1.3.3 Substrate Recognition by HIV-1 Protease

Structural biology has also provided insights into recognition by HIV protease. Aspartic protease

sequence specificity depends on the biological function. The most extensively studied human

aspartic protease is renin. Renin cleaves angiotensinogen to generate the angiotensin I peptide that

is next cleaved by ACE to creating the active hormone angiotensin II, which lowers blood

pressure.73 Renin can only efficiently cleave a single substrate.74 Conversely, the digestive protease

pepsin must break down dietary protein by cleaving many sites. Its specificity is limited, but

prefers cleaving between hydrophobic residues. 75 HIV-1 protease is semi-specific and can

efficiently cleave the variety of substrates found in the HIV polyproteins.

Crystal structures of numerous substrates bound to HIV protease revealed recognition of

the sidechains is based on sterics instead of specific donor-acceptor interactions with the

subsites.65, 76 Most protease substrates utilize at least a single hydrogen bond; however, they are

not well conserved between substrates. 65 The residues in protease substrates are named based on

the position relative to the scissile bond and a prime denotes residues after (that is, C-terminal to)

the scissile amide. The most frequently observed polar interaction occurs between Asp30 and a

glutamate or glutamine residue in the P2' position.77 Curiously, bulky hydrophobic groups like

leucine and isoleucine can also occupy the S2' subsite. Though hydrophobic groups are most

commonly found at the P1 and P1' positions, substrates come in two types. Most substrates employ

36

a pair of primary amino acids where both, either, or neither are aromatic rcsiducs and constitute

type 1 sequences. Type 2 sequences utilize a P1 aromatic residue and proline at the P1' position. 78

The PI/Pi' residues further dictate the selection of P2/P2' residues and add additional features to

the two types of cleavage sites. 79-80 Nonetheless, the bulk of recognition comes from hydrogen

bond interactions with the main chain of substrates.81-82 Insights into substrate recognition by HIV

protease have been critical in tailoring the specify of protease inhibitors.

37

1.4 Inhibition of HIV-1 Protease

Protease inhibitors are an important class of antiviral drugs that have dramatically improved

clinical outcomes for HIV patients.83 As of 2018, the FDA has approved ten small molecules

inhibitors of HIV protease for the treatment of HIV, eight of which still find clinical utilization.

The standard of care utilizes protease inhibitors as part of ART. Initial implementation of ART

required patients to take a combination of pills, but an emerging trend is a single pill containing

multiple drugs to simplify dosing. Recent interest in reducing daily intake of drugs has motivated

studies demonstrating protease inhibitor monotherapy can manage viremia after initial suppression

by ART.84 Though lack of adherence is still a major driver of treatment failure, decades of use

have selected for viruses with proteases that are no longer inhibited by even the best of clinical

inhibitors.85 Drug resistance and reducing the pill burden continues to motivate the development

of improved members of this class of drug.

1.4.1 Natural Product-inspired Inhibitors of Aspartic Proteases

Clinical inhibitors are a classic example of rational drug design where nature provided a lead

compound. In 1970, a nature product secreted by actinomycetes was discovered to inhibit the

digestive protease pepsin. 86 This inhibitor, named pepstatin, is a peptide that contains the non-

canonical amino acid statine (Figure 1.4.1 .A). An analog of leucine, statine when incorporated into

peptide substrates at the P1 position, generate potent competitive inhibitors of aspartic proteases. 87

Statine is a y-amino acid with a single S-configured hydroxyl group substituted on the P-position

(Figure 1.4.2.B). Investigations revealed the stereochemistry of the hydroxyl is important for

inhibition. The S-enantiomer is 100-fold more potent than the R-enantiomer.88 This moiety works

38

by mimicking the tetrahedral gemdiol intermediate (Figure 1.4.1.C). Jncorporation of this

substrate into substrates of renin and later HIV- 1 protease generated potent inhibitors with values

of Ki near 10-10 M.89-90 These seminal studies identified the pharmacophore employed in the

majority of clinical inhibitors.

1.4.2 The First Generation of Clinical Inhibitors

Following the success of peptide models employing the hydroxyethylene tetrahedral-intermediate

isostere, research pushed forward to identify peptidic scaffolds with enhanced stability in vivo.

The first protease inhibitor to gain regulatory approval was saquinavir. Its design was based on the

unusual property of HIV protease being able to accommodate a proline residue at the P1' position. 91

The second approved inhibitor was ritonavir. Though ritonavir is an efficient inhibitor of HIV-1

protease, its current role in treatment is a pharmacokinetic enhancer by inhibiting metabolic

enzymes that degrade protease inhibitors.92 Indinavir was the third FDA approved PI; however, its

use is no longer recommended due to side effects.93 Further elaboration of saquinavir improved

solubility of the P2 targeting group leading to the improved the potency of nelfinavir. 94 Finally,

amprenavir was designed to avoid bulking hydrophobic groups and amide linkages to make one

of most compact protease inhibitors.95 -96 The first generation of HIV protease inhibitors suffered

from poor pharmacokinetics, several side effects, and high susceptibility to drug resistance

mutations.97-98 These setbacks motivated the second generation of inhibitors.

39

1.4.3 The Second Generation of Protease Inhibitors

Taking lessons learned from the first generation, the design of the second generation of inhibitors

sought to enhance stability and maintain affinity in the presence of resistance mutations (Figure

1.4.3.). A key issue with amprenavir was the lack of solubility and poor bioavailability. This

limitation was remedied by appending a phosphate ester to the hydroxyethylene pharmacophore

to make the prodrug fosamprenavir. 99 Fosampreanvir dramatically reduced the pill burden of

amprenavir.1 00 Redesign efforts to address drug resistance of ritonavir lead to the discovery of

lopinavir.101

Moving away from amide linkages, atazanavir was designed with a hydrazine group

connecting the Pl' and P2' targeting groups.' 02 All clinical inhibitors of HIV protease utilize the

hydroxyethylene isostere with the exception of tipranvir. Tipranavir was discovered through an

extensive SAR campaign following the discovery that a hydroxyl-substituted coumarin derivative

inhibited HIV-1 protease.1 03 Tipranavir is effective against viruses resistant to other protease

inhibitors.1 04 The best-in-class and best-characterized protease inhibitor is the most recent to

achieve approval by the FDA. Darunavir is an analog of amprenavir with a modified P2-targeting

moiety.' 05 Initially studied in a saquinavir analog, the bis-THF group can accept a pair of hydrogen

bonds from the main chain amides of Asp29 and Asp30.1 06-107 Darunavir also has the unique ability

to inhibit protease dimerization.' 08 Inhibition of HIV-1 protease, by darunavir in particular, has

provided an exceptional model system to study drug resistance.

40

1 A A Th Resistancead the Fture of Pr-otes Inbhbtars

Several strategies are operative in the manifestation of drug resistance through HIV protease

mutations. 109 The most easily understood variety are the result of perturbation of the interface

between the protease and the ligand. Expansion of the active site can make an unfilled cavity

between the inhibitor and the subsite, which is disfavored by entropy.' 1 0 Alternatively, contraction

of the active site leads to steric occlusion of the inhibitor. These ideas combined with structural

analysis led to the substrate envelop hypothesis that posits inhibitors designed to closely match the

volume occupied by substrates are least susceptible to drug resistance mutations.76 Mutations of

polar side chains can also disruption non-covalent interactions with ligands. Such mutations can

be combated by targeting the main chain instead of side chains as exemplified by darunavir. Drug

resistance mutations frequently reduce viral fitness and compensatory mutations are required to

maintain infectivity." 1-112 These distal mutations can help restore protease activity lost in the

selection of primary mutations." 3 Substrates in the polyproteins also change to enhance catalysis

of the mutant protease.11 4

Despite conforming to contemporary design paradigms aimed at reducing drug resistance,

treatment failure has been observed in patients taking darunavir over an extended period." 5 The

protease flaps play a prominent role in darunavir resistance.116 An isolate named MDR 769 has

numerous mutation in the hinge region that connects the flap to the core domain. This variant was

the first protease crystallized with open flaps and provided insights into targeting the

conformation."1 7 A highly darunavir-resistant protease named PR20 has a 1000-fold reduced

affinity for darunavir."1 8 This variant has been crystallized in both open and semi-open forms and

includes similar hinge mutations to MDR769. EPR studies of MDR769 and PR20 revealed that

41

mutations in the hinge leads to changing the thermodynamically favored conformation from the

closed form to the semi-open form.1 9 MD simulations and NMR studies provided additional

structural details how the flap dynamics of PR20 are altered by hinge mutations. 120-121

Additionally, the variant P51 has been observed to bind darunavir in an alternative conformation

with open flaps.1 22 An emerging path to targeting these variants is enhancing interactions with the

flaps.1 2 3 New subsite targeting groups and core scaffolds are needed to continue fighting drug

resistance.

42

1.5 Versatile Recognition by Boronic Acids

Found to the left of carbon on the periodic table, boron exhibits features of both metals and non-

metals.1 2 4 As a p block element, the structure and bonding configuration of boron are similar to

those of organic compounds. Boron can, however, form stable ions like metals. Boron is neutral

when it forms three sigma bonds and becomes negatively charged with the addition of a fourth

bond that fills the octet. Boric acid is the thermodynamically favored form of boron in aqueous

solution. Oxidized species of boron with a single organic substituent are called boronic acids.

Boronic acids have emerged as an important intermediate in chemical synthesis.' 2 ' The unique

reactivity has also attracted the attention of medicinal chemists. Of note, boronic acids have been

extensively studied as protease inhibitors.1 26

1.5.1 Chemistry of Boronic Acids

The chemistry of boronic acids provides unique opportunities for medicinal chemists. Boronic

acids form a single bond with a carbon atom and two to three bonds with oxygen atoms. When

trivalent, a vacant p orbital exists on the boron leading to sp2 hybridization and a planar geometry.

This vacant orbital can form bonds with nucleophilic species including primarily 0 and but also

N, F, and S atoms. The formation of the tetravalent species creates an anionic, sp3 hybridized boron

with a tetrahedral geometry. In aqueous solution, boronic acids are in equilibrium between the

trigonal and tetrahedral states as described by the pKa (Figure 1.5).

Boronic acid pKa values are typically between 7 and 10.124 Functionalities substituted on

the organic component that act as electron withdrawing groups will limit donation to the vacant p

43

orbital on the boronic acid and reduce the pKa.127 Conversely, electron donating groups will add

to the p orbital and compete with hydration, thereby increasing the pKa. The exchange between

trigonal and tetrahedral states is fast and the two states are equally populated when the pH is equal

to the pKa.1 28 In addition to water, boronic acids can form bonds with alcohols and carboxylic acids

(Figure 1.5).129 Complexes with these ligands are referred to as boronic esters. The formation of

esters in aqueous contexts depends both on the solution pH and the pKa's of the boronic acid and

the ligand.13 0 The Kd of boronic esters is a function of pH. At pH near the boronic acid pKa, the

tetrahedral state and boronic ester formation is favored. Ester formation drops the pKa of the

boronic acid complex favoring the tetrahedral species. Planar esters typically have diminished

equilibrium constants relative to tetrahedral ones.' 3' Finally, ligands with greater acidity form

more stable complexes.

A special class of boronic acids form intramolecular esters. Phenylboronic acid with an

ortho benzyl alcohol group forms a cyclic boronic ester as a 5-membered ring. This ring is called

a "benzoxaborole". Benzoxaboroles have attracted substantial attention from medicinal and

carbohydrate chemists due to the enhanced sugar-binding capabilities. 3 2 The intramolecular ester

of benzoxaborole reduces its pKa to 7.4, which favors complex formation with ligands. Sugars

present many diols, which can form esters with boronic esters. Benzoxaboroles and boronic acids

in general, have the greatest affinity for 1,2-diols, specifically in the cis configuration, such as

those found on fructose and ribonucleotides. 33

44

1 < I DfD +- 1,U , A ,.AqI .. J ./-. %-%JU V CtlI%.4L %J L J'L J1 I VJL I L1 Uy 1.JL ji I%, I %ALI.a

The intriguing properties of boronic acids have found utility in the covalent recognition of

proteins. 134 A class of drugs that rely on conventional recognition strategies to position drugs at a

target site and, once bound, evoke the formation of a covalent bond are known as covalent

inhibitors. 3 5 Covalent inhibitors carry a distinct advantage: the dissociation rate constant of these

molecules is limited by the rate at which the covalent bond between the drug and the target breaks.

However, these types of molecules infrequently find clinical utility due to off-target reactivity. A

special class of covalent inhibitors forms bonds with active site residues in a reversible manner.

Boronic acids typify this group.

Several amino acids present side chains that can form complexes with boronic acids. The

majority of reported protein-boronic esters formed with hydroxyl groups of serine and threonine

sidechains. These residues are often employed as nucleophiles during catalysis.1 36 The formation

of complexes between serine and threonine sidechains has served as the basis of covalent

inhibitors. 3 7 In an extreme case, a tetravalent complex between two serine sidechains and a lysine

side chain was observed in inhibitors of penicillin-binding protein. 138 These groups typically only

form tetrahedral complexes as boronic ester formation reduces the pKa.

A classic example of boronic acid-based inhibitors are covalent inhibitors of proteases.1 39

A number of protease families employ serine or threonine as a nucleophile in amide hydrolysis.

The attack leads to a tetrahedral intermediate, which collapses as the nascent amino termini group

leaves to form an acyl-enzyme intermediate. Next, water hydrolyzes the ester linkage between the

intermediate and the active site serine through another tetrahedral intermediate. This transition

state is isosteric with a tetrahedral boronic acid ester. 4 0 The first FDA-approved boronic acid,

45

bortezomib, targets the 20S proteasome for the treatment of multiple myeloma. The boronic acid

is key to activity as the potency is dramatically reduced upon deboronation, a common form of

metabolism in vivo.141 In 2015, the orally administered boronic acid ixazomab received FDA

approval.1 4 2 As of 2018, delanzomib is in cancer clinical trials.143 These boronic acids utilize the

same mode of inhibition as bortezomib.1 4 4 Peptidic inhibitors of HIV-1 protease have been

reported; however, whether they form covalent bonds with the active site remains to be

determined. 145

Benzoxaboroles and esters thereof are an emerging class of clinical boronic acid inhibitors.

These compounds form boronic esters with the ribose ring of nucleotide cofactors. This

complexation in the active site prevents dissociation of the product, thus reducing the off rate to

trap the enzyme in an intermediate state. The first demonstration of this class came from inhibitors

of tRNA synthetases.1 46 Fungal variants of these enzymes are sufficiently orthogonal to humans

resulting in selective inhibition as exemplified by tavaborole which received FDA approval in

2014.147 Benzoxaboroles have also been employed as ligands for the metal center of PDE4 to act

as inhibitors.1 4 8 This work lead to the crisaborole, which received FDA approval in 2016 for the

treatment of psoriasis.1 4 9

1.5.3 Non-covalent Recognition of Proteins by Boronic Acids

In biological contexts, boronic acids present two hydroxyl groups. No other functional group can

so economically present this many hydrogen bond donors and acceptors. Molecular encapsulation

studies have revealed boronic acids more efficiently form hydrogen bond mediated dimers than

46

amides and carboxylates. 15 0 A be7oxaborole can present a hydroxyl grp nA kriAi ng xy

This group has been employed as a P-strand mimic in an inhibitor of rho-activated kinase 2.151

Non-covalent recognition is, however, an underutilized application of boronic acids. 3 4

47

1.6 Ribonucleases as Antiviral Therapeutics

The cells of vertebrates secrete a unique family of ribonucleases called pancreatic-type

ribonucleases (RNases). RNases exhibit a number of desirable features making them an attractive

protein to serve as the basis of chemotherapeutics.15 2 First, RNases share a conserved set of

disulfide bonds that confer resounding stability. Second, they possess cytotoxic ribonucleolytic

activity. Third, RNases do not require delivery agents, as they have the endogenous ability to gain

entry to the cytosol. These properties would make RNase highly cytotoxic, but cells are

safeguarded by the cytosolic ribonuclease inhibitor (RI) protein. Diminishing interactions with RI

without detriment to the desirable properties has been the focus of extensive work on the

engineering RNases as cancer chemotherapeutics.153

1.6.1 Structure of RNases

The RNase homolog found in the bovine pancreas, RNase A, is among the best studied

proteins.1 54 RNase A was the subject of seminal protein folding studies that demonstrated the

primary amino acid sequence contains enough information to direct the spontaneous folding of the

tertiary structure. Key to the folding of RNases is disulfide bond formation. This family of enzymes

have four conserved disulfide bonds that are important for maintaining the remarkable stability of

these proteins as well as organizing the active site residues.' 5 5 -15 6 Overall, RNases have a kidney

shape with a central P-sheet with three a-helices and several loops and turns (Figure 1.6.A).1 57

Cellular production of RNases occurs by translation of the protein into the ER where PDI can assist

in the formation of disulfide bonds prior to secretion.' 58 As indicated, these disulfides can also be

efficiently formed in vitro enabling preparative scale production of engineered variants.' 59

48

1.6.2 Catalysis by RNases

Catalysis by RNase A is also historically significant, as it was the first enzyme to have its

mechanism deduced.1 60 The active site consists of two key histidine residues and an accessory

lysine. Catalysis begins with His12 acting as a base and deprotonating the 2'-hydroxyl group of

the substrate RNA. RNases bind RNA to preorganize them for an intramolecular nucleophilic

attack by the ensuing 2'-alkoxide in-line with the 5'-oxygen of the phosphodiester, which becomes

the leaving group. His 119 protonates the nascent group to generate the 5'-hydroxyl group of the

product. The negative charge developing in the transition site is stabilized by a hydrogen bond

with Lys41. The first step produces a cyclic phosphodiester between the 2' and 3' oxygens that can

be hydrolyzed to a 3' monophosphate in a second step.' 61 There is limited sequence specificity,

however, including a preference for pyrimidine nucleotides upstream of the scissile phosphoryl

group. 162

RNase A and many of the homologs, including human RNase 1, are potent catalysts and

have catalytic efficiencies around kcat/KM = 10' M's-1, which is near the limit of diffusion. 163

Several intriguing RNases have limited ribonucleolytic activity. RNase 5, also known as

angiogenin, exhibits a second order rate constant of 102 M-Is-1.164 Remarkably, this relatively low

activity can stimulate neovascularization. Recent studies have identified angiogenin specifically

interacts with the promoter associated RNA of ribosomal RNA.1 65 Another weakly active RNase

is ranapirnase, also called Onconase* (ONC), from the oocytes of the northern leopard frog. This

unique variant employs an N-terminal pyroglutamate in the active site. Additionally, this enzyme

49

is among the most stable with a Tm value of 900 C, likely needed to withstand harsh terrestrial

conditions. 166

1.6.3 Internalization of RNases

RNase A helped pave the route to understanding the role of charge in the cellular uptake of

proteins. The glycocalyx is a 0.5-3.0 ptM think coat of glycans and glycoproteins that protects the

10-nm thick plasma membrane.1 67 This admixture of functional groups prominently features

carboxylates and sulfates and imparts a negative charge to the glycocalyx. Ribonucleases and other

cationic proteins bind the cell surface through Coulombic interactions. 168 -169 This property can also

be conferred to proteins through the addition of a terminal tag by recombinant expression or

chemical means.1 70-171 Once associated with the cell surface, endocytosis brings cationic proteins

into the cell through one of a variety of processes, including macropinocytosis and clathrin-

dependent endocytosis.' 72 The contents of the endosome are destined for destruction as the vesicle

matures into a lysosome.1 73 Acidification is thought to enable RNases and other cell-penetrating

proteins to escape the endosome through a process that is not well understood.1 74 Unlike many

therapeutically desirable proteins, RNases have the innate ability to access the cytosol. Unchecked

RNase activity in the cytosol leads to apoptosis.17 5

1.6.4 Evasion of Ribonuclease Inhibitor

Cellular RNA is safeguarded from cytotoxic RNase activity by RI.1 76 Ribonucleases form among

the tightest known protein-protein interactions with RI. Human RNase 1 and RI form a complex

50

with a Kd value of 10-16 NM (Figure 1.6.B).1 77 Sties of n s s affinities riwealed that

differences in RNases have led to compensatory changes in RI to maintain high affinities.78179

Two key properties explain the remarkable affinity between RNases and RI: shape

complementarity and charge distribution. An edge of the horseshoe-shaped RI fits snugly in the

active site of RNases to competitively inhibit substrates binding.' 80 Onconase, a remote homolog,

has a unique shape and is only weakly inhibited under low salt conditions which enhances

Coloumbic interactions.181 In addition to the complementarity of shape, RI and RNase have pairs

of charged residues arranged to enhance binding. 8 2 Disruption of these interactions has been a

prominent approach in efforts to make mammalian RNases cytotoxic to evade RI and engender

cytotoxicity. 153, 183

1.6.5 Antiviral Activity of RNases

The biological functions of RNases are emerging, but their biology remains a fertile area of

research.1 84 RNases are involved in innate immunity. 185 Human RNases 2, 3, and 7 are employed

by the immune system as cytotoxic agents toward invading pathogens.18 6-187 Cells secrete

angiogenin and RNase 4 to suppress HIV infection.1 88 Additionally, antiviral activities of several

RNases has been observed.' 8 9-'90 These encouraging results make RNases promising agents

serving as the basis of novel antivirals.

51

Figure 1.3

52

A

HIV-1 protease-JG-365B

renin-aliskirenC

pepsin-pepstatin

Figure 1.3. Structures of Aspartic Proteases Bound to Ligands. Retroviruses like HIV utilize a

homodimeric protease has part of their lifecycle. A. The structure of HIV-1 protease is shown in

complex with an early inhibitor (pdb: 7hvp).1 91 Alternatively, eukaryotes possess monomeric

aspartic proteases. Renin cleaves angiotensin and subsequent activation leads to increased blood

pressure in human. B. The drug aliskiren inhibits renin an acts to lower blood pressure (pdb:

2vOz). 192 C. Pepsin is responsible for digestion and can be inhibited by the natural product,

pepstatin (pdb: Ipso).1 93 Inhibitors of aspartic proteases will be discussed in greater detail in

Chapter 4.

53

Figure 1.4.1

A

H0 OH0 H OH O

N N NJ-KN NyQIQ OHpeptat H 0

pepstatin

B OH OH2N OH

statine

HO OH

NH

tetrahedral intermediate

54

Figure 1.4.1. Structure of Pepstatin, Statine, and a Tetrahedral Intermediate. A. Pepstatin is a

natural product that contains two statine residues. B. Statine is resembles leucine but contains an

additional hydroxylethlyene group. C. The hydroxyethylene group mimics the tetrahedral

intermediate formed after the first step of catalysis by HIV protease.

55

Figure 1.4.2

First generation

H2N 0

H0 H PH H,, H

Nz N NNHH0

0 HHb 0;

saquinavir

N:N OH H OHOH

Ndv

indinavir

OH

H "HHO-L%

0/ +

H OH H

nelfinavir

H 1 0N N Ny< H)~

amprenavir r n vritonavir

Second generation

HOH r

0 00 _y\ NNK NHO N N

0 N W NH& 2 0 0

H 0

-/ \ lopinavirca2

+

fosamprenavir H 0 0 _

0 -,, N NH2H H OH(,r

0 OH 0ONN

H 0

0 Nz N0

atazanavir

darunavir

. tipranavir

OH H I F

*tipranavir F

56

Figure 1.4.2. FDA Approved Inhibitors of HIV Protease. As of 2018, ten protease inhibitors have

been approved for the treatment of HIV. Arrows indicate the progression in design of inhibitors

with a common core scaffold.

57

HO, BOH

RBoronic Acid (sp9)

+H 20 HO OHHO-B-

RBoronate (sp)

II +HOR'

HO 0'B' 0R'R

Boronic Ester (sp2)

+H 20 HO 0HO-B- R'

R

Boronate Ester (sp3)

58

Figure 1.5

Figure 1.5. Equilibria of Boronic Acids in Aqueous Solution. Boronic acids are planar at pH below

the pKa. Boronates are favored at pH above the pKa. Boronic acids can also accept organic oxygen

species to form esters and the coordination state is driven by the boronic ester pKa which is

typically lower than the corresponding boronic acid.

59

Figure 1.6

A scissilebond

Lys41

His118

Hisl2

RNase A-d(ApTpApA)

B

RNase 1-RI

60

Figure 1.6. Structures of RNases Bound to Ligands. RNases bind nucleic acids with a cationic

active site cleft. A. This is demonstrated by a structure of RNase A bound to a substrate analog,

DNA (pdb: lrcn).1 94 RNases are potently inhibited by RI. B. Human RNase 1 and RI form a highly

complementary complex that blocks the substrate from binding the active site.1 82

61

Chapter 2

Fluorogenic Assay for Inhibitors of HIV-1 Protease

with Sub-picomolar Affinity

Contribution:

I designed and performed all experiments. I wrote the initial manuscript and contributed to the

revision of the final manuscript.

This chapter has been published in part, under the same title. Reference:

Windsor, I. W.; Raines, R. T., Fluorogenic assay for inhibitors of HIV-1 protease with sub-

picomolar affinity. Sci. Rep. 2015, 5, 11286.

62

Abstract

A fluorogenic substrate for HIV- 1 protease was designed and used as the basis for a hypersensitive

assay. The substrate exhibits a kcat of 7.4 s-1, KM of 15 tM, and an increase in fluorescence intensity

of 104-fold upon cleavage, thus providing sensitivity that is unmatched in a continuous assay of

HIV- 1 protease. These properties enabled the enzyme concentration in an activity assay to be

reduced to 25 pM, which is close to the Kd value of the protease dimer. By fitting inhibition data

to Morrison's equation, Ki values of amprenavir, darunavir, and tipranavir were determined to be

135, 10, and 82 pM, respectively. This assay, which is capable of measuring Ki values as low as

0.25 pM, is well-suited for characterizing the next generation of HIV- 1 protease inhibitors.

63

2.1. Introduction

HIV-l protease is a dimeric, aspartic acid protease. This enzyme is not only an important target

for chemotherapeutic agents but also has been a key model for the development of structure-based

drug design and in studies of drug resistance.195-197 Hundreds of small molecules that bind to the

enzymic active site and inhibit proteolyic activity have been characterized, ten of which have

achieved regulatory approval and clinical relevance for the treatment of HIV/AIDS. Three

inhibitors in particular, amprenavir, darunavir, and tipranavir, have garnered significant attention

due to their picomolar inhibition constants and reduced susceptibility to drug-resistance mutations

in the viral genome. 96, 103, 118, 198-200

Different workers have used different assays to assess inhibition by tight-binding inhibitors

of HIV-l protease. The ensuing values of inhibition constant (Ki) for the same inhibitor range over

several orders-of-magnitude (Table 2.1). These discrepancies, along with the pending emergence

of even more potent inhibitors 2 01 requiring even more sensitive characterization techniques,

provided an impetus for our work.

Solution-phase assays based on enzymatic activity are the oldest and still the most popular

approach for characterizing the inhibition of HIV-I protease.48-4 9 The loss of enzymatic activity

upon addition of inhibitor is employed as a metric to assess the formation of an enzyme inhibitor

complex. Classical methods employ graphical analysis to estimate a value of IC5 o and its

conversion to a value for the inhibition constant, Ki.202 This approach is limited to assay conditions

in which the Ki value is above or near the enzyme concentration.

High-affinity inhibitors possess Ki values below the usable enzyme concentrations of

traditional assays. As little free inhibitor is present under sub-saturating conditions, the observed

64

PnfzyfmtiC ctivity deC linrly with inhibitcr etrtion. This hgh- 1t inh;itor

problem was remedied by curve fitting as described by Morrison, 203 but a computational analysis

has revealed that the reliability of his methodology is limited to the determination of Ki values that

are no less than 100-fold below the enzyme concentration. 204

An alternative assay for characterizing high-affinity inhibitors of HIV-1 protease is

isothermal titration calorimetry (ITC). 20 -20 6 ITC is made powerful by its label-free nature and

provision of a full thermodynamic characterization. Still, ITC has notable disadvantages compared

with activity-based assays. For example, ITC has a theoretical limit in the low nanomolar range

for the direct measure of an equilibrium disassociation constant (Kd).

The key to assessing the next generation of HIV-1 protease inhibitors is reducing the

enzyme requirement in activity-based inhibition assays. Towards that end, we report here on the

design and characterization of a novel fluorogenic substrate for HIV-1 protease. Its attributes-

high kcat and kcat/KM values and high signal-to-noise ratio-are unprecedented, and enable the

rapid, facile determination of sub-picomolar values of Ki.

2.2. Results

2.2.1. Substrate Design

The first major design criterion for an improved fluorogenic substrate was identifying a peptide

that was bound by HIV protease with high affinity and cleaved rapidly in a catalytic manner. To

meet this criterion, we employed a peptide substrate that had been selected by phage display. The

sequence GSGIFLETSL was reported to have kcat/KM = 1.3 iM-s-1 for cleavage between the

phenylalanine and leucine residues.207 These values were determined by a discontinuous HPLC

65

method. Under the same conditions, an endogenous cleavage site in the HIV polyprotein has

kcat/KM = 0.022 pM-Is-1. 2 07

The second major design criterion was employing a sensitive method to detect substrate

turnover in a continuous manner. We focused on the loss of Fbrster resonance energy transfer

(FRET), which underlies many useful assays,2 08 and considered three donor/acceptor moieties.

Appending the fluorophore p-aminonitrobenzoic acid (Abz) and installing a nitro group in the para

position of phenylalanine as a quenching chromophore is the basis for a popular HIV protease

substrate.49 This pair, however, lacks sensitivity, as Abz is a weak fluorophore and its use provides

a relatively low signal-to-noise ratio. FRET between fluorescein and rhodamine is the basis for

some of the most sensitive known assays for enzymatic activity, 209 but protonation of a fluorescein

moiety at pH 5, which is optimal for catalysis by HIV-l protease,4 8 compromises the utility of this

pair for our purpose, and others.2 10-2 11 We chose 5-((2-aminoethyl)amino)naphthalene-l-sulfonic

acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) as a FRET pair.4 8, 209

We installed these moieties into GSIFLETSL by replacing the glycine residue at the N-terminus

with a glutamate-EDANS conjugate and by replacing the leucine residue at the C-terminus with a

lysine-sDABCYL conjugate.48 Finally, we added an arginine residue to each terminus to enhance

aqueous solubility at pH 5, thereby generating substrate 1 (Figure 2.1).

2.2.2. Assay Design

Our assay was designed to minimize the enzyme concentration while maintaining a high signal-

to-noise ratio. An inherent complexity is that HIV-1 protease is an obligate dimer. Significant

attention has been paid to the Kd value of the HIV- 1 protease dimer, and conflicting ideas abound

66

regaruing an appropriaL Cnzymev CInCInRLaL11 f01 aCtivity assays. 1 re1ir anu coworkers

employed a rigorous thermodynamic approach to determine a dimer Kd value of 23 pM.2 13 We

used this value as a lower limit for the enzyme concentration in our assays. Additionally, initial

velocities require measurements from <10% substrate turnover, and a convenient upper limit for

the enzyme concentration in our assays was determined to be 6.5 nM. Upon cleavage by HIV-1

protease, the fluorescence intensity (I) of substrate 1 increases by I/Jo = 104.

2.2.3. Michaelis-Menten Kinetics

Michaelis-Menten kinetics were used to evaluate the performance of substrate 1 as a substrate for

HIV-1 protease. The initial velocity was directly proportional to enzyme concentration at a fixed

substrate concentration (Figure 2.2A), and increased with substrate concentration at a fixed

enzyme concentration (Figure 2.2B). The latter data fitted well to the Michaelis-Menten equation

(Figure 2.2C). The observed Vmax value of 1.58 nM s- 1 for substrate 1 at an enzyme concentration

of 214 pM afforded a kcat value of (7.4 0.2) s-1; the KM value was (14.7 1.0) [tM (Table 2).

2.2.4. Determination of Ki Values with Morrison's Equation

Substrate 1 was used as the basis for assays of the inhibition of HIV-1 protease by amprenavir,

darunavir, and tipranavir (Figure 2.3). The data fitted well to Morrison's equation, and afforded

values of Ki values (Table 2.1). Darunavir and tipranavir exhibited time-dependent inhibition;

amprenavir did not. Pre-equilibrium data were omitted from the initial-velocity data fitted by linear

regression.

67

2.3. Discussion

An assay of high sensitivity is critical for assessing the efficacy of high-affinity inhibitors of

enzymatic activity. The HIV- 1 protease substrate described herein provides initial velocity data of

unmatched quality and sensitivity. Substrate 1 has a 1.5-fold higher kcat value, 7-fold lower KM

value, and higher signal-to-noise ratio than does the parent substrate developed by Matayoshi and

coworkers (Table 2.2).48 The Abz-based substrate developed by Toth and Marshall has a similar

KM value, though substrate 1 provides a 17-fold greater signal-to-noise ratio.49 These

improvements in kinetic parameters have enabled us to reduce the concentration of enzyme in

standard assays to values close to the Kd value of dimeric HIV- 1 protease.

To quantify the utility of a substrate, we define the sensitivity (S) of an assay as the increase

in fluoresence intensity brought about by the action of an enzyme on a low concentration of

substrate. We express sensitivity (S) as the product of the kinetic parameter kcat/KM and the

spectroscopic parameter If/Io: S= (kcat/KM)(If/Io). By this measure, the sensitivity of an assay that

uses substrate I is >I 0-fold greater than any fluorescence-based assay for HIV-I protease activity

(Table 2.2).

The values of Ki for amprenavir, darunavir, and tipranavir derived from initial velocities

for the cleavage of substrate 1 are consistent with literature values (Table 2.1). Notably, the Ki

value for darunavir determined herein is comparable to literature values and much closer to values

reported by other activity-based methods than is the value determined by an indirect, competitive

displacement method, 201 which is two orders of magnitude lower. The high signal-to-noise, low

variation in initial velocity measurements and low standard error for fits of inhibition data by

Morrison's equation makes substrate 1 a useful probe for assaying high-affinity inhibitors.

68

LO a co11puLaL1na1 assessment, Morrison's equaL1in ca11n Ue useU Lo determine

values of Ki that are up to 100-fold lower than the concentration of enzyme in an assay.204 Because

enzyme concentrations as low as 25 pM provided valuable data herein, we believe that our assay

can be used to determine Ki values that are >250 fM. We anticipate the use of substrate 1 in the

development of the next generation of HIV- 1 protease inhibitors.

2.4. Materials and Methods

2.4.1. Materials.

The HIV-1 protease inhibitors darunavir (from Tibotec, Inc), amprenavir, and tipranavir were

obtained through the AIDS Reagent Program, Division of AIDS, NIAID, NIH. Substrate 1 was

synthesized and purified by HPLC to 99.5% by Biomatik (Wilmington, DE). All inhibitors and

peptides were used without further purification.

2.4.2. Plasmid Preparation.

Double-stranded DNA encoding a pseudo-wild-type HIV-1 protease and flanked by regions of

homology near the T7 promoter and terminator found in the pET32b vector was obtained from

IDT (Coralville, IA). This HIV-1 protease had Q7K, L331, L631, C67A, and C95A substitutions. 2 14

Linear pET32b was prepared by PCR using primers that were the reverse complements of the

DNA encoding HIV-1 protease. Gene and plasmid fragments were combined with Gibson

assembly. 215

69

2.4.3. Protein Purification.

BL-21 codon-plus RIL from Agilent Technologies (Santa Clara, CA) was transformed freshly with

the pET32b-HIV protease. A single colony was used to inoculate 1 L of Luria-Bertani medium

containing ampicillin (200 [M) in a Fernbach flask shaken at 37 'C. Expression was induced by

the addition of IPTG (to 2 mM) upon reaching saturation (OD600 nm 2.8-3.4), and the culture was

grown for an additional hour. HIV-1 protease was purified and folded as described previously. 2 16

Cells were pelleted, resuspended in 20 mM Tris-HCl buffer, pH 7.4, containing EDTA (1 mM)

and lysed at 18 kPSL using a cell disruptor from Constant Systems (Kennesaw, GA). Inclusion

bodies were isolated by centrifugation at 1 0,000g for 10 min. The pelleted inclusion bodies were

washed with resuspension buffer containing urea (1.0 M) and Triton X-100 (1% v/v), and again

with resuspension buffer. Inclusion bodies were isolated by centrifugation and lyophilized.

Inclusion bodies were dissolved by sonication in aqueous acetic acid (50% v/v) at a

concentration of 5 mg/mL. The solution was clarified by centrifugation, and soluble protein was

applied to a Superdex 75 gel-filtration column from GE Healthcare Bio-Sciences (Pittsburgh, PA)

that had been pre-equilibrated with aqueous acetic acid (50% v/v). Unfolded HIV- 1 protease that

eluted as major peak near one column-volume was pooled and lyophilized. HIV-1 protease was

folded at a concentration of 0.1 mg/mL in 100 mM sodium acetate buffer, pH 5.5, containing

ethylene glycol (5% v/v) and glycerol (10% v/v). The solution of folded HIV-1 protease was

clarified by centrifugation and concentrated with an Amicon stirred-cell concentrator equipped

with a 10K MWCO membrane from EMD Millipore (Billerica, MA). The concentrated protease

was applied again to a Superdex 75 gel-filtration column that had been pre-equilibrated with the

folding buffer. A new major peak containing dimeric HIV- 1 protease was pooled and concentrated.

70

The folding buffer was exchanged for 1 mM sodium acetate buffer, pH 5.0, containing NaCl (2

mM) using a PD-10 desalting column. A solution (~1.5 mg/mL) of purified HIV-1 protease was

flash-frozen in liquid nitrogen and stored at -80 'C until use.

2.4.4. Enzymatic Activity Assays.

Substrate 1 was dissolved at a concentration of 1.0 mM in DMF containing TFA (0.1% v/v).

Fluorescence of the EDANS moiety was measured on a M1000 Pro plate reader from Tecan

(Maennedorf, Switzerland) by excitation at 340 nm and observation of emission at 490 nm. A

fluorophore calibration was performed to enable quantitation of assay data. The product exhibits

a fluorescence of 70 RFU/nM at a gain setting of 216, and all assays were performed at this gain

setting unless indicated otherwise. Assays were performed in a Coming black, flat bottom, non-

binding surface, 96-well plate. Assays were conducted at room temperature in 200 tL of 50 mM

sodium acetate buffer, pH 5.0, containing NaCl (0.10 M), DMF (2% v/v), substrate 1 (1-40 pM),

and HIV-1 protease (25 pM-6.5 nM). Assays with 30 and 40 pM of substrate 1 required 3% and

4% v/v DMF, respectively. Inhibition assays were conducted with picomolar-nanomolar inhibitor

(depending on the enzyme concentration and Ki value) and 10 pM substrate 1. Inhibition assays

were monitored for until <7% of the substrate was converted to product. Initial velocities were

measured in quadruplicate.

Solution concentrations of HIV-1 protease (10.7 kDa) was determined by measuring the

absorbance at 280 nm and estimating the extinction coefficient as 12,500 M-cm-1 with software

from ExPASy.2 17 The fraction of active enzyme was determined by active-site titration and found

to be 76% with respect to the value based on the A 280 nm. Fluorescence was monitored over the

71

linear range of the detector, which corresponds to 700 nM of product formation at a gain setting

of 216.

2.4.5. Data Analysis.

The velocity (v) of all enzyme-catalyzed reactions was obtained by linear fit of initial-velocity data

using Prism 6 software from Graphpad (La Jolla, CA). Pre-equilibrium values from the beginning

of data sets were removed to provide fluorescence measurements that were linear as a function of

time (Figure 2.3).

Values of v in the absence of an inhibitor were fitted to the Michaelis-Menten equation

(eq 2.1) by non-linear regression using Prism 6 software.

k [E] [S] (2.1)KM + [S]O

In eq 2.1, [S]. refers to the concentration of substrate 1 prior to the addition of enzyme.

Values of v in the presence of an inhibitor were fitted to Morrison's equation (eq 2.2) by non-linear

regression using Prism 6 software.

[E]+[I]+K. 1-+ IO [E]+[I]+K. 1+ ISO -4[E] [1]V KM KM (2.2)

v_ 2[E]

72

In eq 2.2, v0 refers to the reaction in the absence of inhibitor. Enzymatic activity measured

in the absence of an inhibitor was used to determine the enzyme concentration for data obtained

in the presence of an inhibitor. These enzyme concentrations, which agreed ( 10%) with values

estimated by active-site titration, were used as constraints for the non-linear regression analysis.

2.5. Acknowledgments

I.W.W. was supported by BiotechnologyTraining Grant T32 GM008349 (NIH). Inhibitors were

obtained from the NIH AIDS Reagent Program. This work was supported by Grant RO1

GM044783 (NIH).

73

Table 2.1

74

Table 2.1. Inhibition Constants (pM) Reported for Amprenavir, Darunavir, and Tipranavir

Assay type Amprenavir Darunavir Tipranavir Reference

Enzymatic activity 100 8 88 198

(Unknown) 600 96

Enzymatic activity 8 103

(Unknown) 16 199

ITC (competitive displacement) 390 4.5 200

fluorescence (competitive displacement) 36 0.147 - 201

Enzymatic activity 57 - - 206

Enzymatic activity 135 6 10 1 82 6 This work

Table 2.2

75

Table 2.2. Kinetic Parameters of Popular HIV- 1 Protease Substrates and Substrate 1

kcat/Km

Substrate kcat (S-1) Km (pM) (pM'-s-') If/io (pM-'s)-1

DABCYL-SQNYPIVQ-EDANSb 4.9 0.2 103 8 0.048 0.004 40 2

Abz-TINleF(p-NO2)QR 8.2 0.4c 13 1 0.63 0.06 6d 4

Substrate iY 7.4 0.2 14.7 1.0 0.50 0.04 104 52

aSensitivity: S = (kcat/KM)(I/Io). bValues ( SD) are from ref. 48. Values ( SE) are from ref. 206.

dValue is from ref. 49. 'Values ( SE) are from this work.

Figure 2.1

-0 3 S

NH O

NHHN

0RESGI FLETSKR

N-

N"N

HIV-1 protease,

-03 S

NH

HN0

RESGIF

N-

NN

+ ONH

LETSKR

Substrate 1

76

Figure 2.1. Structure of Substrate 1. Substrate 1 includes EDANS and DABCYL on opposite sides

of the scissile bond. Hydrolysis catalyzed by HIV-1 protease relieves quenching by the DABCYL

moiety, enabling quantitation of the product (which contains the EDANS moiety) with

fluorescence spectroscopy.

77

Figure 2.240-

A20-

10-

200- 0 200

[HIV-1 Protease] (pM)

IL100-

0-00 2 4 6

[HIV-1 Protease] (nM)B 50000 -

40 pM

30 pM

20 pM30000 - 15 pM

10 pM2of7.5 pM

5.0 pM10000 ........ 2.5 pM

1.o pM0

0 60 120 180 240 300Time (s)

C

1.0-

Cl)

0.5 -

0.00 10 20 30 40

[Substrate 1] (pM)

78

Figure 2.2. Catalysis of the Hydrolysis of Substrate 1 by HIV-1 Protease. Initial velocities were

measured in 50 mM sodium acetate buffer, pH 5.0, containing NaCl (0.10 M), DMF (2% v/v),

TFA (0.002% v/v), substrate 1, and HIV-1 protease. (A) Plot of initial velocities at 10 pM substrate

1 and 130 pM-6.5 nM HIV-1 protease (3 replicates with a gain setting of 180). Inset: Plot of initial

velocities at 10 pM substrate 1 and 25 pM-250 pM HIV-1 protease (4 replicates). Data were fitted

by linear regression. (B) Progress curves at 1-40 pM substrate 1 and 214 pM HIV-1 protease (4

replicates). Data were fitted by linear regression to give initial velocities. (C) Plot of initial

velocities from panel B. Data were fitted by non-linear regression to the Michaelis-Menten

equation (eq 1) to derive the kinetic parameters listed in Table 2.2.

79

Figure 2.3

0 pM

15000 100 pM

250 pM

10000- 500 pM

1000 PM1500 PM

50001 2000 pM

0 120 240 360 480 600Time (s)

0 pM

20 pM40 pM

70 pM90 pM 100 pM

110 pM120 pM150 PM200 pM

40000 -

30000-

20000-

1200Time (s)

1800600

30000 -

20000-

10000-

0 pM50 pM

100 pM200pM300 pM

500 pM750 pM

1000 pM2000 pM

480120 240 360Time (s)

1.04

I /V0

0.5-

0.0-0

1.0-

0.5-

0.0-

1.0-4

V/V0

0.5 -

0.0 -

500 1000 1500[amprenavir] (pM)

2000

S

) 50 100 150[darunavir] (pM)

2200

IS

S

0 500 1000 1500 2000[tipranavir] (pM)

80

A

U-

B

DLL

C

LL

Figure 2.3. Inhibition of HIV- 1 Protease by Amprenavir, Darunavir and Tipranavir. Plots showing

the inhibition of HIV-1 protease in 50 mM sodium acetate buffer, pH 5.0, containing NaCl (0.10

M), DMF (2 % v/v), TFA (0.002% v/v), and substrate 1 (10 tM) by (A) amprenavir at 120 pM

enzyme (R2 = 0.99), (B) darunavir at 100 pM enzyme (R 2 = 0.96), and (C) tipranavir at 200 pM

enzyme (R 2 = 0.98) (4 replicates). Data were fitted by non-linear regression to Morrison's equation

(eq 2) to derive the values of Ki listed in Table 2.1.

81

Chapter 3

Substrate Selected by Phage Display Exhibits

Enhanced Side-chain Hydrogen Bonding with HIV-1

Protease

Contribution:

I performed all experiments and wrote the initial manuscript. I contributed to structural analysis

and revision of the final manuscript.

This chapter has been published in part, under the same title. Reference:

Windsor, I. W.; Raines, R. T., A substrate selected by phage display exhibits enhanced side-chain

hydrogen bonding to HIV-1 protease. Acta Crystallogr. 2018, D74, 690-694.

82

Abstract

Crystal structures of inactive variants of HIV- 1 protease bound to peptides have revealed how the

enzyme recognizes its endogenous substrates. The best of known substrates is, however, a

nonnatural one that was identified by directed evolution. We report the crystal structure of the

complex between that substrate and the D25N variant of the protease at a resolution of 1.1 A. The

structure has several unprecedented features, especially the formation of additional hydrogen

bonds between the enzyme and the substrate. This work expands the understanding of molecular

recognition by HIV- 1 protease and informs the design of new substrates and inhibitors.

83

3.1. Introduction

Elaboration of how HIV- 1 protease recognizes its endogenous substrates has been a triumph of

structural biology. 65',218-220 The homodimeric protease is known to bind peptidic substrates between

its core and flaps through formation of a mixed P-sheet-like motif. These conserved interactions

with the main chain diminish reliance on substrate side chains for recognition. The side chains of

bound substrates are buried in subsites (Figure 3.1A) through hydrophobic and non-conserved

hydrogen bonding interactions. Accordingly, HIV-1 protease substrates lack a rigid consensus

sequence (Table 3.1). This variability could provide spatial and temporal regulation of proteolytic

processing.221

Endogenous substrates exhibit modest affinity for of HIV- 1 protease, having values of KM

in the millimolar to high micromolar range. Despite extensive efforts, few good substrates for HIV-

1 protease have emerged from rational design.222 In contrast, directed evolution has generated

excellent substrates. 70, 207 In previous work, we employed a substrate for HIV-1 protease with a

low micromolar KM value, SGIFLETS, as the basis for a hypersensitive assay of catalytic

activity. 223 Here, we report on the high-resolution X-ray crystal structure of the complex of that

substrate with an inactivated protease variant.

3.2 Results and Discussion

3.2.1 SGIFLETS Binds in Alternative Orientations

Unlike analogous complexes, the substrate in the D25N HIV- 1 protease-SGIFLETS complex lies

in two antiparallel orientations (Figures 3.2a and 3.2b). These orientations are not of equal

occupancy (0.6 and 0.4 for A and B, respectively). Chemical symmetry (Table 3.1) of the residues

84

i" the P3 through P+' pksii n aa n rsiu at hrth the PA and the P4' 'positions areillI LJLL% I. .J tLJ LXJL1 . X..J yF.J Lamla~& "L1%_& ". a3,1LL I L ' L X'L LJLA'% X. T ".LII'.4 LIIJ% _F 1-"-'XI 1,%

characteristics of SGIFLETS that could have led to this redundancy. Moreover, the protease flaps

in the complex with SGIFLETS are in a previously unreported conformation in which a bridging

water molecule (wat254) accepts hydrogen bonds from the main-chain amides of both Ile50 and

Gly5 1, which are residues in the flaps. In other HIV- 1 protease structures, an intersubunit hydrogen

bond forms between main-chain atoms of Ile50 and Gly51. Despite unique interactions with its

side chains, SGIFLETS is recognized by the protease through conserved interactions (Figure

3.2C).

3.2.2 SerlA and Gly2A at the P4 and P3 Positions Occupy Alternative Conformations

Elder and coworkers used directed evolution (i.e., phage display) with the intent of diversifying

the P3 to P3' residues. 207 Instead, they found that the residues of SGIFLETS varied in the P2 to

P4' positions. Elder and coworkers postulated that a marked preference for serine in the P4 position

led to a high frequency of serine and glycine at the P4 and P3 positions in the selected substrates.

Yet, both of these residues occupy alternative conformations in the major substrate orientation

(conformation A) of the protease SGIFLETS structure.

Few endogenous substrates exhibit alternative binding modes for P3 and P4 residues. 65

Unlike the conserved recognition strategy wherein the side-chain of Asp29 accepts a hydrogen

bond from the P3 main-chain N-H and the N-H of Gly48 donates a hydrogen bond to the P4 main-

chain carbonyl oxygen (Figure 3A), the P3/P4 amide of the pl/p6 substrate interacts with the

carbonyl oxygen of Gly48 and the side-chain N12-H of Arg8 (Figure 3.3B). The

protease-SGIFLETS complex employs both recognition strategies (conformations A/B and C).

85

Though found in opposite orientations relative to the protease, conformations A and B of Serl and

Gly2 share the conserved P-sheet mode of main-chain recognition with the side-chain hydroxyl

group of SerlA/B forming a unique hydrogen bond with Lys45 in the protease flap (Figure 3.3C).

The alternative conformer (conformation C) of the major orientation is reminiscent of the

alternative recognition mode observed in the p l/p6 complex with SeriC instead forming a unique

hydrogen bond with the carbonyl oxygen of Gly49 (Figure 3.3D). Alternative recognition of the

P4 main-chain carbonyl group in the p I/p6 and selected substrates occur through both direct and

water-mediated interactions with Arg8. The unique hydrogen bonding exhibited by Serl provides

a structural explanation for the preference of serine and glycine at the P4 and P3 positions.

The tips of the protease flaps were also resolved in a previously unreported interaction

where a bridging water molecule accepts hydrogen bonds from the main-chain N-H of both Ile50

and Gly51 (Figure 3.3D). The occupancy of this novel water-bridge correlates with the previously

unreported hydrogen bond between the side-chain of SerC and the carbonyl oxygen of Gly49B.

Rotation of Gly49B to accept the hydrogen bond appears to move the tip of the flap into a

conformation that is incompatible with the inter-flap hydrogen bond, thus enabling water-bridge

formation.

3.2.3 Glu6 and Ser8 Form a Network of Hydrogen Bonds

Weber and coworkers identified hydrogen bonds between the side-chain carboxyl group of a

glutamic acid residue at position P2' of the CA/p2 substrate and the side chain of Asp30 of the

protease. 77 This interaction is also apparent in the protease-SGIFLETS complex (Figure 3.4A).

Given the pH of 5 at which these crystals were grown, a plausible explanation for the 2.7 A

86

interatomic distance between of Glu6 (P2') and 02 of Asp3(chain A) is fnrmat;in F n

intraresidue hydrogen bond (Figure 3.4B). Such a hydrogen bond is consistent with the substantial

increases in the Michaelis constant (KM) of the peptide substrate and the inhibition constant (Ki)

of an analogous inhibitor upon increasing the pH from 5.6 to 6.7.207 The different interatomic

distances (2.7 A and 3.3 A) between 081 and 0,2 of Glu6 and 062 of Asp30 suggest a single

hydrogen bond with the proximal oxygen and not a bifurcated hydrogen bond.224

Serine residues at P4/P4' also form hydrogen bonds with the carboxyl group of Asp30

(Figure 3.4A). Few polar interactions have been revealed between Asp30 and residues in the P4/P4'

positions, including arginine and serine. Neither of these interactions occur alongside a P2/P2'

interacting side chain (Figure 3.4B). In the P-strand conformation of bound substrates, the side

chains of adjacent residues (i ... i + 1) are farther from each other than are the side chains of two

residues with an intervening one (i . i + 2).225 Bulky groups can lead to a steric clash between side

chains and only spatially compatible amino acids are found at the i and i + 2 positions. The

structure of the protease -SGIFLETS complex reveals the interdependence of P2' and P4' residues

where, in addition to sterics, the identity of the residue is constrained by donor-acceptor

interactions.

3.2.4 Thr7 Plays a Limited Role

Endogenous protease substrates employ 2-5 polar residues in the core recognition sequence. Yet,

only some of these side chains participate in hydrogen bonds, with an average utilization of about

60%.226 In the protease SGIFLETS complex, three of the four polar side chains of SGIFLETS

form hydrogen bonds. The exception is Thr7. The protease buries little of the P3/P3' side chain,

87

leaving residues in this position largely exposed to solvent. Although threonine is a residue in the

P3' position of HIV- 1 protease substrates identified by phage display,207 that position seems to

have a limited role in substrate specificity 69 and could be a site for further optimization.

3.3 Materials and Methods

3.3.1. Protein

The expression plasmid for D25N HIV-1 protease was prepared as described previously 223 with

modifications. An initiating methionine codon was placed directly before the native N-terminal

proline residue, and an AAC codon was used for residue 25. D25N HIV protease was produced

heterologously in Escherichia coli cells grown in Luria-Bertani medium. Expression was induced

when the OD reached 1.5 at 600 nm, and cells were grown for an additional 4 h. Cell pellets were

suspended in 20 mM Tris-HCl buffer, pH 7.4, containing EDTA (1 mM), lysed with a cell

disrupter from Constant Systems, and collected by centrifugation at 10,500g for 30 min. The cell-

pellet was dissolved in 20 mM Tris-HCl buffer, pH 8.0, containing urea (9 M), and this solution

was clarified by centrifugation at 30,000g for 1 h. The supernatant was flowed through a 0.2-tm

filter and a Hitrap Q column from GE Healthcare, which removes anionic contaminants 227 . To fold

the protease, the resulting solution was diluted 20-fold by dropwise addition into 50 mM sodium

acetate buffer, pH 5.0 containing NaCl (100 mM), ethylene glycol (5% v/v), and glycerol

(10% v/v). The solution of folded protease was concentrated by using a stirred cell concentrator

from Amicon and applied to a G75 gel-filtration chromatography column (GE Healthcare) that

had been equilibrated with the folding buffer. The protease, which eluted near 0.5 column-

volumes, was concentrated to 10 mg/mL. The purity of the ensuing protein was verified by SDS-

PAGE.

88

3.3.2. Peptide

The SGIFLETS peptide with free N and C termini was synthesized and purified to be >99% pure

by Biomatik (Wilmington, DE). Stock solutions in DMSO containing TFA (0.1% v/v) were

prepared at a concentration of 1 mM for crystallization.

3.3.3. Crystallization

Protease and peptide stock solutions were mixed at a volume ratio of 4:1, respectively. Crystals

were grown by vapor diffusion in 2-jiL drops hanging over a mother liquor of 100 mM sodium

acetate buffer, pH 5.0, containing NaCl (1.0 M). Crystals, which grew within 24 h, were

cryoprotected in mother liquor containing glycerol (10% v/v) by flash-freezing with N2(l).

3.3.4. Data Collection and Processing

Single-crystal diffraction data were collected at the Advanced Photo Source at Argonne National

Laboratory in Sector 21 (LS-CAT) at Station G. Data were indexed, integrated, and scaled using

HKL-2000 (HKL Research). Details regarding diffraction and data reduction can be found in Table

3.2.

3.3.5 Structure Solution and Refinement

Molecular replacement was conducted with Phaser implemented in Phenix228 using PDB entry

lkjf as a starting model. Model building was conducted with COOT2 29 . Refinement with Phenix

following initial substrate placement revealed an additional anti-parallel orientation of the

89

substrate. Subsequent refinement estimated occupancies of approximately 0.6 (conformation A)

and 0.4 (conformation B) for the major and minor orientations and revealed other alternative

conformations for residues Serl and Gly2 in conformation A (Figures 3.2a and 3.2b). Because of

the complexity of constraining alternative conformations of some residues simultaneously with

other residues that occupy subsites fully (i.e., 1.0), occupancies were set manually. The residues

in conformation A with alternative conformations were assigned occupancies of 0.4 (conformation

C) with the original conformer retaining 0.2 of the total occupancy (0.6) of conformation A. Details

regarding refinement and the statistics of the final model are listed in Table 3.2.

90

3.4 Conclusion

The endogenous substrates of HIV-1 protease represent but a small subset of sequences that can

be cleaved by the enzyme. Among the best of known substrates, SGIFLETS, was derived by phage

display. Its structure bound to the protease reveals the formation of many hydrogen bonds with its

glutamic acid and serine side chains. Thus, hydrogen-bond formation could serve as the basis for

the design of optimal substrates and, perhaps, inhibitors of HIV-1 protease.

3.5 Acknowledgements

I.W.W. was supported by Biotechnology Training Grant T32 GM008349 (NIH) and a Genentech

Predoctoral Fellowship. This work was supported by Grant ROl GM044783 (NIH).

91

Table 3.1

Table 3.1. Endogenous and Optimized HIV-1 Protease Substrate Sequences

Substratea P4 P3 P2 P1 P1' P2' P3' P4'

MA/CA S Q N Y P I V Q

CA/p2 A R V L A E A M

p2/NC T A I M M Q K G

NC/pt R Q A N F L G K

P1/p6gag P G N F L Q S R

NC/TFP R Q A N F L R E

TFP/p6pol N L A F Q Q G E

p6pol/PR S F S F P Q I T

PR/RTp51 T L N F P I S P

RT/RTp66 A E T F Y V D G

RTp66/INT R K V L F L D G

Nef D C A W L E A Q

Phage displayb S G I F L E T S

aRef 230bResidues with a grey background areby phage display.

shared with the substrate identified

92

Table 3.2

Table 3.2. Crystallographic Data Collection and Refinement Statistics

PDB Code 6bra

Data CollectionX-Ray SourceDetectorWavelength, AResolution, A (last shell)Space groupa,b,c in Aa,p8, y in 0

No. of ReflectionsNo. of Unique Reflections (last shell)Redundancy (last shell)Mean I/- (last shell)Completeness (last shell)Rmeas (last shell)Wilson B-factor

RefinementWorking Set (last shell)Test Set (last shell)Rwork (last shell)Rfree (last shellRMS deviation bond lengths, in ARMS deviation bond angles, in

Total Number of AtomsProtein ResiduesProteinLigandWater

Avg. B-factorProteinLigandWater

Ramachandran Favored, Allowed, Outliers, in %(MolProbity)

LS-CAT 21-ID-GMAR 300 CCD0.9785726.0-1.11 (1.15-1.11)P212i258.033, 85.767, 46.1390,90,9061299690088 (8193)6.8 (3.9)33.6 (1.5)98.63 (91.12)0.065 (0.691)10.99

90066 (8181)1998 (181)0.1708 (0.2536)0.1840 (0.2732)0.0040.78

210120618224275

15.2513.4619.5827.04

99, 1, 0

93

Figure 3.1

Flaps

(a)

P2

P3

PI, PT,

0%P4'

Q

(b)

94

P4

P5

Figure 3.1. Structure of the D25N HIV-1 Protease CA/p2 Complex. Protease residues from chain

A are shown in white, chain B in grey, and substrate CA/p2 in ball-and-stick representation (PDB

entry 1f7a). (a) Substrate CA/p2 binds in the active site of the protease (white and grey) in an

extended conformation between the two flaps and core domain. (b) Substrate side chains are

numbered relative to the scissile bond.

95

Figure 3.2

lle3A/Glu6B Leu5A/Phe4B

Ser1N/Ser8B * "Thr7A

Gly2NCGly2B

Thr7B

Ser8AISerlBSeriC Phe4AILeu5B Ile3AIGlu6B

(a)

Ile3AJGlu6B Leu5AIPhe4B

Ser1N/Ser8B Thr7A

Gly2 IAC Gly2B

Thr7B

Ser8A/SerlBSeriC Phe4AILeu5B lle3A/Glu6B

(b)

Ile50 11950Gly48

Gy4 Ile4711047 X

Gly49 GSer8

i Gly2 4 l3 Wat102 Glu6

'A& Th7

SPhe4 WtSWat242 Wat259

Ala28 4An5 Gy7Asp29Aw6 Gly27

As29 AAla28

Wat234 Gy27 Wat240

Arg87hr26

Arg87 T s2

(C)

96

Figure 3.2. Electron Density and Interactions of SGIFLETS Bound in the Active Site of D25N

HIV-1 Protease. Protease residues from chain A are labeled in white, chain B in grey, and

SGIFLETS in black. Maps of 2Fo - F, (contoured at 1 a) (a) and Fo - Fc after simulated-annealing

refinement with the substrate excised (contoured at 3a) (b) are depicted as a mesh around the

substrate in the final structure. (c) Conformation A of SGIFLETS showing hydrogen bonds with

HIV-1 protease residues (yellow, direct hydrogen bonds; magenta, water-mediated hydrogen

bonds).

97

Gly51

Gly51

li11950110

Gly48 Gy4O

of 2 3

'AI I

Ala2 Ap29(P4)

(a)

Gly51

IleSO Gy1

G"y48

Pro2

G y3(P3) if)

WatS37

Arg8

(b)

Wft2S4A

GGySlA yA

Gb,4A

G y2A

SerlA

(N)4

(P3)

*00

(c)

X GlyGl1B

Ile50BXw,

IleSOBC GlyOBWat254B

G y40B

Serl C(PN)

Gly2C(P3) ?'%I

Wat1O9

Arg8

(d)

98

Figure 3.3

Figure 3.3. Alternative Conformations of P3 and P4 Residues. Protease residues from chain A are

shown in white, chain B in grey, and substrates in black. (a) CA/p2 complex (PDB 1 f7a). Ala2

(P4) and Arg3 (P3) form P-sheet-like interchain hydrogen bonds with Asp29 and Gly48. (b) p l/p6

complex (PDB lkjf). Gly48 and Arg8 alternatively recognize the P3/P4 amide. (c) Alternative

orientations A and B of SGIFLETS exhibit the conserved P-sheet conformation and a unique

hydrogen bond between the side-chains of SerlA/B (P4) and Lys45. (d) Alternative conformation

C is similar to that in panel b and has a unique hydrogen bond between the side-chain hydroxyl

group of SerC (P4) and the main-chain oxygen of Gly49.

99

Thr7B(P3')

(PT) ( )r8B(P4')

(a)

6n AS(P3)

(P4)

(b)

100

Figure 3.4

Figure 3.4. Role of Asp30. (a) Network of hydrogen bonds formed by Glu6 (P2') and Ser8 (P4')

of SGIFLETS. Protease residues from chain A are shown in white and SGIFLETS in orientation

B in black. (b) Analogous hydrogen bonds formed by Ser2 (P4) and Asn4 (P2) of the MA/CA

substrate (Ikj4), though these residues interact with each other and only Ser2 interacts with Asp30.

101

Chapter 4

An n-+r* Interaction in the Bound Substrate of

Aspartic Proteases Replicates the Oxyanion Hole

Contribution:

I performed all bioinformatics analysis and computational experiments, and prepared the initial

manuscript. I contributed to the design of computational experiments and revision of the final

manuscript.

This chapter has been published in part, under the same title. Reference:

Windsor, I. W.; Gold, B.; Raines, R. T., An n-+* interaction in the bound substrate of aspartic

proteases replicates the oxyanion hole. ACS Catal. 2018, In press.

102

Abstract

Aspartic proteases regulate many biological processes and are prominent targets for therapeutic

intervention. Structural studies have captured intermediates along the reaction pathway, including

the Michaelis complex and tetrahedral intermediate. Using a Ramachandran analysis of these

structures, we discovered that residues occupying the P1 and P ' positions (which flank the scissile

peptide bond) adopt the dihedral angle of an inverse y-tum and polyproline type-IL helix,

respectively. Computational analyses reveal that the polyproline type-I helix engenders an n--z*

interaction in which the oxygen of the scissile peptide bond is the donor. This interaction stabilizes

the negative charge that develops in the tetrahedral intermediate, much like the oxyanion hole of

serine proteases. The inverse y-turn serves to twist the scissile peptide bond, vacating the carbonyl

7r* orbital and facilitating its hydration. These previously unappreciated interactions entail a form

of substrate-assisted catalysis and offer opportunities for drug design.

inverse

N NI'-WH dinteraction interaction

H

103

4.1 Introduction

Aspartic proteases (EC 3.4.23) have played a central role in the history of protein science. The

human digestive enzyme pepsin was the subject of seminal studies in mechanistic enzymology

and protein crystallography.5 6,23 ' Fueled by aspartic proteases being therapeutic targets,

endothiapepsin and HIV- 1 protease emerged as model systems and together account for over

1,000 entries in the Protein Data Bank. In accord with a common reaction mechanism, aspartic

proteases share susceptibility to inhibition by the peptidic natural product pepstatin. Its

hydroxyethylene pharmacophore, which mimics an intermediate in the enzyme-catalyzed

reaction, forged the route to clinical inhibitors of aspartic proteases. 90, 195

Aspartic proteases employ a pair of aspartic acid residues to activate a water molecule for

nucleophilic attack on a peptide bond. This catalytic dyad arises from a pair of "DTG" motifs at

the interface between two globular domains (Figure 4. 1A).232 Eukaryotic aspartic proteases are

monomeric and consist of two unique domains,52 whereas retroviral homologs are homodimeric

(Figures 4.1B and 4.1 C). 233 -234 In addition to the DTG motifs, aspartic proteases share other

structural elements, including a p-barrel domain and flap that close upon polypeptide substrates.

The enzymes vary, however, in their substrate promiscuity and biological niche. For example,

renin cleaves angiotensinogen with exquisite specificity to elicit vasoconstriction, 23 5-2 36 whereas

HIV- 1 protease recognizes a variety of substrates to enable maturation of new virions.237

The catalytic mechanism of retroviral proteases has been informed by the use of substrate

mimetics8 1 and, especially, inactive variants. In seminal work, Wlodawer and coworkers employed

site-directed mutagenesis to inactivate FIV protease and then determined the structure of the

inactive variant bound to an actual substrate. 238 Schiffer and coworkers extended this strategy to

104

HJV-1 protease while developing their shape-complementary model of side-chain reognition.65'

82

Typically, the main-chain conformation of the substrates of aspartic proteases is thought to

resemble that of a P-strand.239 We have examined the structures of inactivated protease substrate

complexes, focusing on the main chain. We have discovered that the substrates do not resemble a

P-strand near the scissile peptide bond. Instead, the substrate adopts a conformation in which the

oxygen of the scissile peptide bond donates an n--+ir* interaction to the next carbonyl group in the

main chain. The discovery of this interaction, as well as the formation of an inverse y-turn within

the substrate, suggests that aspartic proteases rely on substrate-assisted catalysis 240 to effect the

cleavage of peptide bonds.

105

4.2 Results and Discussion

We began our investigation of the role of main-chain conformation in the mechanism of aspartic

proteases by gathering structures of substrates bound to inactivated enzymes. 24 1 Since 1997, 35

structures of inactivated retroviral proteases with flaps closed on peptidic substrates have been

deposited in the PDB (Table 4.S 1).65, 82, 218, 222, 238, 242-247 Next, we measured the # and qL angles of

residues in the P1 and P1' position to generate a Ramachandran plot (Figure 4.2). The peptide bond

between these two residues is the one cleaved by the enzymes. We found distinct clusters of P1

and Pl' residues, with mean values of #= -95.5' 10.10 and / = 44.9' 10.4' for P1 residues,

and # = -56.3' 14.5' and v = 140.0' 7.5' for Pl'residues.

4.2.1 Conformation of the P1 Residue

The P1 residue of bound substrates adopt conformations in the broad region of the Ramachandran

plot associated with f-strands. An analysis by Rose and coworkers identified a subset of this region

with # angles between -100' to -70' and V angles between 50' to 100' populated by members of

a coil-library that contain an inverse y-turn, called the "y-basin" (Figure 4.3A).248 Its signature

inverse y-tum motif was described nearly a half-century ago 249 and is characterized by a hydrogen

bond between the i - 1 main-chain oxygen and i + 1 main-chain nitrogen, forming a 7-membered

ring.250 The donor-acceptor angle deviates from linearity, making the hydrogen-bond energies of

inverse y-turns weaker than those of other secondary structural elements, such as P-turns and p-

sheets. The inverse y-turn centered on the P1 residue results from the P2 (i - 1) residue accepting

a hydrogen bond from the P1' (i + 1) residue (Figure 4.3B). 1-Branched amino acids are excluded

from inverse 7-turns, as their side-chain would clash with the main-chain nitrogen of residue i.248

106

Notably, the incorporation of fp-branched residues at the P1 position yields inefficient substrates

for HIV- 1 protease, consistent with the importance of an inverse y-turn conformation for

catalysis.2 51

An inverse y-turn is incompatible with the /4 of endogenous HIV protease substrates that

contain a P1' proline residue, which lacks the requisite main-chain N-H.2 0 Segregating the P1

Ramachandran plot based on sequence revealed to us that substrates with a P1' proline residue

have expanded # angles that move the conformation of the P1 residue out of the y-basin (Figure

4.3A). These structures have intramolecular hydrogen bonds between the side chains of the P2 and

P4 residues (Figure 4.3C). Their proline-containing substrates all have P2 and P4 residues with

polar groups, which could serve to increase the q angle and move the P2 carbonyl group away

from the P1' pyrrolidine ring. An asparagine residue is found most commonly at the P2 position

of substrates with a P1' proline residue, but is also found at the P2 position of those substrates

known as "p l/p6". In p I/p6 substrates, the side-chain oxygen of the asparagine residue, rather than

its main-chain oxygen, accepts a hydrogen bond from the i + 1 main-chain nitrogen (Figure

4.3D).242 In addition to an increased # angle, the P1 residue of pI/p6 substrates exhibits a reduced

y angle, which serves to rotate the main-chain N-H towards the side-chain of the asparagine

residue in the P2 position (Figure 4.3A).

The inverse y-turn in the main-chain of P1 residues has not gone unnoticed. Medicinal

chemists, guided by knowledge that proline is often found in inverse y-turns, discovered that

replacing the main-chain of substrates at the P2 to P1' positions with a seven-membered ring can

generate potent inhibitors of HIV protease.2 5 2 Although this strategy was intended to mimic the

107

inverse y-turn, structural analyses revealed that the ring shifted by a "half' residue relative to that

in substrates. 2 -2

4.2.2 Conformation of the Pl' Residue

The main chain of the P1' residue occupies a region of the Ramachandran plot associated with the

polyproline type-II (PPII) helix (Figure 4.4A) and exhibits structural features conducive for

preorganization (Figure 4.4B, Table 4.S1). Comparing the main-chain dihedral angles of Pl'

residues (Figure 4.4C) with the structure of a PPII helix solved by Wennemers and coworkers

using direct methods reveals a striking similarity (Figure 4.4D).25 The lack of hydrogen bond-

donating groups in the main-chain of polyproline entices donation of oxygen electron density to

an alternative acceptor-the r* orbital of the i + 1 carbonyl group.25 1-25 1 (For reviews of the n-**

interaction, see refs. 258-259.) In a-helices, donor carbonyl groups employ n-+w* interactions with

the si face of the adjacent carbonyl group, whereas in PPII helices the interaction is with the re

face. Previous systematic, energetic analysis by our group estimated that a residue occupying the

dihedral angle near the average for residues in the Pl' position ( = -55' and V = 140.0') has an

n-T* interaction with an energy of 1.0 kcal/mol.260

The PPII-helical conformation is not limited to proline-rich sequences. Unfolded protein

sequences likewise occupy this conformation. 2 61 Protease substrates must be sufficiently

disordered to avoid adopting a secondary structural element like an a-helix or P-sheet, which

would preclude binding, but must adopt a conformation complementary to the enzymic active

site. 2 62 An analysis by Brown and Zondlo identified proline, leucine, and alanine as the residues

with the greatest propensity to form PPII helices when flanked by pairs of proline residues. 263

108

These three amino acids constitute half of the Pl' residues in endogenous substrates. Thir analysis

also identified P-branched and small polar residues as being disfavored in a PPII helix, and those

residues lead to inefficient substrates of HIV- 1 protease when in the P1' position.66

4.2.3 Mechanistic Insights

Informed by our bioinformatics analysis, we sought insight on the catalytic mechanism of aspartic

proteases. The catalytic mechanism of amide hydrolysis proceeds first via nucleophilic attack of

water at the P1 carbonyl group to generate a tetrahedral intermediate that is a geminal diol (Figure

4.5). Proteolytic cleavage is completed upon protonation of the P1' nitrogen and subsequent C-N

bond scission. To investigate this mechanism, we performed quantum chemical calculations on

coordinates extracted from high-resolution co-crystal structures of a substrate and a gem-diol

intermediate bound to HIV- 1 protease. To represent the Michaelis complex, we chose a recent co-

crystal structure of a highly efficient substrate bound to an inactivated protease (PDB entry

6bra2 64), which has the highest resolution of any such structure. To represent the gem-diol

intermediate, we chose a structure by Weber and coworkers that employed threonine as the Pl'

residue (PDB entry 3b80 66).

When extracting coordinates, we sought to preserve noncovalent interactions between the

P2/P 1 and the P1'/P2' carbonyl groups near the scissile peptide bond. To do so, we extracted the

coordinates of the P1 and Pl' residues to the P-carbon of their side chains, the distal amides and

C' atoms from the P2 and P2' residues along with a conserved water molecule, the main-chain

amides of the protease flap that form hydrogen bonds with the conserved water molecule, and the

aspartic acid or asparagine side chains of protease residues 25 and 25'. Next, we optimized the

109

positions of hydrogen atoms with density functional theory (DFT) calculations. Hydrogen atoms

were added to the structure to create a neutral structure of the Michaelis complex and a

monoanionic structure of the tetrahedral intermediate. There was no ambiguity in assigning

hydrogen atoms in the substrate structures. The protonation of the gem-diol intermediate was

modeled to match neutron diffraction structures in which both hydroxy groups form hydrogen

bonds with an aspartate residue,265 as is proposed for the tetrahedral intermediate. Finally, we

performed Natural Bonding Orbital (NBO) analysis of the ensuing models to estimate the strength

of noncovalent interactions. 266-267

In the Michaelis complex, the inverse y-turn-PPII-like motif activates the n* orbital of the

P1 carbonyl group for nucleophilic attack by water. The two flanking carbonyl groups (i.e., those

of the P2 and P1' residues) form hydrogen bonds, either with a conserved water molecule in

retroviral proteases or with the flap of monomeric proteases. The ensuing constraint increases the

electrophilicity of the P1 carbonyl group by both enhancing the n-+* interaction with the Pl'

carbonyl group and preventing n->*7r donation from the P2 carbonyl group, which would

otherwise raise the energy of the ;r* orbital of the P1 carbonyl group and diminish its

electrophilicity. 268

As the reaction proceeds to the tetrahedral intermediate, the n--+x* interaction grows

stronger, increasing from 0.66 to 2.01 kcal/mol. This latter energy, which is remarkably high,

serves to delocalize developing negative charge (Figure 4.6A and 6B). Thus, the n-+-** interaction

between the P1 and P1' carbonyl groups effectively acts like the renowned oxyanion hole of serine

proteases, which delocalizes developing negative charge through the formation of a pair of

hydrogen bonds with enzymic N-H groups. 269 -273 As has been postulated for hydrogen-bonding

110

1 ih the xninbn hr1l,273-274 fh - tit-rcticon is ctrnnar in the te-tr1hedral intermediate thanoxyLI an1on kiJ"A%"L 1J% Lll% It IILI iJiL%1LL%'L1J1Ai 1k.1 AX J A-

in the Michaelis complex. This differential binding of the Michaelis complex and tetrahedral

intermediate is likely to enhance catalysis.2 75 2 8 0

Like the n--7c* interaction, the inverse y-turn is important in the enzymatic reaction

mechanism. In the first step, amidic resonance must be overcome. At first glance, a hydrogen bond

to the main-chain nitrogen of the P1' residue might be thought to increase amidic resonance and

thereby decrease the electrophilicity of the scissile peptide bond. Upon forming the inverse y-tum,

however, the scissile peptide bond is twisted out of plane, decreasing amidic resonance and

increasing electrophilicity. 281 -28 s In the Michaelis complex, the amide nitrogen is pyramidalized

slightly and the lone pair hybridizes to a small degree (~3% s-character, versus <1% s-character

in adjacent amides) because of the inverse y-turn hydrogen bond, which has an energy of a 0.66

kcal/mol (Figure 4.6C). This interaction is absent in substrates that have proline in the P1' position,

and these substrates are cleaved only slowly by the protease. 286 Similarly, the asparagine side chain

in the P2 position of the p I/p6 substrate competes for the y-turn, resulting in efficient cleavage. 79

In the tetrahedral intermediate, the energy of the inverse y-tum hydrogen bond is

maintained at 0.67 kcal/mol (Figure 4.6D). Although the scissile peptide bond is now hydrated, its

nitrogen lone pair still contains only ~7% s-character, which is much less than the 25% s-character

of an sp3-hybridized orbital. We find that the low hybridization is due to the presence of the

neighboring gem-diol, in which a higher energy p-rich lone pair of the nitrogen is a better donor

of electron density to the two o*c-o orbitals. Nonetheless, the inverse y-tum also aligns the nitrogen

lone pair towards the protonated oxygen of Asp25, resulting in a hydrogen bond with an energy of

0.79 kcal/mol (Figure 4.6E). This aspartic acid residue also maintains a hydrogen bond of energy

111

5.36 kcal/mol with the gem-diol (Figure 4.6F). As the proton transfer to the N-H proceeds, the

energies of the two nN-*cy*C-O interactions likely decrease and the energy of the no--+U*C-N

interaction likely increases, promoting C-N bond scission to form the products.

Computational chemists have devoted much attention to the catalytic mechanism of

aspartic acid proteases, especially that of HIV-1 protease. 287 -290 These studies have tended to

employ a hybrid quantum mechanical/molecular mechanical (QM/MM) approach in which the

higher level of theory is used to describe molecular fragments that are smaller than those analyzed

herein. 7 2 , 291-292 Our findings suggest that previous analyses have missed critical details: the n-*

interaction and inverse y-tum. New computational strategies are needed to select fragments that

capture all of the interactions that make significant contributions to catalysis by aspartic acid

proteases and other enzymes. 293

4.3 Materials and Methods

4.3.1 Structural Analyses

The atomic coordinates of X-ray crystal structures that were deposited in the Protein Data Bank as

of August 9, 2018 and that contain an inactivated retroviral aspartic proteases were downloaded.

The structure SOWJUL25 5 was downloaded from the Cambridge Structural Database. Angles and

distances in these structures were measured to the nearest 0.1 A and 0.1 0, respectively, with the

program PyMOL from Schr6dinger (New York, NY). Structures containing multiple protease

dimers in the asymmetric unit or alternative conformations within a single protease molecule were

measured individually and given equal weight in calculations of the mean. PDB codes and chain

112

idntfiers are listed, An Qal .1 alo ' N + ith the mesred AvA I1- L-s Sftuctures we~re de-picte-d wtMI~L1LLL11..'1_a "4L.v 1L L 11 I. .L L I~ "]ionrr uft. IIIIItL11'lA.L V "L4%L'.3.. "JLI LL%'LL4I% .) VV

the program PyMOL.

4.3.2 DFT Optimization

Atoms important for determining the conformation of the scissile peptide bond in the Michaelis

complex and gem-diol intermediate were extracted from PDB entries 6bra264 and 3b80,66

respectively. All termini were made into amides. Hydrogen atoms were added to extracted

structures in idealized geometries with the program GaussView 6 from Gaussian (Wallingford,

CT) and optimized at the M06-2X/6-311 +G(d,p) level of theory along with the integral equation

formalism variant of the polarizable continuum model (IEFPCM) model for water-solvation 294 by

using Gaussian 16, Revision A.03 software from Gaussian.29 s Optimized structure coordinates are

listed in Tables S3 and S4.

4.3.3 NBO Analysis

Optimized structures were subjected to Natural Bonding Orbital analysis using the NBO 6.0

software from the Theoretical Chemistry Institute of the University of Wisconsin-Madison

(Madison, WI). 2 96 Orbital interaction energies were calculated by second-order perturbation

analysis. Orbitals were depicted with the program NBOView 1. 1.297

113

4.4 Conclusions

Ramachandran analysis of the residues in substrates bound to aspartic proteases has revealed

previously unappreciated noncovalent interactions. When bound, the substrate adopts a

conformation with an n--+7c* interaction and an inverse y-tum, both of which activate the scissile

peptide bond for nucleophilic attack by a water molecule. To our knowledge, the use of these

interactions in substrate-assisted catalysis had not been described previously. Notably, these

findings extend the reach of n--7r* interactions between adjacent main-chain carbonyl groups to

enzymatic catalysis. 298

4.5 Acknowledgements

B.G. was supported by an Arnold 0. Beckman Postdoctoral Fellowship. This work was supported

by Grant RO 1 GM044783 (NIH).

114

M1

I L6 67 ZEI 9*Et- 8*6E 97,6- A 18SOA/AN~d96 87 L*9E1 I*8t,- 6*9E 9'101- a dAHSOA/JN~d4I 09*1 9!bt

9*E6 87Z Z*6EI Wt'- t717 E7001- D daHNO-4/ANDcNI ~99 1 qoj7

CtV6 v*z 6 ! Z*1Vt- 17L 101- D dXNO'1AN~dH 9vi I qot

El16 87 9*S1 1*9t,- 8'H 9*L01- A n"sO&J/ onl -FE6 87Z '9E1 WLA'- 8*8E 1*101- 3l dNISOdANDd" 98* 1 qqot

1*001 67Z t,*M 8*tl- 91't 9'E6- A 'dsol/AN4cfI -

17,6 87 9*9E 1 E~t- 68 E 9*011- 3 d"SO1/ANDcfl 8' 5qot

9*0O1 67 91 ! 6'6E- 7 E86- A 'HxoiNOtAND--FE6 87 9*t I F*it- Ot, Z'01- 3 d)Jo'1/ANDd) 89*1 jqot'

V6 87 E71 91 t- LV9E I*E01- A NJN6A/JINDd --

VE6 87 ~99c Z*9- 6 V901- 3 JaN0d/AN~d 06*1 pqoj7

L'86 WE 8L'M E 09- LVtg &06- Ui WAV3A/IA)1 O17 JtlJ11l tl* E C'Ml 98- E'978- Ul D-owII/I'Nl OE*Z ;DJ17

L17,6 0' 9,1 1 L EL- 9*Jt7 E'gL- Ul 1UXHV/IAIUN117,6 WE 9*191 L EL- VLI7 1~L- D) IUNHV/IAUN 98*I1 AIA

V9~6 WE 6'9t1 Z*8- Z*9 96- d "1S0'71/ANtD 087 IREtZ*001 V E 9*Vl E*ZL~- 09 tM~O!- 9 ;D13ooAtw 09*1 j~xqE

9'L01 WE V9ZI 17,9- 9*Lt Vi10l- d DUIAA/JIOV gzz aixuz

670 1 WE LEI Z*99- 9*19 6- d VDUA1k/AA3V 007 Ixuz

E'101 Z' Z*fI g*E9- 6'89 866- d DU1AJAlIv ooz pxu:

9*8L 87 'L9 I 6*9t- L'8E 6*06- VIIV'd Dr1A/NAON ltJ li978 67 9*tg I 8*t79- 9*E1 t8- d DIA/NVO1 98'1 SUJZ8*6L 87 LZ9 V09- WV t E, 19- d N)IA/NqVO) ovz nsil

601 t" E 9*ti L76- L L W,8/J- d NDID-I/NAO' 007 bslIi

788 87 V6N' E"89- 91717l 676- d dHlSO1/JNDd-J 007 61w IE796 67 Lt, 6*89- 179 9,88- d WAVHV/1A)IV 98*1 81tH!IViol1 WE 9,8 t,1 9*L- L*8t' 9'LO I- d Ald/ANOSA 06*1 LilU IV96 WE ~991 L89- V6t' 976- d lIUA/l11x4J 007 4I)11

1*901 FE 9,8EI 6*tL- S*9 Z6- d VDUAA/AI3V 07 1 I9'06 87Z Vtl*9,I 8*S- t'Lt, 6*L6- d d)JSO'1/ANtDdA% 07 JfI

01 67Z L*8E1 Z89- 8Otl p*88- d D-owI'/w'Nv 00-z L[31Z'011 0W 8 1 U CL- 8717, V801- S Ald/ANOSA

9'E01 WE VLI7I 6*6L- L'8 Lt d OA~d/ANOsA 067 t[FNI

VL6 67 Lt, 6'69- U*19 Wl88- d VIV/I7A)IV-N 007 tLJI

(O)o (yp (0) 1 (O)q5 (0) 1h (O)o 1~ 3oumnbm (Y) UT

11d Id *S;D>J LGd

SOUJ13 Ud UJOJJ SJ;Dlwu~htd poJflsuoW, IS-t' alqtjL

IS1', alq'u

4ij7 1.67 PGNF/LQSS E -91.6 -43.5 138.7 2.8 96.3

- PGNF/LQSSP F -101.5 39 -41.1 130.4 2.7 97.6

4cj8 2.00 RPGNF/LQSRL E -102.2 42.2 -41.6 130.2 2.7 98.3

RPGNF/LQSRL F -105.3 36.4 -40.9 132.4 2.7 95.7

4qj9 1.83 RPGNF/LQSSP P -93.7 44.2 -45 131.2 2.8 97.1

4qja 1.54 RPGNF/LQSRL P -99.5 36.1 -40.7 135.3 2.7 95.3

6bra 1.11 SGIF/LETS S, AltA -95.4 43.2 -48.7 137.1 2.9 96.8

- SGIF/LETS S, AltB -96.3 49.1 -52.9 139.9 2.9 96.9

Mean -95.5 44.9 -56.3 140.0 2.9 96.8

SD 10.1 10.4 14.5 +7.5 0.2 7.0

116

Table 4.52

Table 4.S2. Measured Parameters from CSResidue 0 (0) y (0)

1 -67.1 146.52 -65.7 138.43 -73.1 163.94 -72.8 152.05 -72.5 165.76 -69.0 152.5

Mean SD -70.0 3.2 153.2 10.4

D Entry SOWJULd (A)

3.02.93.13.13.23.0

3.1 0.1

117

O (0)

102.9106.589.898.988.298.2

97.4 7.2

Table 4.S3

Table 4.S3. Coordinates Extracted from PDB Entry 6bra and Optimized Hydrogen Atoms

-7.24337900-5.80699300-4.80021800-5.29355300-3.59684100-6.27593900-7.93901300-5.61028800-4.68357900-7.46746800-5.59438500-7.459219004.503157005.34987000

6.512720004.342846003.535911005.054321004.779132005.560863005.955380003.936433006.399491004.91144200

-7.17082300-5.71364400-4.76266000-3.48856200-5.15569000-3.20799100-7.41367500-5.51747800-2.75724100-7.38023900-5.47457500-7.826774004.523789004.931442005.581182003.993352005.421174003.881802004.55309600

2.591745003.004540002.070421001.028135002.255594000.914223003.291071003.034978000.414384001.603380004.003120002.59959500-2.66943900-2.76866700-3.17583400-3.67535700-2.19653500-2.10169600-2.39460500-2.34637600-3.33569000-1.82897900-1.65184500-2.02229800-2.71314600-3.06179800-2.06995400-2.42722900-0.99479400-3.31462600-1.73125900-3.09730000-1.79518600-2.69378500-4.05755700-3.451077001.885302002.646338003.690052000.960250001.666490002.529634002.13944900

1.992045002.280060001.671150000.995418001.808227000.817659002.458716003.354135000.467424002.397042001.891608000.921921002.179544000.932732000.996107002.571492002.004803002.92973800-0.21005500-1.43920100-1.67071400-0.17010400-1.34663600-2.24986800-1.97665500-2.23923800-1.63863300-1.59960800-1.19167900-1.98804600-2.38298500-3.31616500-1.28692500-0.90596000-1.85569700-2.44070200-2.31648200-1.07263700-1.17225000-2.09544400-2.89670200-2.919578000.10396200

118

4.869995003.982854004.449488004.444743005.950142000.370234000.552342001.58140000

-0.642979000.631456001.06865400

-0.44006000-0.28425800-0.78651700-1.45469300-0.87481900-1.262926000.79572600

-0.70112300-1.95233900-0.39749100-0.46524300-0.512834000.209295001.280318000.202904000.15368400

-1.55446100-0.289750000.180511001.24559700

-0.388558000.28427600-1.271922000.466960001.23860500

-0.352689002.894091002.208896002.43829800

2.833933001.293499002.264310003.839478002.91615100

-0.69843300-0.77105800-0.34060000-0.944206000.30418000

-1.40411200-1.32007100-1.67021900-0.59667800-0.87688100-3.04365700-1.65377400-1.71863800-3.31902500-3.05404000-3.779020000.657054001.713998001.259976000.649419002.943051000.801576001.946168003.275553003.757392002.698814001.548763001.300476002.035685000.232823001.827520001.65364200

-0.051398000.20227300

-0.14724500

1.35 1446000.119268002.178185001.352682001.484382004.534080003.026134002.484609004.846854004.869502004.987407002.329057000.91765700

-0.03793000-1.038016000.637096002.811519000.73918500-0.402329000.817011001.284236000.26996700

-0.72645700-1.98784400-1.92448200-0.164639001.06044800

-0.958493000.74989100

-0.888952000.05629500

-3.13939800-4.40653100-3.13055400-4.53155200-4.43859500-5.21321200-0.03173400-0.690199000.82216300

119

Table 4.S4. Coordinates Extracted from PDB Entry 3b80 and Optimized Hydrogen Atoms

7.144451005.695564004.740019005.185072003.512748005.524412007.770721005.506653007.260118007.44646700

-4.88670300-5.84044200-7.01061400-4.93341000-5.16320200-3.87256400-5.24390200-6.09137500-5.91998700-4.24254800-7.13316700-5.866430007.104292005.622926004.664937005.153619003.447049005.436858007.206119005.431581007.552681007.61095500

-4.58914100-5.38652300-6.34267800-3.68157000-5.19536800-4.32201100-4.95617200-5.54338400-4.84241500-4.20955300

2.434650002.775215001.821774000.742139002.158737002.729958002.825849003.774763001.352509002.88159300-2.90194400-3.11528900-3.45335100-1.86292900-3.55427200-3.13656200-2.88638700-2.81548900-3.68547000-2.76818000-2.79615400-1.90824500-2.47808900-2.76242500-1.81667600-0.78549200-2.16615000-2.69045600-1.57263600-3.75738300-3.31695200-2.340040002.265796002.911550003.647032001.816458001.496048003.026130002.608335003.231697003.170744001.93674000

-2.11890900-2.39618600-1.71029600-1.22960900-1.64786200-3.47221500-2.91984800-2.00507900-2.06190000-1.17077000-1.80800600-0.66000300-0.79196300-2.13138800-2.63690200-1.485252000.496587001.644241002.277720000.565882001.325370002.205352002.056258002.302639001.587710001.054748001.601225003.374169001.458295001.900409001.525010003.012137001.621902000.488661000.717745001.218740002.100535002.35696300

-0.70598800-1.88395000-2.71595700-0.81436400

120

-5.76467600-6.46494400-0.07444500-0.21829900-1.296113000.539257000.39939600

-1.060163000.78858200

-0.32451500-1.372059000.880864001.383127001.430523001.446433002.732498000.578092000.53812600

-0.03958500-0.89314300-0.308163002.078173000.72722500

-0.248185001.779410001.686431003.337859001.60183200

-0.356181000.753610002.310595000.26264200

-0.69001100-0.576747001.24667100

-1.70767400-0.48838800-2.68350000-2.11674400-2.146388002.90752400

4.277773002.71239500

-1.49178600-1.23103700-0.81880500-2.37894500-0.63248900-1.64990100-1.609922001.315565000.69171300

-1.48066500-0.36281400-2.95758000-0.75256400-0.083158000.835287001.828426003.251922003.159121000.73948100

-1.469218003.86119100-1.63137000-1.57861400-2.92308000-0.451630001.950569003.72308800

-3.79904400-3.094527001.663363001.706071000.807367001.868908001.757740002.584689000.03434700

-0.265968000.252187001.66595800

-2.14718700-4.33948300-2.84087100-2.38899200-4.49753300-4.81469400-4.77640400-0.626792002.053819001.92693100

-2.087069000.08419500

-0.231871001.44040900

-0.31069000-0.198445000.872663000.55723400-0.11389600-0.762195001.538743000.07996200-0.29043400-2.537316000.826873000.337195001.075789001.48799300

-0.38138200-0.860496003.245102004.370811004.977190003.340202003.984439004.98273100-0.30178000-1.016062000.46350200

-1.08886900

121

Figure 4.1

C

)

122

Figure 4.1. Structural Features of Aspartic Proteases. (A) Aspartic proteases share the "DTG"

motif with an inter-domain hydrogen bond. Here, the residues from a eukaryotic pepsin (PDB

entry ipsol 93) and HIV-1 protease (PDB entry 5hVp2 99) are aligned with an RMSD of 0.272 A. (B)

The eukaryotic pepsin consists of two unique domains and has only one flap. (C) HIV- 1 protease

consists of identical monomers that associate with two-fold symmetry

123

135 -

90

451l

O)o

--45

-0

-135

18Q-180 -135 -90 -45 U

o(o)

124

Figure 4.2

40 VU 135 IOU

Figure 4.2. Ramachandran Plot of P1 and P1' Residues in Substrates Bound to The Active Site of

Inactivated Aspartic Proteases.

125

Figure 4.3

100- r -- --- ---- --n -- -A 100y-basin

80-

60- Pl'Pro 4 :

y() P2Asn,,, - -.' -- ----40 - a

P2 Asn20

0-120 -100 -80 -60

B P1'P2

P4

P3 APi

C P4P2 P'

P3

P1

P2 i

P3

P4 01

126

Figure 4.3. Main-chain Conformation of the P1 Residue. (A) Ramachandran plot of P1 residues

in substrates bound to the active site of inactivated aspartic proteases. Data points are colored by

the presence of a proline residue at the P1' position and an asparagine residue at the P2 position

(red), an asparagine residue at the P2 position (blue), or neither (black). All data points are shown

relative to the y-basin. (B) The P2, P1, and Pl' residues engage in an inverse y-turn (NC/pl

substrate; PDB entry ltsq).244 (C) A proline residue at the P1' position precludes the formation of

an inverse y-turn but is found with polar residues at the P2 and P4 positions that form hydrogen

bonds (MA/CA substrate; PDB entry lmt7).218 (D) The side chain of an asparagine residue at the

P2 position forms a hydrogen bond with the nitrogen of the scissile peptide bond (pl/p6 substrate;

PDB entry 4obf).242

127

Figure 4.4

A 170.

160-160 polyproline

150- . 0

(0) . P1'

140 0 00 * 0

130-

-90 -70 -50 -30

B CP2

R P1l

P1i

D

polyproline0

128

Figure 4.4. Main-chain Conformation of the P ' Residue. (A) Ramachandran plot of P1' residues

substrates bound to the active site of inactivated aspartic proteases (black) and the residues in a

PPII helix (red).25 5 (B) Parameters used to assess n-+rw* interactions between adjacent main-chain

carbonyl groups in a protein (Tables SI and S2). (C) Structure of a P1 residue bound to an

inactivated HIV-1 protease and showing its n-*r* interaction (PDB entry lkjh; d = 3.0 A, 0 =

95.4 ).65 (D) Structure of a polyproline fragment showing its n--+7w* interactions (CSD entry

SOWJUL; d = 3.1 A 0.1 A, 0= 97.40 7.2 ).2

129

Figure 4.5

Inversey-tumn 00--mH NL" N "

N H-n0H P, interaction- 0 H

% OH, 0

0 >

Asp25 Asp23'

Michaelis Complex

H P

4--.O\

C H H

0 ",b01-

Asp25 Asp25'

gem-Dial Intermediate

0--- HH, 4N

H pfjK HO\ HH

0- 0-'

0 bASp25 ASp25'

gem-Diol Intermediate(N-protonated)

-4-

0

0H2Nij0 H7

H pHHOH

0- 0

'0 0Asp25 Ap25'

Product Complex

130

4-

Figure 4.5. Putative Mechanism of Catalysis by HIV-1 Protease. The hydrolysis reaction proceeds

through at least three discrete steps. Upon binding the peptidic substrate and lytic water, Asp25 of

one monomer deprotonates the water while Asp25' of the other monomer transfers a proton to the

oxygen of the scissile peptide bond. This oxygen also forms an n-+7* interaction (red dashed line)

with the main-chain carbonyl group of the next residue in the substrate. The n--7r* interaction is

even stronger in the tetrahedral intermediate. The inverse y-turn (blue dashed line) disturbs the

planarity of the scissile peptide bond and orients the nitrogen lone pair for protonation by Asp25.

131

Figure 4.6

R

P1 P1

Asn25'

Michaelis Complex(n-r* interaction)

P1

P1,

Asp25'

Asp25

gem-Diol Intermediate(inverse y-turn)

gem-Diol Intermediate(n-+r* interaction)

E

P1

P1',

Asp25

Asp25

gem-Diol Intermediate(0-H - N hydrogen bond)

gem-Diol Intermediate(0-H ---0 hydrogen bond)

132

D

CP1,A I

P1P1

Asn25'

Michaelis Complex(inverse y-turn)

F

iP

Asp25'

Asp25

Figure 4.6. Orbital Interactions of the Scissile Peptide Bond During Catalysis by HIV-1 Protease.

An n--+r* interaction occurs within the substrate (A) and tetrahedral intermediate (B). An inverse

y-turn likewise forms within the substrate (C) and tetrahedral intermediate (D). The hydrogen bond

of the inverse y-turn enforces a hydrogen bond between the lone pair of the nitrogen of the scissile

peptide bond and an active-site aspartic acid residue (E), despite a competing hydrogen bond of

the aspartic acid residue with the gem-diol (F).

133

Chapter 5

Sub-Picomolar Inhibition of HIV-1 Protease with a

Boronic Acid

Contribution:

I performed all experiments, prepared all materials accept for the synthetic compounds, and

wrote the initial manuscript. I contributed to the inhibitor design, preparation of the final

crystallographic model, and revision of the final manuscript.

This chapter has been published in part, under the same title. Reference:

Windsor, I.W.; Palte, M.J.; Lukesh 1II, J.C.; Gold, B.; Forest, K.T.; Raines, R.T., Sub-picomolar

inhibition of HIV-l protease with a boronic acid. J. Am. Chem. Soc. 2018, 140, 14015-14018.

134

Abstract

Boronic acids have been typecast as moieties for covalent complexation and are employed only

rarely as agents for noncovalent recognition. By exploiting the profuse ability of a boronic acid

group to form hydrogen bonds, we have developed an inhibitor of HIV-1 protease with

extraordinary affinity. Specifically, we find that replacing an aniline moiety in darunavir with a

phenylboronic acid leads to 20-fold greater affinity for the protease. X-Ray crystallography

demonstrates that the boronic acid group participates in three hydrogen bonds, exceeding that of

the amino group of darunavir or any other analog. Importantly, the boronic acid maintains its

hydrogen bonds and its affinity for the drug-resistant D30N variant of HIV-1 protease. The

BOH - OC hydrogen bonds between the boronic acid hydroxy group and Asp30 (or Asn30) of the

protease are short (ro ...o = 2.2 A), and density functional theory analysis reveals a high degree of

covalency. These data highlight the utility of boronic acids as versatile functional groups in the

design of small-molecule ligands.

HHHN'

NH

H B

H

135

5.1 Introduction

Clinical inhibitors of HIV- 1 protease are quintessential triumphs of structure-based drug design.97 ,

300 The protease cleaves diverse sequences that connect individual domains of viral polyproteins,

recognizing four substrate residues on each side of the scissile bond.65 The components of most

effective inhibitors-a tetrahedral-intermediate mimetic flanked by subsite-targeting groups

have undergone iterative optimization for 30 years. 30 1 The discovery of the bis-THF moiety of

darunavir, which targets the enzymic S2 subsite, was a major breakthrough.1 05 Its two bis-THF

oxygen atoms accept hydrogen bonds from the main-chain amides of Asp29 and Asp30, leading

to low picomolar affinity (Table 5. 1).199, 302 Mutations that overcome such main-chain interactions

are rare, 11, 122, 303-304 and darunavir is among the most resilient of protease inhibitors.3 05 -3 07

136

5.2 Results and Discussion

Despite countless attempts at optimization, an ideal functional group for the S2' subsite has been

elusive. Inspection of structures of complexes between substrates and darunavir analogs (Table

5.1, Figure 5.1) in conjunction with biochemical characterization revealed opportunities to us. Half

of endogenous substrates occupy the S2' subsite with a glutamine or glutamic acid residue.65, 7 7,2 07

These side-chains have been observed to form hydrogen bonds with both the main-chain N-H and

the side-chain carboxylate group of Asp30 (Figure 5. 1A). The aniline nitrogen of darunavir and

the methoxy group of an anisole analog form only a single hydrogen bond (Figures 5.1 B and 5.1 C).

Benzyl alcohol and cyclopropyl-amino-benzothiazole groups can form two hydrogen bonds with

Asp30, one with the main-chain N-H and another with the side-chain (either via a water-bridge or

directly) (Table 5.1, Figures 5.11D and 5.1E). Other aryl sulfonamide substituents, including

benzoic acid and benzamide, form a water-bridge with Gly48 in addition to accepting a hydrogen

bond from the main chain of Asp30, but again exhibit a <2-fold increase in affinity, but provide

<2-fold increases in affinity (Table 5.1, Figure 5.1 F). This water-bridge with Gly48 is another

interaction that can be exploited to recognize the main chain. Yet, no extant protease inhibitor

interacts with all three of these targets: main chain and side chain of Asp30, and a water molecule

that bridges to the main chain of Gly48.

We reasoned that an optimal functional group for targeting the S2' subsite would serve as

both a donor and an acceptor of hydrogen bonds. We were aware that the two hydroxy groups

presented by boronic acids are versatile in this manner. 13 4 , 308-309 These hydroxy groups display

four lone pairs and two hydrogen-bond donors. No other functional group provides six

opportunities to form hydrogen bonds so economically. We anticipated that one hydroxy group of

137

a boronic acid could form both interactions with Asp30 while allowing the other hydroxy group to

form a water-bridge with Gly48. Accordingly, we synthesized boronic acid 1, in which the

4-sulfonylaniline moiety of darunavir is replaced with a 4-sulfonylphenylboronic acid (Table 5.1,

Scheme 5.S 1).

Boronic acid 1 is a competitive inhibitor of catalysis by HIV-1 protease. By using a

hypersensitive assay of catalytic activity,2 23 we found its inhibition constant (Ki) to be 0.5 0.3

pM, which is indicative of 20-fold greater affinity compared to darunavir itself (Table 5.1, Figure

5.S ID). Because the boronic acid moiety of 1 is anticipated to interact with Asp30, we suspected

that D3ON HIV-1 protease, which is a common variant that endows resistance, could compromise

the affinity of boronic acid 1. For example, the D30N substitution entices darunavir to form a

water-bridge between its aniline nitrogen and the nascent asparagine, diminishing affinity by 30-

fold.310 Remarkably, the affinity of boronic acid 1 for the D30N variant (Ki = 0.4 0.3 pM) is

indistinguishable from that for wild-type HIV- 1 protease.

To understand the basis for the extraordinary affinity and resiliency of boronic acid 1, we

determined the X-ray crystal structures of its complexes with both wild-type HIV- 1 protease and

the D30N variant. The two structures were solved at resolutions of 1.60 A (Rfree = 0.1967) and 1.94

A (Rfree = 0.2203), respectively (Table 5.S1, Figure 5.S2). True to its design, the boronic acid

participated in all three hydrogen-bonding interactions (Figures 5.1G and 5.1H). Of special note

are BOH... OC hydrogen bonds observed in both structures (Figures 5.1G and 5.1H). The

interatomic distance of 2.2 A between the boronate oxygen and side-chain 06 of residue 30 is

reminiscent of a low-barrier hydrogen bond (LBHB).311312

138

We analyzed the atypically short hydrogen bonds between boronic acid 1 and HIV-1

protease with computational methods. First, we optimized the hydrogen atoms by applying density

functional theory (DFT) to a simple model extracted from the crystal structure. We examined the

electronic structure by using Natural Bonding Orbital (NBO) analysis. 266 NBO analysis revealed

an interaction energy of 69.8 kcal/mol between boronic acid 1 and the wild-type protease. The

typically non-hybridized p-type lone pair of the carboxylate oxygen hybridizes to sp3 -99 in the

hydrogen-bonded complex. This large interaction energy and hybridization suggest a large degree

of covalency in the BOH OC hydrogen bond. Next, we assessed the covalency of the short

hydrogen bond between boronic acid 1 and the wild-type protease with quantum theory of atoms

in molecules (AIM). 313 AIM calculations-specifically, structural elements at the bond critical

point (BCP)-enable quantification of the covalency between neighboring atoms. At the

BOH.-. OC BCP, we calculated an electron density (p) of 0.174 eV-3, a Laplacian (V 2p) of -0.08

e- 5 , and a bond index of 0.22. Typical OH - OC hydrogen bonds display p < 0.2 eA 3 , positive

V2p values, and a bond index <0.1.314 Instead, the attributes of BOH OC are consistent with the

attributes of an LBHB."-31

An LBHB arises from functional groups with closely matched pKa values.311-312, 315 This

requirement can be met by a carboxylic acid and a boronic acid,1 8 which are isoelectronic. In the

enzyme- inhibitor complex (Figure 5.1G), the boronic acid group of 1 displays an no,p-p3B

interaction (i.e., resonance) of 89.1 kcal/mol, and the carboxylic acid group of Asp30 in HIV- 1

protease displays a comparable nop--+r*c=o interaction of 87.9 kcal/mol. The ensuing

hyperconjugative interaction between a boronic acid and carboxyl acid is reminiscent of a

139

resonance-assisted hydrogen bond.3 1 6-3 18 Such hyperconjugation is absent in other inhibitors, such

as the benzyl alcohol analog of darunavir (Figure 5.1 D).

Boronic acids possess attractive properties beyond their versatile hydrogen bonding.

Boronic acid 1, like darunavir, is not toxic to human cells at concentrations up to 1 mM (Figure

5.S3). In vivo, aniline moieties can exhibit problematic genotoxicity as a result of metabolic

activation. 319-321 In contrast, the major metabolite of boronic acids is the oxidative deboronation

product, an alcohol, which is modified further in phase II metabolism for efficient excretion.14 1'

322,323

We conclude that a boronic acid group in a ligand can be profuse and versatile in forming

hydrogen bonds with a protein. These attributes are especially valuable in the design of ligands for

proteins that are under the selective pressure of drug resistance. In those instances, the ability of

boronic acids to form multiple hydrogen bonds enhances affinity, and the admixture of hydrogen-

bond acceptors and donors enables adaption to mutations.

140

5.3 Materials and Methods

5.3.1 General

Commercial reagents were used without further purification. (2S,3S)-1,2-epoxy-3-(Boc-

amino)-4-phenylbutane was from Sigma-Aldrich (St. Louis, MO). All glassware was oven- or

flame-dried, and reactions were performed under N2(g) unless stated otherwise. Dichloromethane

was dried over a column of alumina. Triethylamine was dried over a column of alumina and

purified further by passage through an isocyanate scrubbing column. Flash chromatography was

performed with columns of 40-63 A silica, 230-400 mesh (Silicycle, Quebec City, Canada). Thin-

layer chromatography (TLC) was performed on plates of EMD 250-pim silica 60-F2 54.

The term "concentrated under reduced pressure" refers to the removal of solvents and other

volatile materials using a rotary evaporator at water aspirator pressure (<20 torr) while maintaining

the water-bath temperature below 40 'C. Residual solvent was removed from samples at high

vacuum (<0.1 torr). The term "high vacuum" refers to vacuum achieved by a mechanical belt-

drive oil pump.

NMR spectra were acquired with a Bruker DMX-400 Avance spectrometer at the National

Magnetic Resonance Facility at Madison (NMRFAM) and referenced to TMS or residual protic

solvent. Electrospray ionization (ESI) mass spectrometry was performed with a Micromass LCT

at the Mass Spectrometry Facility in the Department of Chemistry at the University of Wisconsin-

Madison.

All procedures were performed in air at ambient temperature (~22 'C) and pressure (1.0

atm) unless indicated otherwise.

141

5.3.2 Chemical Synthesis

0

0 N NHH OH

SI O

11 11i Br

Et3 N, C Br Br

O< S0 N N

DCM H OH

S2

A round-bottom flask containing compound S1 (1.791 g, 5.323 mmol; synthesized as described

previously 324) was dissolved in 60 mL of DCM, and the resulting solution was cooled to 0 'C.

Triethylamine (1.2 mL, 8.6 mmol) and 4-bromobenzenesulfonyl chloride (1.362 g, 5.330 mmol)

were then added, and the reaction mixture was left to stir overnight under an atmosphere of dry

N2(g). After 16 h, the reaction mixture was concentrated under reduced pressure, and the product

was purified by column chromatography (silica, 30% v/v EtOAc in hexanes), resulting in

compound S2 as a white solid (2.543 g, 86%). 'H NMR (400 MHz, CDCl 3, 6): 7.67-7.62 (m, 4H),

7.33-7.22 (m, 5H), 4.64 (d, J= 8.4 Hz, 1H), 3.87-3.76 (m, 3H), 3.11 (d, J= 6.1 Hz, lH), 3.03-

2.84 (m, 4H), 1.91-1.81 (m, 1H), 1.36 (s, 9H), 0.91 (d, J= 6.6 Hz, 3H), 0.88 (d, J= 6.6Hz, 3H).

13C NMR (100 MHz, CDCl 3, 6): 156.2, 137.8, 137.7, 132.5, 129.6, 128.9, 128.6, 127.9, 126.6,

79.9, 72.7, 58.4, 54.9, 53.4, 35.6, 28.4, 27.2, 20.2, 20.0. HRMS-ESI (m/z): [M + Na]+ calcd for

C25H35BrN205SNa, 577.1343; found, 577.1364.

N' N Br

0 N NH OH

S2

KOAc, Pd(dppf)C1 2-CH 2C 2 ,

o 0B-B

Dioxane, 80 0C

B90

'N BNH OH

S3

142

Compound S2 (0.262 g, 0.472 mmol), KOAc (0.139 g,1.416 mmol), bis(pinacolato)diboron

(0.708 g, 2.78 mmol), and Pd(dppf)C12-CH2Cl2 (34.54 mg, 0.0472 mmol) were placed in a dry

Schlenk tube. The reaction flask was then evacuated and backfilled with N2(g) five times. Freshly

degassed 1,4-dioxane (5 mL) was then added, and the reaction mixture was heated to 80 'C and

stirred for 24 h under a N2(g) atmosphere. After 24 h, the reaction was vacuum filtered through a

pad of Celite and concentrated under reduced pressure, and the product was purified by column

chromatography (silica, 30% v/v EtOAc in hexanes), giving compound S3 as a white solid (0.253

g, 89%). 1H NMR (400 MHz, CDCl 3, 6): 7.94 (d, J= 8.2 Hz, 2H), 7.75 (d, J= 8.2 Hz, 2H), 7.3 1-

7.28 (m, 2H), 7.25-7.21 (m, 3H), 4.67 (d, J= 8.6 Hz, 1H), 3.93-3.91 (m, 1H), 3.84-3.81 (m, lIH),

3.78-3.74 (m, 1H), 3.13-3.06 (m, 2H), 3.01-2.81 (m, 4H), 1.89-1.81 (m, 1H), 1.36 (s, 12H), 1.35

(s, 9H), 0.89 (d, J= 6.6 Hz, 3H), 0.86 (d, J= 6.6 Hz, 3H). 13C NMR (100 MHz, CDC13, 6): 156.1,

140.6, 137.9, 135.5, 129.7, 128.6, 126.5, 126.4, 84.6, 79.8, 72.8, 58.6, 54.9, 53.7, 35.5, 29.8, 28.4,

27.2, 25.0, 20.2,20.0. HRMS-ESI (m/z): [M + Na]' calcd for C3IH47BN207SNa, 624.3126; found,

624.3151.

9 1) HCI, DioxaneB- H 0 0 0 H 0 9

0 N NN2) E'SHB

OH S4 OHS3 DCM S5

To a round-bottom flask containing compouind S3 (0.327 g, 0.543 mmol) was added 15 mL of 4.0

M HCl in dioxane. After stirring for 4 h, the reaction mixture was purged with N2(g) to remove

excess HCl(g). Once the evolution of HCL(g) ceased, the reaction mixture was concentrated under

143

reduced pressure and dried overnight under high vacuum. The residue was then dissolved in 10

mL of DCM and placed under an inert atmosphere. Triethylamine (0.38 mL, 2.7 mmol) and

compound S4 (0.147 g, 0.543 mmol; synthesized as described previously 324 ) were then added, and

the reaction mixture was stirred overnight. After reacting for 16 h, the reaction was concentrated

under reduced pressure, and the product was purified by column chromatography (silica, 5% v/v

MeOH in DCM), yielding compound S5 as a white solid (0.293 g, 82%). 'H NMR (400 MHz,

CDCl 3, 6): 7.94 (d, J= 7.6 Hz, 2H), 7.75 (d, J= 7.6 Hz, 2H), 7.30-7.26 (m, 2H), 7.22-7.20 (m,

3H), 5.65 (d, J= 5.1 Hz, 1H), 5.04-4.99 (m, 1H), 4.95-4.92 (m, 1H), 3.96 (t, J= 8.2 Hz, IH),

3.88-3.83 (m, 3H), 3.72-3.68 (m, 2H), 3.63-3.59 (m, 1H), 3.20-3.14 (m, 1H), 3.09-3.05 (m, 1H),

3.02-2.97 (m, 2H), 2.93-2.87 (m, 1H), 2.83-2.78 (m, 2H), 1.87-1.79 (m, 1H), 1.49-1.43 (m, 1H),

1.36 (s, 12H), 1.26-1.24 (m, 1H), 0.93 (d, J = 6.5 Hz, 3H), 0.87 (d, J= 6.5 Hz, 3H). '3 C NMR

(100 MHz, CDCl 3, 5): 155.4, 140.1, 137.5, 135.4, 129.3, 128.6, 126.6, 126.3, 109.3, 84.5, 73.4,

72.7, 70.7, 69.6, 58.0, 55.1, 53.7, 45.3, 35.6, 27.3, 25.8, 24.9, 24.8, 20.1, 19.8; HRMS-ESI (m/z):

[M + NH4] +calcd for C33H51BN309S, 676.3431; found, 676.3440.

HH OH

0,11,-a NaIO 4, NH 4OAc N j

0 H HOH Acetone/H 20 K/H H OHS5 1

A round-bottom flask was charged with compound S5 (0.150 g, 0.228 mmol), which was dissolved

in acetone (10 mL) and H20 (10 mL). The resulting solution was placed under an atmosphere of

dry N2(g), and sodium periodate (0.195 g, 0.911 mmol) and ammonium acetate (70.2 mg, 0.911

mmol) were added. After stirring for 12 h, the reaction mixture was concentrated under reduced

144

nresseir, and the nroiot wvs pirifuar by ncliim-n t-hrnmatngrn- (si1a 20% v/v MeOH in

DCM), giving rise to compound 1 as an off-white solid (0.113 g, 86%). An analytically pure

sample of compound 1 was obtained by reverse-phase HPLC using a preparatory C18 column and

a linear gradient of 10-80% v/v acetonitrile (0.1% v/v TFA) in water (0.1% v/v TFA) over 45 min.

Compound 1 eluted at 38 min and, after lyophilization, was isolated as a white powder. 1H NMR

(400 MHz, methanol-d4, 6): 7.81 (s, 4H), 7.26-7.21 (m, 4H), 7.20-7.15 (in, 1H), 5.59 (d, J= 5.0

Hz, 1H), 4.93 (q, J= 6.4 Hz, 1H), 3.93 (dd, J= 9.9, 6.2 Hz, 1H), 3.84-3.65 (m, 5H), 3.46-3.43

(m, 1H), 3.21-3.10 (m, 2H), 2.99-2.84 (in, 3H), 2.53 (dd, J= 14.3, 10.4 Hz, 1H), 2.07-1.99 (m,

1H), 1.54-1.46 (m, 1H), 1.37-1.32 (m, 1H), 0.94 (d, J= 6.6 Hz, 3H), 0.88 (d, J= 6.6 Hz, 3H). "C

NMR (100 MHz, methanol-d4, 6): 157.7, 140.3, 135.2, 135.1, 130.5, 129.3, 127.4, 127.2, 110.8,

74.6, 74.5, 72.1, 70.6, 58.9, 57.4, 53.9, 46.9, 37.2, 28.0, 27.0, 20.5, 20.4. HRMS-ESI (m/z): [M +

OMe]- calculated for C3 0H4 4BN20 10S, 635.2814; found, 635.2821.

5.3.3 Protein Preparation

Wild-type HIV-1 protease and its D3ON variant were produced heterologously in Escherichia coli

and purified as described previously. 223, 264 This HIV-1 protease had Q7K, L331, L631, C67A, and

C95A substitutions. Protease solutions in 1 mM sodium acetate buffer, pH 5.0, containing NaCl

(2 mM) were concentrated to 4.0 mg/mL (wild-type) or 15 mg/mL (D30N), flash-frozen in N2(l),

and stored at -80 'C. The flash-freezing was done within 30 min of elution from the gel-filtration

column. On the day of an assay, a concentrated stock solution was thawed rapidly, incubated on

ice and diluted with room-temperature assay buffer (50 mM sodium acetate buffer, pH 5.0,

145

containing 0.10 M NaCi) to create a 20x stock solution, which was then used immediately for

assays.

5.3.4 Enzyme Kinetics

5.3.4.1 Instrumentation and Materials

Assays for the catalytic activity of wild-type HIV-1 protease and its D30N variant were

conducted with a fluorogenic substrate, RE(Edans)SGIFLETSK(Dabcyl)R, and an M1000 plate

reader (Tecan) as described previously.2 23 In these assays, all reactions were initiated nearly

simultanteously, that is, within a few seconds. All linear and non-linear regression analyses were

performed with Prism 6 software from GraphPad (La Jolla, CA).

5.3.4.2 Michaelis-Menten Kinetics

Michaelis-Menten kinetics were performed to assess the impact of the D30N substitution

on the catalytic activity of HIV-1 protease. As observed previously, 3 the D30N substitution

decreases the value of kcat by about an order of magnitude, from 7.4 s-1 to 0.50 s-1, and increases

the value of KM slightly, from 15 ptM to 16 ptM. 223 These values were used as parameters in the

fitting of data acquired in the presence of inhibitors. Reaction progress curves were fitted by linear

regression to determine reaction velocities (Figure 5.S lA). Non-linear regression analyses of the

resulting data were performed with the mean reaction velocity, its standard deviation, and the

number of reactions per data point (n) (Figure 5.S1B). Reactions were performed in quadruplicate

(n = 4).

146

5.3.4.3 Inhibition Ki netiCS

The Morrison equation 203-204, 325 was used to evaluate values of Ki that were lower than the

concentration of enzyme used in assays, essentially as described previously.223 In these assays, the

initial fluorescence of assay buffer containing substrate and inhibitor in a combined volume of 190

[pL was measured. Measurement was paused briefly for the addition of the 20x stock solution of

enzyme. Data collected immediately after the addition of enzyme and prior to achieving enzyme-

inhibitor equilibrium were omitted from the analyzed data set. The mean velocity and its standard

deviation (derived from linear regression) at a particular concentration of inhibitor were

normalized to the velocity in the absence of inhibitor. These normalized values were fitted with

the "Morrison Ki" subroutine in Prism 6 software. The Morrison equation can be used to determine

the enzyme concentration, but doing so led to wild-type enzyme concentrations that differed by

>20% from concentrations measured by active-site titration with darunavir. Accordingly, wild-

type enzyme concentrations were set to the value derived from the Michaelis-Menten equation in

the absence of inhibitor, as described previously.223 These values agreed well (<10% variation)

with those measured by active-site titration. In contrast, the D3ON enzyme concentrations provided

by the Morrison equation agreed well (<10% variation) with those measured by active-site titration

with darunavir. Accordingly, D3ON enzyme concentrations were fitted simultaneously with values

of Ki by using the "Morrison Ki" subroutine in Prism 6 software.

5.3.5 Cytotoxicity

MT-4 cells (catalog #120) and darunavir (catalog #11447) were obtained from the AIDS

Reagent Program of the NIAID (NIH). Cells were grown at 37 'C in RPMI 1640 containing fetal

147

bovine serum (10% v/v) and Hyclone antibiotic/antimycotic solution (1% v/v) from GE Healthcare

(Chicago, IL). Cells were plated at a density of 2.5 x 104 cells per 100 pLL in a flat-bottomed, 96-

well plate from Coming (Coming, NY). Concentrated protease inhibitor stocks were prepared at

200 mM in DMF and diluted by >200-fold with final a solvent content of 0.5% by volume in each

condition. After incubation at 37 'C for 48 h, cells were assayed for viability using the CellTiter

96® AQueous One Solution Cell Proliferation MTS Assay from Promega (Madison, WI) by

monitoring absorbance at 450 nm with a GloMax multidetection plate reader from Promega. Each

condition was assayed in triplicate.

5.3.6 X-Ray Crystallography

5.3.6.1 Protein Crystallization

Compound 1 was dissolved in DMF at a concentration of 15 mg/mL. Hanging drop vapor-

diffusion plates were setup by mixing a protease stock solution with the ligand stock solution at

ratios of 1:9 (wild-type:1) or 1:4 (D3ON:1) in 2-pL drops over a reservoir of 100 mM Tris-HCl

buffer, pH 7.4, containing NaCl (200 mM for wild-type; 400 mM for D3ON). Crystals grew within

2-3 days. Crystals were cryo-protected in mother liquor containing 25% v/v glycerol and flash-

frozen in N2 (l).

5.3.6.2 X-Ray Diffraction

Diffraction experiments were conducted at the Advanced Photon Source of Argonne

National Lab with the Life Sciences Collaborative Access Team at sector 21. Frames were indexed,

148

Intrted and scaled with HKL2000 (HKL Researh) Details ofthe diffraction experiment are

listed in Table 5.S1.

5.3.6.3 Structure Solution and Refinement

Molecular replacement (MR) was conducted using Phaser software as implemented in

Phenix using 3nu3 as a reference model with solvent, ligand, and alterative conformations

removed.228 The MR solution was refined with Phenix Refine, and further model building was

conducted with Coot. 229 The atomic coordinates of boronic acid 1 in an idealized conformation

were prepared using WebMO (WebMO, LLC). The final geometry was optimized by the AMI

method and restraints were prepared with eLBOW in Phenix. Compound 1 bound to the wild-type

protease in two antiparallel conformations with unequal occupancy (Figure 5.S2A). The boronic

acid was only well resolved in the major conformation with 0.7 occupancy. Two conformations of

Asp30 were also resolved with major and minor conformers. The conformers of the ligand and

Asp30 that had similar occupancies are believed to be the pair that participate in a hydrogen bond

(Figures 5.1G and 5.S2A). There was sufficient density only in the lower-resolution structure of

the D30N protease complex to place a single molecule of boronic acid 1 and determine the

conformation of Asn30 (Figure 5.S2B). The major conformers in the wild-type structure are nearly

identical to the single conformation in the structure of the D30N protease complex (Figures 5.2G

and 5.2H). Details of the final refined structures are listed in Table 5. S1.

149

5.3.7 Computational Analysis

5.3.7.1 DFT Optimization with Gaussian

Simplified models of the low-barrier hydrogen bonding (LBHB) interactions were

extracted from the X-ray structures of compound 1 bound to wild-type HIV-1 protease and its

D3 ON variant. Average coordinate errors for the structures can be found in Table 5.1. The ligand

was truncated after the sulfonamide, leaving behind two methyl groups on the nitrogen to maintain

sterics. The entire Asp30 or Asn30 residue along with flanking main-chain amide bonds with the

distal C' as a methyl group were included. DFT calculations were used to optimize hydrogens with

Gaussian 16, Revision A.03 software from Gaussian (Wallingford, CT).326 Proton optimization

was conducted at the M06-2X/6-3 11 +G(d,p) level of theory employing the IEFPCM solvation

model.

5.3.7.2 Natural Bonding Orbital Analysis

NBO analysis was conducted with NBO 6.0 software, 296 using the $CHOOSE keyword to

give the Lewis structure where the boronic acid acts as the proton donor (the proton is bonded to

the boronic acid oxygen). When the $CHOOSE keyword is not utilized, the NBO program

generates a low valence proton sandwiched between two oxyanions. NBO analysis shows the

dominant interaction is with the in-plane, predominantly p-type lone pair of the carboxylate

oxygen. The strength of the LBHB interaction is likely largely overestimated at 69.8 kcal/mol

(taken as the 2nd-order perturbation energy minus the steric exchange). The D30N substitution

weakens the interaction to 58.3 kcal/mol. To model attenuation of the carboxylate charge via

external H-bonding, the strength of the interaction with the acid was investigated and found to be

150

40.7 keal/nol The highly covalent nature of this hydrogen bond is apparent. In addition to large

interaction energies, hybridization of the carboxylate lone pair from p to sp3.99 (sp 3-93 for the D30 N

amide) is observed in the NBO analysis. Lone pairs involved in hydrogen bonding on each oxygen

hybridize when $CHOOSE is not used.

5.3.7.3 Atoms in Molecules (AIM) Analysis

To investigate further the covalency of the SSHB, we turned to an alternative description

of chemical bonding-the bond critical point (BCP) concept stemming from the topological

quantum theory of atoms in molecules (AIM) developed Bader. Here, structural elements are

identified by critical points of electron density (Vp = 0), with atoms at maxima and bonds at

minima. The sign of the Laplacian (V 2p) at the BCP gives insights into bonding character, with

covalent bonds showing a negative value and ionic bonds positive. Previous investigations have

found that LBHB have a substantial degree of covalency and in extreme cases can exist as a 2-

center, 3-center bond.3 ' 27

AIMAll (Version 17.01.25) was used for atoms in molecules (AIM) calculations (Table

5.S2). We found a covalent character between the proton and both oxygens, with a calculated

electron density of 0.270 eK-3 and 0.174 e^ 3 at the BCP for the BO-H and BOH - OC bonds,

respectively. Additionally, the V2p was negative for both bonds at -1.38 eA-5 and -0.08 e^-,

respectively. Bond indices were 0.31 and 0.22, respectively. Typical 0... H hydrogen bonds

display bond orders <0.1, p < 0.2 e^ 3 , and positive Laplacian values.327 Thus, we report a short,

asymmetric hydrogen bond with substantial covalent character. The bond in the D30N variant

gives similar values, although the covalency is slightly diminished.

151

5.4 Acknowledgments

I.W.W. was supported by Biotechnology Training Grant T32 GM008349 (NIH) and a Genentech

predoctoral fellowship. M.J.P. was supported by Molecular and Cellular Pharmacology Training

Grant T32 GM008688 (NIH) and American Heart Association predoctoral fellowship

09PRE2260125. B.G. was supported by an Arnold 0. Beckman Postdoctoral Fellowship. This

work was supported by Grants RO1 GM044783 (NIH) and MCB 1518160 (NSF), and made use

of NMRFAM (University of Wisconsin-Madison), which is supported by Grant P41 GM103399

(NIH). This research used resources of the Advanced Photon Source, a U.S. Department of Energy

(DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne

National Laboratory under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21

was supported by the Michigan Economic Development Corporation and the Michigan

Technology Tri-Corridor (Grant 085P1000817).

152

5.5 Hydrogen Optimization Coordinates

Wild-Type HIV- 1 Protease H-opt

2.252662003.239154004.338821002.879147003.787691003.195697002.006527004.104311004.376624003.556846005.415608004.064402003.72329800

-5.14279700-5.24254700-5.01267200-4.93421600-4.99906600-5.934297000.559584001.56085700

-3.40535400-3.24347700-2.03415200-0.85659700-0.95695400-2.206390000.460161002.082072001.301968002.708561004.702729003.282810005.002961005.029310003.375396004.610381002.93628400

-5.77261400

-0.622126000.114190000.480363000.288976000.847874002.107283002.40464800

-0.15093900-1.55330000-2.45545100-1.750136002.844520004.07932400-0.399971000.735726000.484126001.988689001.73728200

-0.49749600-3.61106600-1.51851600-0.20819400-1.58515100-2.19963700-1.49815900-0.075894000.52101800

-2.25397700-0.03053600-0.81852900-1.567725001.09645200

-0.166495000.183136002.550038004.831473004.44901800

3.90230900-0.22908800

3.276686002.385971002.819532001.117288000.13252900

-0.47630900-0.33961800-0.98900400-0.47286100-0.755904000.19012900

-1.14662100-1.831904002.033238001.16671100

-0.453539001.76162400

-1.10641600-0.87425200-0.94613300-0.86930700-0.62101900-0.69608700-0.79049300-0.80740500-0.71027600-0.62403800-0.899470004.176084002.781522003.573726000.67047500-1.70546900-1.51185000-1.16676900-1.12171300-2.33967400-2.566167002.90786100

153

H

HH

HHHHHH

H

HH

HF:

-5.50228500-4.11114200

-5.61718600-5.07506100-3.905559001.476512002.47420200

-4.13898700-2.00887000-0.06258900-2.257050002.02704500

-1774.6501795

-1.29879500-0.565428002.152591002.797266002.02556000

-3.90241200-2.00591500-2.20073300-3.282640000.537568001.60540500

-0.13026100

154

1.532355002.370066002.597985001.047427002.14457500

-0.94910500-0.94438700-0.69582600-0.85591300-0.71249800-0.566821000.75827100

2.130450003.122923004.237561002.738151003.627842003.068097001.872796003.815788004.404887005.652155003.751143003.961081003.60732200

-5.16375400-5.33696700-5.05922900-5.09386600-5.04022900-5.990624001.833807000.67580300

-3.39102100-2.19298300-0.91487300-0.74862100-1.94851600-3.186401000.646445004.574788002.918115006.176150006.093556004.578111001.166575001.876775002.572106001.992808004.934199004.500931002.850584003.21443400

-0.683385000.07919400

0.399970000.357029000.887266002.185041002.46825100

-0.11316000-1.43411700

-1.40038800-2.483348002.957408004.20749400-0.468337000.673690000.468091001.897309001.74737400

-0.45708700-1.59797000-3.63479900-0.251439000.48039300

-0.12499600-1.52913700-2.24427900-1.63261600-2.260064000.32773700

-0.22740300-0.54069300-2.252551001.08958300

-0.83388000-0.04808900-1.65124200-0.130113002.691498004.615050004.027241004.92568700

3.268275002.392798002.829036001.148194000.12987600

-0.43967400-0.36044000-1.02318200-0.57497500-0.10038800-0.67465300-1.04330800-1.705279002.016608001.14702600

-0.503535001.76276900

-1.13490700-1.03186700-0.87131800-0.88195700-0.61264200-0.49372600-0.56782000-0.73696700-0.83438900-0.77080900-0.82806100-1.68218000-1.60345000-0.051890000.214942000.627195002.782784000.804409003.508741004.19686600

-1.02315100-2.17066600-2.46940300-0.98405600

155

D310N HII-1I P r ot e ase H- optt

H -5.43906800H -4.12879000H -5.82713500H -5.25551800H -4.07429700H -5.79401500H 2.61656500H -0.17255900H -2.23509200H -0.04674800H -1.95850700H -4.06516600

HF: -1755.2029447

-1.38981800-0.54718800-0.347898002.725047001.977045002.02211400

-2.23063300-4.074524001.558648000.52472300

-3.31934100-2.26481000

1.502207002.371259002.873904001.073508002.168285002.59327000-0.93202500-0.80524400-0.36903200-0.49893700-0.97807700-0.86938500

156

Scheme 5.S1. Route for the Synthesis of Boronic Acid 1. Overall yield: 54% (unoptimized).

Et3N, CI I-& Br0 0

N NH DCM

S1 OH 86%

KOAc, Pd(dppf)C 2-CH 2C 2,

Br 0__B-B B0_9

H Dioxane, 80 0C HH OH 89% H OH

S2

S3

1) HCI, Dioxane

2) Et3N, H ,, 0S4 0

DCM

82% (2 steps)

O

0 , 0,'C

0 N N Na1O 4, NH40AcH H OH Acetone/H 20

S5 86%

OHH

OH

H0 N OH H O

157

Table 5.1

Table 5.1. Values of Ki for Inhibition of HIV-1 Protease

Asp30,T,, 0 0

O NH--- "0O N N'S R

S 2 N OH S2,Asp29 NH H--- 81ZS

R

NH2

darunavir

01

- OH

OH

0

NSI

HN-<

NH2

0

OH

OH

Ki (pM)

10 1

14

12

12.7

10

8.9

0.5 0.3

Relative Affinity'

1.0

1.2

1.3

1.6

1.8

20

a Values of Ki can depend on assay conditions. Here, values are compared by using darunavir

as a benchmark with Relative Affinity = Ki,danavir /Ki,analog as reported in the indicated

reference.

158

Ref.

223

97

97

328

329

328

This work

Tnhe 5.SI

Table 5.S1. Crystallographic Data Collection and Refinement StatisticsComplex Wild-Type D30NPDB code 6c8x 6c8y

Data CollectionX-ray sourceDetectorWavelength, AResolution A (last shell)Space groupa, b, c (A)a,f8, y (0)

No. of ReflectionsNo. Unique ReflectionsRedundancy (last shell)Mean /- (last shell)Completeness (last shell)Rmeas (last shell)Rsym

Rpim

Wilson B-factor

RefinementWorking set (last shell)Test set (last shell)Rwork (last shell)Rfree (last shell)RMS deviation bond lengths (A)RMS deviation bond angles (0)Coordinate error (maximum likelihood

Total number of atomsProtein residuesProteinLigandSolvent

Mean B-factorProteinLigandSolvent

21-ID-DMAR 300 CCD0.9785327.0-1.60 (1.63-1.60)P 2 121258.7, 86.2, 46.29 90, 90437,30930,670 (1507)14.3 (13.2)38.3 (3.5)100 (100)0.095 (0.667)0.092 (0.642)0.025 (0.181)15.5

30,625 (2,779)1,508 (132)0.174 (0.196)0.197 (0.206)0.0080.940.14 A

1867198158788192

18.517.613.428.2

21-ID-FMAR 225 CCD0.9787248.8-1.94 (1.97-1.94)P 2 1212

59.1, 86.4, 45.890, 90,90128,39117,966 (874)7.1 (7.0)13.9 (2.7)100 (100)0.119 (0.671)0.111 (0.621)0.044 (0.250)21.7

17,927 (1,760)877 (88)0.1733 (0.190)0.2203 (0.270)0.0080.920.22 A

1776198156149166

23.122.224.231.5

159

Ramachandran favored, allowed, outliers (%) 100, 0, 0 98.8, 1.0, 0

160

Ramachandran favored, allowed, outliers (%) 100, 0, 0 98.8, 1.0, 0

Table 5.S2

Table 5.S2. Interaction Energies for Boronic Acid 1 and HIV-1Protease Residue 30

HIV- 1 Protease Bond p (eA-3) V2p (eA-5) BondIndex

Wild-type BO .H 0.270 -1.38 0.31BOH OC 0.174 -0.08 0.22

D30N BO --H 0.295 -1.68 0.32BOH- OC 0.172 0.03 0.20

161

Figure 5.1

CAsp3 Asp3O Asp3O

Asp3OAsp3s Asp3O

Asp3 A Asn30

u2.2 At2.2 A

48

162

Figure 5.1. Interactions with a Substrate, Darunavir, or its Analogs and the S2' Subsite of HIV-1

Protease. (A) A substrate (PDB entry lkj7). (B) Darunavir (4hla). (C) Anisole analog (2i4u). (D)

Benzyl alcohol analog (3o9g). (E) Cyclopropyl-amino-benzothiazole analog (5tyr).

(F) Benzamide analog (4i8z). (G) Boronic acid 1 bound to wild-type HIV-1 protease (6c8x). (H)

Boronic acid 1 bound to D3ON HIV-I protease (6c8y). Major conformers are shown for inhibitors

that bound in non-symmetry-related conformations.

163

A

(~..

w *

164

Figure 5.2

Figure 5.2. Orbital Interactions in a Model of Boronic Acid 1 and Residue 30 of HIV-1 Protease

Derived from X-ray Crystal Structures (PDB entries 6c8x and 6c8y). NBO rendering of the

hydrogen bond between a boronic acid hydroxy group and 06 of Asp30 (A) and Asn30 (B) with

hydrogen atoms optimized at the M06-2X/6-311 +G(d,p) level of theory employing the IEFPCM

solvation model.

165

Figure 5.S1

-milE

U U - -

:e*O eas:O 6;

40 pM

30 pM

20 pM

15 pM

10 pM

5 pM

1 pM

A15000-

10000-

U--

5000-

0

C50000-

40000-

L, 30000-

20000-

500 1000 1500

Time (s)2000 2500

0 pM2WPM

40 pM

70 pM*0 ~9 Px0M

YU~AI~1100 pM

2000

B 20-

1 5-

V 110-(nM/s)

0.5-

0.0-

D

1.0-

0.0

F1.0-

V0V0.5-

2500 3000

Time (s)

10 20[Substrate] (pM)

50

[1] (pM)

50

(1] (pM)

166

1100 1200 1300 1400 1500 1600 1700 1800

Time (s)

0 pM

20 pM

40 pM

70 pM90 pM100 pM110 pM

E

D-

.3,0 . . . . . . .

8000-

7000

6000

500

15 0

100

100

,

Figure 5.S1. Raw and Processed Kinetic Data from Assays of HIV-1 Protease Activity. (A)

Reaction progress curves of 5 nM D3ON HIV-1 protease titrated with a fluorogenic substrate. (B)

Non-linear fitting of reaction velocities to the Michaelis-Menten equation provides values of kcat

= 0.50 0.01 s-1 and KM = 16 1 tM (R2 = 0.99). (C) Reaction progress curves of 85 pM wild-

type HIV-1 protease titrated with boronic acid 1 and 10 VM substrate. (D) Non-linear fitting of

reaction velocities to Morrison's equation, providing a value of Ki = 0.5 0.3 pM (R2 = 0.96). (E)

Reaction progress curves of 100 pM D3 ON HIV- 1 protease titrated with boronic acid 1 and 10 pM

substrate. (F) Non-linear fitting reaction velocities to Morrison's equation, providing a value of Ki

= 0.4 0.3 pM (R2 = 0.98).

167

Figure 5.S2

B

168

r

Figure 5.S2. Depiction of Electron Density of HIV- 1 Proteases with Bound Boronic Acid 1. Fo -

Fe density maps resulting from simulated-annealing refinement with the ligand removed and

residues proximal to the boronic acid replaced with glycine were overlayed on final models from

(A) wild-type HIV-l protease (PDB entry 6c8x) and (B) D3ON HIV-l protease (6c8y). Density is

depicted at 3.0a- within 1.5 A of side-chains proximal to boronic acid 1.

169

Figure 5.S3

A

0.6-

0.4-so

0.2-

0. I I I INo Compound 107 104 10-5 104 10-3 Medium Alone

[darunavir] (M)

B

0.6-

0.4-A450

0.2 -

0.0-

4-0

j-~7 4--- 0--

0

I I I I

No Compound 10-7 10- 10-5 10-4

[boronic acid 1] (M)

0

S

-*44-

10-3 Medium Alone

170

Figure 5.S3. Toxicity of Darunavir and Boronic Acid 1 for Human Cells. Graphs show the

absorbance at 450 nm from a tetrazolium dye-based assay of the viability of MT-4 cells (adult T-

cell leukemia) after incubation with darunavir (A) or boronic acid 1 (B) for 48 h. A high A450 value

is indicative of viable cells.

171

5.6 NMR Spectra

'H NMR Spectrum of Compound S2 in CDC13

(4.o 55 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0

f, (ppm)

13C NMR Spectrum of Compound S2 in CDC1 3

I V

tO 200 190 180 570 160 150 140 530 120 110 (ppm100 90fl (PP-~)

.'/ I %I I \/' V

80 70 60 5o 4b 30 20 10 0

172

0 '10 10 , 400 -lp AW -k - -wo VOMOdA - ipwfw A -. 1 - I-.-.. , I M , 1044 Mb.OIW-

UL(

'H NMR Spectrum of Compound S3 in CDC13

IiiK.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5

fs (ppm) 2.S 2.0 1.s 1O 0.5 0.0 -0

13C NMR Spectrum of Compound S3 in CDC1 3

I I I

- i~~J59 ~ m..,dh

LO 200 AO d I& 1 ' 140 1 60 ' 10 ' 20 1 60 o10fi (PP-~)

90 so 70 60 so 40 30

173

ofmi inv

also

I~~~1A I

1120 10 0

' ...'

771:7 - -- - - - =-:; I

. M . ! . ! . ! I ! . I I --

4.0o 3.s 3.0

I I

.1II I

H NMR Spectrum of Compound S5 in CDC13

.0 6.5s 9.0 ,8.5 .8.0 .7.5 .7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 .3.0 2.5 20 15 10 05 00 -fi. (PP-)

13 C NMR Spectrum of Compound S5 in CDC1 3

| \~ \ \ -

200 190 180 170 160 150 140 130 120 Oil 100 0fl (ppm)

PR I \

V IiL~

60 70 6b 50 40 30 20 10 0

174

P I i ll -- I ll I 1 -111- 1 - -I

'H NMR Spectrum of Compound 1 in Methanol-d4

V

0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 45fi (ppm)

1 3 C NMR Spectrum of Compound 1 in Methanol-d4

I Iaa se E

I..,'r 'r'' ki-ri.. . . -. . .,-- - - ,,TA-6h.., M

200 19 0 180 170 160 I 150 140 13010

LliD 000 50 80 70 60 50 40 30 20

fl (ppms)

/l"A A I

AiL I Il

a10 -55/ I

175

I 0 1

AMMIE 4w-nnMosskqo-t-" 1 11 6d--, 1-4 A-,,

1.0 0.5 04.0 3.5 3.0 2*5 2.0 1.5

'M I 1 -1 1- .- M PIR r W, qi-- W-Yr ,-r w - , 1 7 - -* - T-, I

90 io 70 6b so 40 30 2010 0

Chapter 6

Benzoxaborolone-A Boronic Acid with Remarkable

Oxidative Stability in Aqueous Solution

Contribution:

I performed kinetic analysis and computational experiments. I contributed to performing kinetic

experiments, designing computational and pH titration experiments, preparing the initial

manuscript, and revising the final manuscript.

This chapter has been prepared for publication as:

Graham, B.J*; Windsor, I.W.*; Gold, B.; Raines, R.T., Benzoxaborolone-A Boronic Acid with

Remarkable Oxidative Stability in Aqueous Solution. In preparation.

*Denotes equal contributions

176

Abstract

Oxidative deboronation can limit the utility of boronic acids in aqueous contexts. We measured

the oxidative stability of phenylboronic acid and two derivatives: benzoxaborole (which has an o-

hydroxymethyl group) and benzoxaborolone (which has an o-carboxyl group). At pH 7.4,

phenylboronic acid and benzoxaborole have similar stability towards oxidation by hydrogen

peroxide. In contrast, benzoxaborolone is 2 x 1 04-fold more stable. Computational analyses reveal

that the resistance to oxidation arises from diminished stabilization of the p-orbital of boron that

develops during the rate-limiting step. Like phenylboronic acid and benzoxaborole,

benzoxaborolone binds to saccharides, albeit with slightly less affinity. These findings establish

benzoxaborolone as a pharmacophore of potential utility in chemical biology, medicinal chemistry,

and related areas.

177

6.1 Introduction

The utility of Suzuki-Miyaura coupling has made boronic acids common reagents in synthetic

organic chemistry. 125,309, 330 The electronic properties of boronic acids also impart a unique ability

to bind covalently, yet reversibly, to proteins and carbohydrates. This attribute has found

widespread application in chemical biology and medicinal chemistry,1 34, 331-333 as well as in

materials chemistry.334-337

Despite its beneficial attributes, only four drugs in clinical use contain boron.33 8 The clinical

implementation of boron began with the proteasome inhibitor bortezomib, which contains an

alkylboronic acid. 339 Arylboronic acids are more stable than alkylboronic acids, and can likewise

form cyclic esters with 1,2- and 1,3-diols. In 2006, Hall reported that benzoxaborole, which is the

cyclic ester of 2-(hydroxymethyl)phenylboronic acid (2-HMPBA), forms more stable esters than

does phenylboronic acid (PBA) (Figure 6.1). 132, 340-341 Subsequently, ixazomib was approved for

the treatment of multiple myeloma and two benzoxaboroles, tavaborole and crisaborole, were

approved for the treatment of fungal infections and dermatitis, respectively. 342

We suspected that the sparsity of boronic acids in the clinic might be attributable to their

having poor metabolic stability. In aqueous solution, boronic acids degrade by two major

pathways: (1) protodeboronation, in which a C-B bond is replaced with a C-H bond, and (2)

oxidative deboronation, in which a C-B bond is replaced with a C-O bond. Protodeboronation

requires an extreme pH, high temperature, or a metal catalyst,343 conditions that are not relevant

physiologically. In contrast, the primary metabolite of boronic acids in vivo is the oxidative

deboronation product, an alcohol,322, 34 reactivity that can be replicated in vitro by using reactive

oxygen species or P450 enzymes.1 41' 345 Boronic acids also react with hydrogen peroxide in

178

cellulZ,346 dA this 1110 1"s been epy in scnensors cf hydrngen nrivde nd ;int~~t, r-L~ LII & "t" L-1" A 113 i~1 sIIIJ re iii c ;on1 sLII I~J'i

the unmasking of potential chemotherapeutic agents.347-349

Herein, we provide the first detailed characterization of the oxidative stability of

phenylboronic acid and two simple derivatives. One is benzoxaborole. The other is

benzoxaborolone, which is the cyclic ester of 2-carboxyphenylboronic acid (2-CPBA) (Figure

6.1). Unlike phenylboronic acid and benzoxaborole, benzoxaborolone has not been utilized in the

context of chemical biology, medicinal chemistry, or materials chemistry.2 10 Our findings

incentivize its use.

179

6.2 Results and Discussion

6.2.1 pKa Values.

An intrinsic property of boronic acids is their pKa value, which is governed by the transition

between the neutral trigonal species and the tetrahedral anionic species (Figure 6.1).350 The pKa

values of phenylboronic acid (pKa = 8.7)133 and benzoxaborole (pKa = 7.3)127 are known. Before

embarking on an analysis of oxidative stability, we sought to determine the pKa values of

benzoxaborolone. An initial potentiometric pH titration revealed that the pKa values of the boronic

acid and carboxylic acid groups of 2-CPBA are extreme. In accord, "B-NMR-titration

experiments showed that a single species is dominant from pH 0.9-12, with a chemical shift

consistent with the monoanion. 2-CPBA was, however, converted quantitatively to the dianion at

high pH, affording a pKa2 value of 13.0 (Figure 6.S1). No 1 B chemical shift consistent with a

neutral species was observed at any pH value, even in 6 M HCl.

To provide further insight, we crystallized 2-CPBA at low pH and used X-ray diffraction

analysis to determine its structure (Tables 6.Sl--6.S7). Crystalline 2-CPBA was a zwitterion in

which the monoanion was protonated on a hydroxyl group (Figure 6.1). Thus, the cyclic ester was

maintained, rather than dissociated into a boronic acid and carboxylic acid. 3 This structure

provides additional evidence for the stability of 2-CPBA species containing a lactone and a

tetrahedral boron atom. We conclude that the predominant form of 2-CPBA in water is its

monoanion (Figure 6.1).

180

6. 2.2 Sus4ceptibility ti Oxidtion.

As an oxidant, we chose hydrogen peroxide. Discovered 200 years ago, 354 hydrogen peroxide is

now known to be the major reactive oxygen species in humans. 355 The oxidation of phenylboronic

acids by aqueous hydrogen peroxide yields a phenol and boric acid.13 2,356-359 As the phenol product

exhibits a significant blue-shift in UV absorbance relative to the boronic acid starting material, we

were able to employ a continuous UV-absorbance assay to monitor the oxidation of PBA, 2-

HMPBA, and 2-CPBA (Table 6.S8; Figure 6.S2).360 We found that the oxidation rates were first-

order with respect to hydrogen peroxide and each boronic acid (Figure 6.S3). The ensuing second-

order rate constants varied with pH (Table 6.S9) and were half-maximal for PBA and 2-HMPBA

when pH = pKa. We found that oxidation rates increase in a pH-dependent manner after and before

a pH-independent regime (Figure 6.2). This observation is consistent with the existence of two

pH-independent rate constants, ki and k2. Whereas the oxidation rates of PBA and 2-HMPBA at

physiological pH are similar to those for biological thiols, the comparative stability of CPBA to

oxidation is extraordinary (Table 6.1).

The reaction mechanism for boronic acid oxidation is consistent with complexation of

peroxide anion to the boron atom, a 1,2-aryl shift to form a borate ester and hydroxide ion, and

hydrolysis of the borate ester to form a phenol and borate salt.357 -359 The nucleophilicity of

hydrogen peroxide in water is high.36' Moreover, the rate constants for oxidation (Figure 6.2) are

much less than those for complexation of ligands to boronic acids (k= 103-10 5 M-1s-1). 3 62 -3 64 Thus,

the complexation of the peroxide is not the rate-determining step in the oxidation reaction. This

conclusion is supported by 'H-NMR spectra, which reveal the accumulation of covalent

intermediates during oxidation (Figure 6.S4).

181

For PBA, 2-HMPBA, and 2-CPBA, the transition from a neutral trigonal state to a

monoanionic tetrahedral state occurs when the pH surpasses the pKa (Figure 6.1). Hence, the

oxidation rate at pH >> pKa is largely that of the tetrahedral species. (The borolone is differentiated

by its existence as a monoanionic tetrahedral species above pH 1.) The second-order rate constants

for the oxidation for the monoanionic tetrahedral states of PBA, 2-HMPGA, and 2-CPBA are listed

in Table 6.2. Whereas the borole moiety provides an order-of-magnitude in resistance to oxidation,

the borolone provides six orders-of-magnitude.

6.2.3 Affinity for Saccharides.

PBA and 2-HMPBA are employed often for their ability to form esters with saccharides. 134 , 331-337

As the affinity of boronic acids for saccharides is generally greatest near their pKa value,35 0 the low

pKai of 2-CPBA portends a low affinity. The results of 'H-NMR titration experiments with D-

fructose, D-glucose, and N-acetylneuraminic acid (Neu5Ac) are consistent with this expectation

(Table 6.3). The loss in affinity of 2-CPBA for saccharides is, modest (~4-fold versus PBA and

- 13-fold versus 2-HMPBA) compared to the gain in oxidative stability (Table 6.1).

6.2.4 Computational Analyses.

We employed computational analyses to elucidate the physical origin of the remarkable oxidative

stability of 2-CPBA. Specifically, we performed density functional theory calculations to model

stationary points along each oxidation reaction coordinate, enabling energetic comparisons with

the pH-independent rate constant, kmonoanion (Table 6.2). We began by modelling the requisite

tetrahedral intermediate of each boronate with hydrogen peroxide and the boric ester products

182

(h 1 Tab1l'c Q1 () an Q 1 1 ; ire' . 5) Next we c1a1cnted transit;on stafte strucituires c'fI .3. A , X %1 3 " XJ'J tI. L W. " I% " L I% X.J ,..) JI VV %L4XiWL4& 4 L XX x "&" LX.I'&

the oxidation reaction, which replicated the results of similar systems (Figure 6.3).358 The

calculated relative rate constants for oxidation match exceptionally well to the experimental ones

(Table 6.2). Optimized transition state structures show a concerted mechanism in which the 1,2-

aryl shift from boron to the proximal peroxide oxygen is concomitant with peroxide

oxygen-oxygen bond cleavage, in agreement with previous mechanistic work.357' 365-366 As the

reaction progresses, the tetrahedral boronate is transformed into a planar, trigonal boric acid.

We turned to Natural Bonding Orbital (NBO) analysis to reveal interactions that are

responsible for the increased activation energies of 2-HMPBA and, especially, 2-CPBA (Table

6.S 12). In PBA, a p-type lone pair (nop) of each hydroxyl groups stabilizes the developing p-

orbital on boron (pB) via a classical (hyper)conjugative no--pB z-bonding interaction (Figure

6.4A). These interactions are absent in the starting tetrahedral boronate (where a much weaker

no-*u*B-C interaction of 8.0 kcal/mol is present) but are worth 35.2 kcal/mol in the transition state

and 69.3 kcal/mol in the product.

Intramolecular boronic ester formation affects both the donor abilities of the no,p lone pairs

and no,p-PB orbital overlap, resulting in a substantially weaker n,p-+PB interaction of 20.4

kcal/mol in the transition state for 2-HMPBA (Figure 6.4B). Despite the presence of only a

relatively weak C-H u-acceptor, a significant nO,p-+-U*C-H anomeric effect of 9.8 kcal/mol is

present in the transition state for 2-HMPBA (Figure 6.4D), reducing the no,p--+PB interaction. The

bulk of the -15 kcal/mol decrease in the no,p+PB interaction, arises from a decrease in orbital

overlap due to geometric constraints enforced by the 5-membered ring. Despite the large decrease

in stabilization of the developing empty p-orbital on boron, the rate of 2-HMPBA oxidation is only

183

slightly less than that of PBA, perhaps because of strain-relief upon ring-expansion, reflected in

the more exergonic reaction (Table 6.S 11).

Much more dramatic effects are observed during the oxidation of 2-CPBA. There, the

no,pPB interaction is diminished to 4.0 kcal/mol. More importantly, the no,p-*PB interaction

competes with no,p-,r*c=o ester resonance of 66.3 kcal/mol (Figure 6.4E). In addition to the large

decrease in its donor ability, the no,p lone pair is almost orthogonal to the pB orbital that develops

in the transition state. As a result, donation from the other lone pair, which resides in a lower-

energy sp2.2 orbital, is worth 9.1 kcal/mol (Figure 6.4D). Altogether, the transition state for 2-

CPBA oxidation is -7 kcal/mol greater in energy than that for PBA oxidation, suggesting that

strain-relief also contributes to the energetics of the 2-CPBA transition state.

We note that analogous arguments regarding donation to the PB orbital have been proposed

to be important in protodeboronation pathways.8,343,367 Indeed, experimental data indicate similar

trends in protodeboronation and oxidative deboronation pathways, with 2-CPBA being

significantly more stable than PBA or 2-HMPBA near physiological pH (Figure 6.S6).

184

6.3 Materials and Methods

6.3.1 General

Commercial reagents were used without further purification. Phenylboronic acid and

2-carboxyphenylboronic acid were from Combi-Blocks (San Diego, CA). 2-

(Hydroxymethyl)phenylboronic acid and aqueous hydrogen peroxide (30% v/v) were from

Sigma-Aldrich (St. Louis, MO).

NMR spectra were obtained with an Avance-400 spectrometer from Bruker (Billerica,

MA). The pH of buffers (10 mM) was determined with an Accumet XL50 pH-meter from Fischer

Scientific (Hampton, NH), calibrated with a pH 3.00 reference standard buffer from VWR

International (Radnor, PA) and pH 12.00, 12.45, and 13.00 reference standard buffers from Ricca

Chemicals (Arlington, TX).

All procedures were performed in air at ambient temperature (~22 'C) and pressure (1.0

atm) unless indicated otherwise.

6.3.2 pKa Determination

A 100 mM solution of 2-CPBA was prepared in 10 mL of 1 M NaOH containing D 20 (5% v/v).

The pH of the solution was measured and adjusted with 10 M NaOH and concentrated HCl, and

0.65-mL samples were transferred at the indicated pH values to 5-mm quartz NMR tubes from

Norell (Morganton, NC)) and analyzed by 'H and "B NMR spectroscopy, referencing to the

solvent peak. For the samples in 6 M DCl in D20 and 5 M NaOH in D20, the pH was outside of

the useful range of the pH probe. The acidic sample gave poor referencing from the solvent peak

due to an extreme chemical shift of the water peak, and was referenced to an external standard of

185

BF3 Et2 0 using a sealed melting-point capillary filled with the reference compound and placed

inside the NMR tube.

6.3.3 X-Ray Crystallography

Crystals of 2-CPBA grew out of the extremely acidic conditions (6 M DCl) in deuterium oxide

employed during NMR-titration experiments. Crystals were roughly 0.5 mm x 0.5 mm x 0.1 mm.

Crystals were mounted in mineral oil. X-ray diffraction data were collected from a single crystal

using a Bruker D8 Venture Kappa Duo diffractometer equipped with a Bruker Photon2 CPAD

detector and MoKa radiation (k = 0.71073 A). Diffraction data were reduced with SAINT software

from Bruker. The structure was solved with SHELXT and refined with SHELXL. Details

regarding the refinement and quality of the final structure are listed in Table 6.S 1.

6.3.4 Chemical Kinetics

6.3.4.1 Instrumentation and Materials

The oxidation reactions were monitored by UV absorbance using a Cary 60 UV-vis

spectrophotometer from Agilent. Some extinction coefficients were determined with an M1000

plate reader from Tecan. The extinction coefficient of PBA in PBS was found to be identical in

both instruments. Buffers were degassed prior to use. Measurements were performed with 1.0-mL

sample volumes in disposable, plastic, UV-transparent cuvettes. All measurements were

performed in triplicate (at least). Stocks of boronic acids and phenolates were prepared by

dissolving the compounds in buffer, assisted by sonication if necessary. All stock solutions were

prepared on the day of their use.

186

6.3. 4.2 Wavelength Optimization and Calibration

Initial wavelength scans in the range of 250-350 nm revealed a bathochromic shift between

boronic acids and phenolate (exemplified at pH 7.4 in Figure 6.S1). Wavelengths with optimal

signal-to-noise ratios were determined from these spectra and used for single-wavelength kinetic

assays. The hydrogen peroxide used as an oxidant also absorbs light at these wavelengths.

Extinction coefficients for the two reactants and the product were determined at each pH (Table

6.S8). Extinction coefficient for the oxidation of boronic acids were calculated as the difference in

absorbance between the product and substrates with the equation:

Acoxidation = phenol - (Eboronic acid - &hydrogen peroxide) (6.1)

Initial boronic acid concentrations were <1 mM, and the final pH of reaction mixtures was

confirmed not to change by more than 0.1 units.

6.3.4.3 Initial Velocity Kinetics

Initial velocity kinetic assays were performed by measuring the UV absorbance of 990 [tL of a

boronic acid solution and then initiating the reaction by the addition of 10 tL of a 100 x hydrogen

peroxide stock solution. Rates were measured in triplicate. Slopes from the first 10% of the

reaction (or from 10 min of a slow reaction: kobs < 10-2 M-s-1) were calculated and converted to

units of M/s by dividing by the value of Acoxidation. Reactions were slow enough not to require a

stopped-flow apparatus, but the initial velocity (vo) for the fastest reactions (kobs ;> 1 M's-1) could

only be calculated from a few dozen data points. Rates at three different concentrations of boronic

187

acid and hydrogen peroxide were measured and determined to be first-order with respect to both

substrates (Figure 6.S3). Second-order rate constants were determined with the equation:

kobs - V" (6.2)[boronic acid] [hydrogen peroxide]

6.3.4.4 Evaluation ofpH-Dependence

The pH-dependence of oxidation reactions was assessed by using the buffers listed in Table 6.S8.

Data were fitted to the equation:

kobs =ki +k2(1+1OpKapH) (6.3)

with pKa values shown in Figure 6.1. Reactions conducted at pH > 11.7 (which is the pKa of

hydrogen peroxide) engendered the evolution of gas. These rates, which were slower than

expected, were omitted from the analysis, though the relative rates of the different boronic acids

at pH >11.7 fitted the expected trend.

6.3.5 Saccharide Binding

The affinity of boronic acids to saccharides was assayed by 1H-NMR-titration experiments as

described previously.368 Due to its low affinity, D-glucose was used at twice the concentrations of

other saccharides.

188

%J-' -J %-%J L L"L-Lk'JL'" vJL "t.Y 1 3%10J

6.3.6.1 DFT Optimization with Gaussian

Optimizations were performed using Gaussian 16 software from Gaussian at the M06-2X/6-

31 1+G(d,p) level of theory and employing the IEFPCM solvation model for water.326 Frequency

calculations were performed to confirm each stationary point as a minimum or first-order saddle

point. Energies for each structure were determined with the freq keyword and are listed in Table

6.S10.

6.3.6.2 Natural Bonding Orbital Analysis

Natural Bonding Orbital (NBO) analysis of the optimized structures were conducted with NBO

6.0 software. 29 6 Interaction energies and the off-diagonal NBO Fock matrix element, which is

proportional to the overlap integral, are listed in Table 6.S 12.

189

6.4 Conclusions

The metabolic stability of a compound is a critical determinant of its utility in chemical biology

and medicinal chemistry. We have found that the oxidative stability of boronic acids can be

enhanced greatly by the formation of a borolone. The endowed stability results from

stereoelectronic constraints imposed by the ring and carbonyl group. The ensuing "twisting" and

"pulling" renders stabilization of the developing p-orbital on boron much less efficient than in the

more reactive phenylboronic acid and benzoxaborole. This functional group, which retains affinity

for saccharides and tightly clusters many hydrogen-bond donating and accepting groups, could

have a broad range of applications.

6.5 Acknowledgments

I.W.W. was supported by Biotechnology Training Grant T32 GM008349 (NIH) and a Genentech

predoctoral fellowship. B.G. was supported by an Arnold 0. Beckman Postdoctoral Fellowship.

This work was supported by Grant RO1 GM044783 (NIH). We are grateful to Dr. Peter Mtller

(Department of Chemistry, Massachusetts Institute of Technology) for determining the crystal

structure of benzoxaborolone.

190

Scheme 6.1. Boronic Acid Oxidation Pathway

HO 0 HR 01HHOB' 'R HOOH 01

OH - OH HO, PH1- 01'3 '0 0'B

HO' 00 H'2Computationally Investigated Pathway

R = H, CHraryl, CO-aryl

191

Table 6.1

Table 6.1. Rate Constants for the Oxidation of Boronic Acids and Biological Thiols at pH ~7.4

Compound kobs (M-s-')

PBA 2.4

2-HMPBA 2.4

2-CPBA 0.00015

Cysteinea 2.9

N-Acetylcysteinea 0.16

Glutathionea 0.89

aValues are from ref. 230, 369.

192

T able 6.2

Table 6.2. Experimental and Computational Kinetic Parameters for the Oxidation of BoronicAcids

experimental computational

kmonoaniona kmonoanion AGI k

(M-s-1) (rel) (kcal/mol) (rel)

PBA 49 1 27.0 1

2-HMPBA 4.2 0.12 28.3 0.085

2-CPBA 0.00016 0.0000033 34.3 0.0000034

aValues of kmonoanion (which is k2 for PBA and 2-HMPBA, and ki for 2-CPBA) are derived from

the data in Figure 6.2 and refer to the monoanionic species depicted in Figure 6.1.

193

Table 6.3

Table 6.3. Values of Ka (M-1) for Boronic Acids and Saccharides

D-fructose D-glucose Neu5Ac

PBAa 128 20 5 1 13 1

2-HMPBAa 336 43 28 4 43 5

2-CPBA 23 2 1.7 0.1 6.0 0.5

aValues are from ref. 368. bEach value is the mean standard deviation (SD) for >5

measurements in 0.10 M sodium phosphate buffer (pH 7.4) containing 2% (v/v) D20.

194

Thi. A.Q1

Table 6.SL Crystal Data and Structure Refinement

Identification code

Empirical formula

Formula weight

Temperature

Wavelength

Crystal system

Space group

Unit cell dimensions

Volume

Z

Density (calculated)

Absorption coefficient

Fooo

Crystal size

Theta range for data collection

Index ranges

Reflections collected

Independent reflections

Completeness to theta = 25.242'

Absorption correction

Refinement method

Data / restraints / parameters

Goodness-of-fit on F2

Final R indices [I> 2sigma(I)]

R indices (all data)

Extinction coefficient

Largest diff. peak and hole

P18065 (In-house, CCDC ID pending)

C7 H7 B0 4

165.94

100(2) K

0.71073 AMonoclinic

P21/c

a = 13.3947(5) Ab = 7.9080(3) Ac = 7.0529(2) A

743.84(4) A3

4

1.482

0.119

a 90'

b = 95.3376(12))

g= 900

Mg/m 3

mm'

344

0.450 x 0.400 x 0.080 mm 3

1.527 to 36.330'

-22 < h < 22, -13 < k < 13, -11 < l < 11

51761

3598 [Rint = 0.0680]

99.9%

Semi-empirical from equivalents

Full-matrix least-squares on F2

3598/3/ 118

1.072

R1 = 0.0365, wR2 = 0.0990

R1 = 0.0388, wR2 = 0.1012

n/a

0.629 and -0.320 e.A-3

195

Table 6.S2

Table 6.S2. Atomic Coordinates (x 104) and Equivalent Isotropic Displacement Parameters

(A2 x 10') for P18065. Ueq is defined as one third of the trace of the orthogonalized Uij tensor.

Atom x y z Ueq

B(1) 1461(1) 3951(1) 4044(1) 9(1)

0(1) 1110(1) 2160(1) 3907(1) 10(1)

0(2) 1382(1) 4478(1) 5992(1) 11(1)

0(3) 741(1) 5015(1) 2725(1) 9(1)

0(4) 796(1) 7021(1) 518(1) 13(1)

C(1) 2511(1) 4345(1) 3192(1) 9(1)

C(2) 2315(1) 5593(1) 1804(1) 10(1)

C(3) 3055(1) 6373(1) 848(1) 13(1)

C(4) 4041(1) 5863(1) 1323(1) 15(1)

C(5) 4263(1) 4622(1) 2713(1) 16(1)

C(6) 3506(1) 3857(1) 3654(1) 13(1)

C(7) 1227(1) 5960(1) 1592(1) 9(1)

196

Table 6. S3

Table 6.S3. Anisotropic Displacement Parameters (A 2 x 10) for P18065. The anisotropic

displacement factor exponent takes the form: --2u2[h2a* U1 + ... + 2hka*b* U12].

Atom U 1 U2 2 U33 U23 U13 U12

B(3) 10(1) 8(1) 9(1) 1(1) 3(1) 0(1)

0(l) 11(l) 8(l) 10(l) 0(1) 4(l)-()

C(2) 15(1) 9(1) 9(1) 0(1) 5(1) 1(1)

0(3) 9(1) 9(l) 10(1) 2(l) 3(l) 0(1)

C(4) 13(1) 11(1) 14(1) 4(1) 3(1) 2(1)

C(7) 9(1) 8(1) 10(1) 0(1) 3(1) 0(1)

C(2) 10(l) 9(1) 10(l) 1 (1) 4(l) 0(l)

C(3) 13(l) 13(l) 15(l) 3(l) 6(l) -1 (1)

C(4) 11(1) 16(l) 19(1) I(1 7(l) -2(l)

C(5) 10(l) 19(1) 19(1) I(1 4(l) 1 (1)

C(6) 10(l) 16(l) 14(l) 3(l) 2(l) 2(l)

C(7) 10(l) 8(l) 10(l) 0(l) 3(l) 0(l)

197

Table 6.S4

Table 6.S4. Bond Lengths for P18065.

Atom Atom Length (A)

BI 02 1.4487(7)

BI 01 1.4934(7)

BI 03 1.5284(7)

BI C1 1.6095(8)

01 HA 0.927(10)

01 HIB 0.890(10)

02 H2 0.819(10)

03 C7 1.3109(7)

04 C7 1.2377(7)

Cl C6 1.3964(8)

Cl C2 1.3976(8)

C2 C3 1.3931(8)

C2 C7 1.4797(8)

C3 C4 1.3914(9)

C3 H3 0.9500

C4 C5 1.3996(9)

C4 H4 0.9500

C5 C6 1.3996(8)

C5 H5 0.9500

C6 H6 0.9500

198

Table 6 .S5

Table 6.S5. Bond

Atom Atom

02 BI

02 BI

01 BI

02 BI

01 BI

03 BI

BI 01

BI 01

HIA 01

BI 02

C7 03

C6 Cl

C6 Cl

C2 Cl

C3 C2

C3 C2

Cl C2

C4 C3

C4 C3

C2 C3

C3 C4

C3 C4

C5 C4

C6 C5

C6 C5

C4 C5

Cl C6

Cl C6

C5 C6

Angles

Atom

01

03

03

Cl

Cl

Cl

HIA

HIB

HIB

H2

BI

C2

BI

BI

Cl

C7

C7

C2

H3

H3

C5

H4

H4

C4

H5

H5

C5

H6

H6

for P18065.

Angle (0)

106.48(4)

108.72(4)

107.76(4)

116.22(5)

116.14(4)

100.97(4)

118.5(7)

111.3(7)

108.4(10)

110.6(8)

111.30(4)

118.09(5)

135.00(5)

106.54(4)

123.81(5)

127.02(5)

109.14(4)

117.17(5)

121.4

121.4

120.46(5)

119.8

119.8

121.27(5)

119.4

119.4

119.19(5)

120.4

120.4

199

C7 03 121.97(5)

04 C7 C2 126.50(5)

03 C7 C2 111.53(5)

200

04

Table 6.S6

Table 6.S6. Hydrogen Coordinates (x 104 ) and Isotropic

Displacement Parameters (A 2 x 103) for P18065.

Atom x y z Ueq

H(1A) 1204(8) 1554(14) 2813(14) 14

H(1B) 468(8) 2077(14) 4129(15) 14

H(2) 1207(9) 5469(13) 6029(16) 16

H(3) 2893 7217 -89 16

H(4) 4567 6361 700 18

H(5) 4940 4292 3024 19

H(6) 3667 3017 4596 16

201

Table 6.S7

Table 6.S7. Hydrogen Bonds for P18065.a

D-H A dD-H (A) dH.- -A (A) dD. A (A) LDHA (0)

O(1)-H(1A)- -O(2)#1 0.927(10) 1.559(10) 2.4860(6) 178.9(11)

O(1)-H(1B) 0(3)#2 0.890(10) 2.569(11) 3.1332(6) 122.0(9)

O(l)-H(lB) - 0(4)#2 0.890(10) 1.734(10) 2.6230(6) 176.7(11)

O(2)-H(2) O(4)#3 0.819(10) 2.082(10) 2.8885(6) 168.0(11)

aSymmetry transformations used to generate equivalent atoms:

#1: x, -y +,/2 z - '2; #2: -x, y -,/2 -z + /2; #3: x, -y +3 /2, Z + '/2

202

Table 6. S8

Table 6.S8. Values of Ac During Oxidation Reactions (M-'cm-')

pH Buffer PBA 2-HMPBA 2-CPBA

3.2 sodium citrate 1069 2 1336 3 3386 15

5.0 sodium acetate 962 2 1448 3 3364 9

6.2 sodium citrate 816 5 1521 7 2830 7

7.3 sodium phosphate 1220 8 1474 9 3332 10

7.3 PBS 1029 33 1516 35 2568 266

9.0 sodium borate 1210 6 2025 9 3147 46

10.5 sodium citrate 1791 9 1940 9 3469 40

203

Table 6.S9

Table 6.S9. Second-Order Rate Constants for the Oxidation of Boronic Acids (M-ls-1)a

pH PBA 2-HMPBA 2-CPBA

3.2 (1.3 0.1) x 10-2 (3.8 0.2) x 10- 3 (2.2 0.1) x 104

5.0 (1.2 0.01) x 10-2 (2.4 0.1) x 10-2 (1.7 0.1) x 1 0 -4

6.2 (1.8 0.01) x 10-' (2.0 0. 1) x 10-1 (1.4 0. 1) x 10-4

7.3 2.4 0.2 2.4 0.2 (1.5 0.1) x 10-4

7.3a 6.5 0.2 3.5 0.1 (2.8 0.5) x 10-4

9.0 24 3 4.5 0.4 (7 .1 2 .6 ) x 10-4

10.5 53 1 4.7 0.2 (4.1 0.1) x 10-2

aEach value is the mean SD from initial-velocity experiments performed in triplicate

with at least three different concentrations of boronic acid and peroxide. bDetermined

in PBS.

204

TabMe I 1m A

Table 6.S10. Calculated Energies of Starting Materials, Transition States,

and Products (Hartrees)

Structure HF Zero Point H G ImaginaryFrequency

PBASM -559.3340606 -559.192210 -559.181182 -559.227363 -

PBATS -559.2857740 -559.147518 -559.135260 -559.184357 748.1166

PBAProd -483.4941140 -483.364085 -483.354294 -483.399097 -

2-HMPBA _SM -597.4252050 -597.275288 -597.263948 -597.311131 -

2-HMPBA _TS -597.3757670 -597.229576 -597.217704 -597.26600 743.2438

2-HMPBAProd -521.5891240 -521.450972 -521.441425 -521.485137 -

2-CPBA _SM -671.4918544 -671.360385 -671.348323 -671.397383 -

2-CPBA _TS -671.4329050 -671.305185 -671.292760 -671.342679 757.4783

2-CPBAProd -595.6372030 -595.517838 -595.507956 -595.552297 -

205

Table 6.S11

Table 6.S11. Calculated Free Energies ofActivation and Reaction (kcal/mol)

Boronic Acid AG+ AGreaction

PBA 27.0 -61.0

2-HMPBA 28.3 -62.4

2-CPBA 34.3 -50.4

206

T tl 6.1

Table 6.S12. Boron-Oxygen and Competing Donor-Acceptor Interaction Energies

Boronic Acid Donor Acceptor E2 (kcal/mol) F1

PBA noip PB 35.2 0.118n02,p PB 37.4 0.122noi,p PB 20.4 0.089

2-HMPBA n02,p PB 43.9 0.131noi,p U*C-H 9.8 0.080

noi,sp2.2 PB 9.1 0.069

noi,p PB 4.0 0.0392-CPBA n02,p PB 47.7 0.136

noi,sp2.2 r*C=O 3.3 0.057nO,p x*C=O 66.3 0.151

207

HO'B.OH + H20+

Hg, OHHO-B-

APhonylboronic Acid (PSA) monoanlon

HO ,BOH HOl *H 20 HO- H20 B-0 H. HOpo

OH pN x73

2-(Hydmxym-thyl)phen lbornlc Acid Benzoxaborole monoanlon(244MP8A)

HO, OHaB 0 - H2 0

~fOH

2-Carboxyphonwbomrnlc Add(2-CPSA)

H O ,H + H20

H O

dianlon

HO, B-0

Benoxaboroloe 81

t

0

3

+H* OilC 2 C1 C7

HO C K

onC4 C3

monoonlon

208

Figure 6.1

Figure 6.1. Structure of Phenylboronic Acid (top), Benzoxaborole (middle), Benzoxaborolone

(bottom), and Hydration and Protonation States that are Relevant in Aqueous Solution. pKa values

are given for phenylboronic acid,1 33 benzoxaborole,' 2 7 and benzoxaborolone (this work). The

ORTEP diagram of the protonated monoanion was determined by X-ray crystallography.

209

PBA

2-HMPBA

physiological pH

- 2-CPBA*

3 4 5 6 7 8pH

9 10 11 12

210

Figure 6.2

102

10

100

10

10-4

2

Figure 6.2. pH-Dependence of the Rate Constant for the Oxidation of Boronic Acids by Hydrogen

Peroxide. Data were fitted to eq 3 with pKa values in Figure 6.1 to give ki = (1.1 0.2) x 10-2 M-

Is- and k2= 49 4 M--'s- 1 for PBA; ki = (3.4 0.4) x 10-' M-'s-1 and k2 = 4.2 0.2 M-'s-' for 2-

HMPBA; and ki (1.6 0.1) x 10-4 M-'s-1 and k2 = 9.7 1.3 M-1s-' for 2-CPBA.

211

Figure 6.3

B C

p

6

212

Figure 6.3. Calculated Structure of the Transition State for the 1,2-aryl Shift During the Oxidation

of (A) PBA, (B) 2-HMPBA, and (C) 2-CPBA by Hydrogen Peroxide. Structures were optimized

at the M06-2X/6-31 1+G(d,p) level of theory using the IEFPCM solvation model.

213

Figure 6.4

A tno

B no,

PBA

B

2-HMPBA

CD

n.2-CPA2kc-HMmP l

2-CPBA 2-HMPBA

nokc.Vmo

2-CPBA

214

Figure 6.4. Images of Key Orbitals in the Transition State for the 1,2-aryl Shift During the

Oxidation of PBA, 2-HMPA, and 2-CPBA. Images were created with NBOView 1.1. The energy

is for the depicted donor-acceptor interaction (Table 6.S12). (A) A hydroxyl oxygen of PBA

donates electron density from a p-orbital into the vacant p-orbital of boron. (B) The bridging

oxygen of 2-HMPBA donates less electron density from its p-orbital than in panel A. (C) The

bridging oxygen of 2-CPBA donates still less electron density from its sp2.2 orbital than in panels

A or B. (D) The bridging oxygen of 2-HMPBA engages in a competing interaction with the U-*

orbital of a benzylic C-H bond. (E) The bridging oxygen of 2-CPBA donates strongly into the

Z* c=o orbital (i.e., ester resonance); its p-orbital is nearly orthogonal to the p-orbital of boron.

215

Figure 6.S1

HO B OHo XOH

e dianion

SF JEt Of

5 M NsOH

pH-13.0

\\ pH=12.8

pH=12.7

pH-12.6

pH=12.0

\\ pH-10 4

pH=8.8

pH-7.3

pH-G I

pH-5,8

pH=4.8

pH-3.2

f1 (ppm)

216

Figure 6.S1. Representative 11B-NMR Spectra Used to Determine the pKa Values of 2-CPBA. A

solution of 2-CPBA (0.10 M) was prepared in 10 mL of 1 M aqueous NaOH containing D20 (5%

v/v). The solution pH was adjusted with 10 M NaOH or concentrated HC1, and 650 PL samples

were removed at the designated pH values and analyzed by 1 B NMR spectroscopy, referencing to

the solvent peak except in the case specified. The pKa was calculated by fitting the fraction of

dianion/monoanion to the expected speciation based on pH. The shift observed in the low pH

samples is likely affected by a strong shift in the solvent reference peak, as the sample with external

reference under extremely low pH conditions does not show the same shift.

217

Figure 6.S2

300Wavelength (nn

- PBA.--- phenol- 2-HMPBA- - -- 2-hydroxymethylphenol

2-CPBA-- -- sialic acid

350

1.0 mM PBA + 5 mM H202

0.5 mM 2-HMPBA + 5 mM H202

1.0 mM 2-CPBA + 25 mM H2O2

2Time (min)

4 6

218

I' ~./I - 'a

A

CD

cc0

B

20Ch

5-

4-

3-

2-

1 -

0-2

1.5 -

1.0-

0.5-

i0

0.0 00

Figure 6.S2. UV-Spectroscopic Basis for Assays of the Oxidation of Boronic Acids. (A) UV

spectra of PBA, 2-HMPBA, 2-CPBA and their phenolic oxidation products. (B) Representative

raw data showing the change in absorbance upon oxidation in PBS buffer.

219

Figure 6.S3

35-

30

25-C(D

20-

0.4

PBA Oxidation (5 mM H 2 0 2)

R2 = 1.000

0.6 0.8 1.0[PBA] (mM)

2-HMPBA Oxidation (5 mM H 20 2)

R2 = 0.9981

0.25 0.35 0.45 0.55[2-HMPBA] (mM)

40-

-30

20-0

Q.10 -

0

PBA Oxidation (1 mM PBA)

2 = 0.9999

2 4 6

[H 202] (mM)

,2-HMPBA Oxidation (0.5 mM 2-HMPBA)

.2 18-

o 16

E 12 R2 = 0.9994

02 10

)1%

6 8[H202] (mM)

10

2-CPBA Oxidation (50 mM H202)

R2 = 0.9982

2-CPBA Oxidation (1 mM 2-CPBA)

R2= 1.000

0

CL>4

10

30 50

[H202] (mM)70

220

0

CL

E

0

10-

8-

6-

A.

0

CL

A)

15-

10-

5-

0.2 0.4 0.6 0.8 1.0[2-CPBA] (mM)

Figure 6.S3. Kinetic Traces of the Oxidation of Boronic Acids by Hydrogen Peroxide at pH 7.4.

Second-order rate constants derived from these data are listed in Table 6.S 1.

221

222

Figure 6.S4

A

8 .5 8.0 7.5 7.0 6 .5 6.0 5.5

f1 (ppm)50 4.5 4.0 3.5 3.0 2.5

223

72 h

Phenol

45 min

Initial

72 h

B

8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0

f1 (ppm)4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6

224

o-Hydroxymethylphenol

45 min

Initial

.....................

8.0 7.5 7.0 6.5 6.0

fi (ppm)

5.5 5.0 4.5

225

C

72 h

Salicylic Acid

45 min

Initial

4.0 3.5

AA 2-CPBA

v Salicylic Acid

1 Week

1 Day

-42 Minutes

-40 Minutes

~38 Minutes

-36 Minutes

-34 Minutes

-32 Minutes

~30 Minutes

-28 Minutes

~26 Minutes

~24 Minutes

-22 Minutes

-20 Minutes

-18 Minutes

-16 Minutes

-14 Minutes

~12 Minutes

-~10 Minutes

-8 Minutes

-6 Minutes

-4 Minutes

~2 Minutes

Initial

8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5

f1 (ppm)

226

Figure 6.S4. 1H-NMR Spectra Acquired During the Oxidation of Boronic Acids: (A) PBA, (B)

2-HMPBA, and (C) 2-CPBA. Solutions (50 mM) were prepared in PBS. A solution of sodium

bisulfite (0.5 M) in D20 was prepared as to quench the oxidation reaction. A 2-mL aliquot of the

boronic acid solution was placed in a 1-dram vial, and 100 pL was added to 600 pL of the quench

solution to provide an initial sample. A 50-ptL aliquot of 30% (v/v) hydrogen peroxide solution

was added to each boronic acid solution and the resulting solutions were stirred for 45 min. A 100-

[tL aliquot of the reaction mixture was added to 600 [IL of the quench solution, and the samples

were analyzed by 'H-NMR spectroscopy. Solutions of phenol, 2-hydroxymethylphenol, and

salicylic acid were subjected to the same quench and analysis. The reaction mixtures were

incubated for 72 h and another aliquot was quenched and analyzed. No 1H-NMR peaks

corresponding to oxidation of the phenols were observed. (D) Reaction conditions: 0.99 mL of 2-

CPBA (40 mM) in 0.10 M sodium phosphate buffer pH 7.3, containing D20 (10% v/v) was added

to an NMR tube, and the sample was locked and shimmed in an NMR spectrometer. Hydrogen

peroxide (10 [pL of a 30% v/v solution) was added to the tube, the contents were mixed by thorough

shaking, and the tube was returned to the spectrometer and analyzed (16 scans, 5-s relaxation

delay) obtaining a spectrum approximately every 2 min. Spectra of the starting material and

product alone were obtained in the same buffer solution.

227

Figure 6.S5

A

B

228

Figure 6.S5. Optimized Structure of Each Hydrogen Peroxide Complex (A) and Product (B)

During the Oxidation of Boronic Acids. Left: PBA; middle: 2-HMPBA; right: 2-CPBA. The

structure of each transition state is shown in Figure 6.3.

229

Figure 6.S6

-0- PBA--- 2-HMPBA

+ 2-CPBA

115

0cc

C

20m0CDcIn

50-

40-

30-

20-

10-

0-0 5

pH10

230

. I .

Figure 6.S6. Graph of the Extent of Protodeboronation of Boronic Acids at Three pHs. Solutions

of PBA, 2-HMPBA, and 2-CPBA (50 mM) were prepared in 1.0 M sodium phosphate buffer

containing D 20 (5% v/v) at pH 2.2, 7.3, and 12.2. A 1.0-mL aliquot of each solution was placed

in sealed 1-dram vials. The vials were heated at 95 'C with shaking at 650 rpm in a VWR

temperature-controlled heating block/shaker. After 2 h, the vials were cooled on ice, and the

solutions were transferred to NMR tubes. Protodeboronation was quantified by "B-NMR

spectroscopy, comparing the area under the boric acid peak with the area under the sum of the

boric acid peak plus the PBA, 2-HMPBA, or 2-CPBA peak.

231

6.6 Atomic Coordinates of Optimized Structures

PBASM

2.61827900

1.23227000

0.44567400

1.11039300

2.49341900

3.25563200

3.20433000

0.73206100

0.51653000

2.98178700

4.33412300

-1.70824700

-1.80290300

-1.58202600

-2.44895400

-1.72543100

-3.15761200

-3.20705000

-1.17147900

1.12521200

1.19868800

0.05242800

-1.17977300

-1.27019400

-0.11300800

2.03264900

2.16218300

-2.07939900

-2.24040600

-0.17649900

-0.91796700

-0.50654400

1.46575300

1.62446400

-0.20430700

-0.28048700

-0.85707400

0.13131400

-0.19495500

-0.05840900

0.09478700

0.10838800

-0.03071300

-0.18366700

-0.30985700

-0.05960600

0.24074700

-0.01828700

-0.29045500

1.16823200

2.02803100

0.69708600

0.31474600

-1.09532100

-0.94207200

-0.16201400

0.24414200

232

2.49121700

1.09967700

0.38654400

1.07729300

2.46838500

3.17775000

3.03998500

0.57097000

0.52012800

3.00043200

4.26020500

-1.67877900

-1.99296900

-1.73479800

-1.69954100

-1.30653200

-3.04840300

-3.07996000

-1.33526700

1.20031600

1.20925000

0.01131700

-1.19829000

-1.21213800

-0.01176700

2.13549300

2.15698900

-2.12904900

-2.15603800

-0.02070800

-1.25390300

-1.10141400

1.10398800

1.90932900

0.05944500

0.10635700

-0.82699600

-0.01736500

0.10719200

0.04165700

0.00406800

0.08020200

0.14777700

0.15992400

0.12162200

0.00923200

0.06568400

0.19375500

0.21479500

-1.12350200

-2.01739700

-1.29646400

-0.77570800

0.87929500

1.51030400

1.73848500

-0.51049100

233

PBA Tq

-2.22122800

-0.90317800

-0.25258900

-0.90317400

-2.22122400

-2.88371500

-2.73216800

-0.36702000

-0.36701400

-2.73216200

-3.91111800

2.56740400

3.29641300

2.56736900

3.29637700

1.04154500

2.08335800

1.20337100

1.20945000

-0.00000300

-1.20945600

-1.20337500

-0.00000200

2.14381700

2.13258900

-2.13259600

-2.14382100

-0.00000100

-1.21056000

-1.18775000

1.21057300

1.18777300

-0.00000400

0.00000300

-0.19541500

0.24789000

0.46768800

0.24787500

-0.19543000

-0.41849200

-0.36487400

0.43154100

0.43151400

-0.36490100

-0.76124800

-0.37254100

-0.99412300

-0.37256200

-0.99414700

0.93510400

0.03430700

234

PBAProd

2-C3n A _NM

0.41434200

1.04169400

2.42042500

3.19465600

2.58908500

1.20401700

2.89200400

4.27135300

3.20184800

0.74758100

-1.17588200

0.01643200

-0.05817500

0.28932000

-1.21183800

-1.80319300

-1.93483900

-1.91029400

-3.23159400

-3.38642600

-0.37053600

0.86602700

0.98129800

-0.17857500

-1.42620900

-1.52100900

1.95151400

-0.11212000

-2.32115000

-2.49947000

-0.08785700

1.97900200

2.44215100

2.77229100

1.39468800

-0.63774800

-0.66346200

-0.05883500

-0.47335800

-0.67604800

0.11937500

-0.04542400

-0.19748200

-0.18016600

-0.01565400

0.12998700

-0.32313500

-0.29106000

-0.00157700

0.25482000

0.25203200

-0.03121800

-1.02562100

0.67474600

0.34513100

-0.98448600

1.37108700

2.11518100

-0.90353800

0.03442300

235

2-CPBATS

0.38149400

0.92588600

2.29987100

3.12209500

2.57446800

1.19349600

2.73241500

4.19592200

3.22592700

0.76283200

-1.30986200

-0.13613600

-0.43168200

0.21352400

-1.23675000

-1.33459300

-1.97462900

-2.28724000

-3.09004200

-2.94131500

-0.39892900

0.86752600

1.02811000

-0.09856700

-1.36686900

-1.52371800

2.01395100

0.01360700

-2.23189900

-2.51075800

-0.00773600

1.94260200

2.18900700

2.86088400

1.42299300

-0.64591800

-0.57191900

-1.44928100

-0.65071900

0.01673600

0.08455300

-0.12218500

-0.25411100

-0.18823700

0.01097400

0.13747600

-0.39344700

-0.28426100

0.06349000

0.27272800

0.52607600

-0.10908600

-1.13779200

0.36923400

0.61291800

-0.71374900

1.63449500

1.40052800

-1.31895600

-1.99482600

236

2CPBA_ DroA

-0.29330200

-0.52874300

-1.84036900

-2.89871000

-2.64392300

-1.34005700

-2.03018100

-3.91480500

-3.46267100

-1.11251500

2.04705400

0.63547600

0.71313500

0.49436300

1.87347000

0.98709900

3.29467300

3.29324300

-0.60220800

0.76917600

1.21960000

0.32098800

-1.04717300

-1.51335300

2.28734300

0.68413200

-1.75477000

-2.57162300

-0.22325500

1.71795800

2.09172100

2.57706000

1.11547100

-1.08468100

-0.74172200

-1.70007100

0.10280000

0.05950300

-0.05803200

-0.11572500

-0.06467300

0.03875000

-0.10224200

-0.20477300

-0.11273800

0.07101200

-0.03707000

0.18050500

1.20750400

-0.47749100

-0.16693600

0.19912500

-0.15154100

-0.09987100

237

2-HMPBASM

C 0.43032800

0.96250100

2.31914100

3.18193700

2.67433000

1.30709300

2.67766600

4.24907700

3.35861700

0.93063000

-1.16769100

-1.28424600

-1.84652700

-1.72503900

-2.67912400

-3.20738200

-3.07902700

-0.12504800

0.04805800

-0.65426600

0.62903900

0.88150800

-0.20812000

-1.50807200

-1.73456000

1.90365000

-0.05348000

-2.34932100

-2.75029000

-0.50145000

1.06743900

-0.91129700

-1.06354700

-1.08773800

-0.46044100

0.49909600

1.66108900

2.86105000

238

0.10103600

0.01405900

-0.14134900

-0.21309300

-0.12696100

0.02932100

-0.20194000

-0.33335900

-0.18207200

0.09748600

0.27329600

0.29394700

-0.96697900

1.46168100

1.35036800

-0.84432200

-0.81299000

0.12093600

0.05721500

2-HMPBATS

0.33428700

0.94073000

2.32028200

3.09347700

2.48660700

1.10088000

2.77370000

4.17135400

3.10292600

0.63262700

-1.35158200

-1.22477200

-1.36384500

-1.99240700

-2.27069500

-3.16610300

-2.97411700

-0.02563400

0.22442000

-0.63323100

0.60943300

0.73247900

-0.42430100

-1.67319400

-1.78881000

1.71175300

-0.35815700

-2.56413800

-2.76175300

-0.14543100

1.31811700

-0.90695700

-0.49048700

-1.40981800

-0.92140200

-0.49538900

1.74270500

2.90250100

0.08100900

-0.07942200

-0.19326300

-0.14757800

0.01298900

0.11644900

-0.29874900

-0.23101700

0.04985100

0.21252000

0.49782900

0.37094100

-0.64321500

1.68258000

1.65485100

-1.22776600

-2.06806000

-0.01058100

-0.24331100

239

2-HMBPAProd

-0.29330200

-0.52874300

-1.84036900

-2.89871000

-2.64392300

-1.34005700

-2.03018100

-3.91480500

-3.46267100

-1.11251500

2.04705400

0.63547600

0.71313500

0.49436300

1.87347000

0.98709900

3.29467300

3.29324300

-0.60220800

0.76917600

1.21960000

0.32098800

-1.04717300

-1.51335300

2.28734300

0.68413200

-1.75477000

-2.57162300

-0.22325500

1.71795800

2.09172100

2.57706000

1.11547100

-1.08468100

-0.74172200

-1.70007100

0.10280000

0.05950300

-0.05803200

-0.11572500

-0.06467300

0.03875000

-0.10224200

-0.20477300

-0.11273800

0.07101200

-0.03707000

0.18050500

1.20750400

-0.47749100

-0.16693600

0.19912500

-0.15154100

-0.09987100

240

241

Chapter 7

Strain Inactivation in a Circular RNase 1 Zymogen

Contribution:

I performed all experiments, designed the constructs, and wrote the initial manuscript. I

contributed to the preparation of all the materials and analysis of the data.

This chapter has been prepared for publication as:

Windsor, I. W.; Graff, C.; Raines, R. T., Strain inactivation of a circular RNase 1 zymogen. In

preparation.

242

A hstr2ct

Latent reservoirs thwart a reliable cure for HIV as viremia rebounds upon halting antiretroviral

therapy, making HIV infection a chronic illness. The strategy nature devised to enable cellular

production of toxic proteins - expression of inactivated, zymogen forms that are proteolytically

activated at the site of action - provides a framework to engineer prodrug variants that target HIV

infected cells. De novo engineering of zymogens has only achieved limited success. Here, we

report the design and characterization of HIV protease-activated zymogens of RNase 1. Following

our initial success of sterically occluding the active site by circularly permuting RNase A to

connect the native termini with a protease substrate, we created a circular variant of RNase 1.

Installing a linker of insufficient length to permit folding of both the N- and C-terminal secondary

structural elements imposes strain on the active site and inactivates RNase zymogens up to 28,000-

fold. Disulfide bonds limit distortion of the fold to the termini, allowing these zymogens to

maintain conformational stability above physiological temperature. Though a moderate reduction

in efficiency is observed, the strained zymogens are still readily cleaved by HIV- 1 protease, which

restores wild-type ribonucleolytic activity. Zymogens of RNase 1 hold the promise to engender

cytotoxicity specifically to HIV infected cells and could find utility as part of a combinatorial viral

eradication therapy.

243

7.1 Introduction

The treatment of HIV remains a fundamental challenge in medicine. Numerous antiviral

compounds that target the many aspects of the viral lifecycle have achieved clinical utility.370

Antiviral compounds can suppress a patient's viral load to below detectable limits, but treatment

must continue as viremia rebounds upon suspension of therapy. 37 1 These treatments have

transformed HIV from a death sentence to a chronic illness; however, a cure remains elusive.3 72 A

key challenge is latency of the virus. Early in HIV infection, a subset of CD4+ cells become

infected with the virus that quiesce to harbor the integrated HIV provirus without producing viral

RNA or proteins. 373 An emerging eradication approach is stimulation of virus production to induce

cytopathic effects; however, activation alone is proving insufficient to kill latently infected cells. 43

Recently, it has been proposed that strategies to target cells that produce viral proteins with

cytotoxic therapeutics are a missing piece of eradication approaches.3 74

Intracellular production of cytotoxic proteins requires precise regulation and often includes

safeguards to attenuate activity until arriving at the intended location. Proteases involved in blood

coagulation and digestion as well as the apoptotic capsases are expressed as zymogens: an inactive

precursor that requires subsequent proteolysis to obtain the mature, active form. The concept of a

zymogen provides a conceptual framework to engineer cytotoxic enzymes as pro-drugs activated

by viral proteins, specifically proteases. Altering caspase 3 for activation by HIV-1 protease has

demonstrated the potential of zymogens as antivirals. 375 Our lab engineered the first zymogen de

novo by circularly permuting ribonuclease (RNase) A to connect the native N- and C-termini with

a linker containing a protease recognition sequence. 376 Several iterations of this strategy produced

RNase A zymogens activated by plasmepsin II, NS3 protease, and HIV-1 protease.377-378 We

244

procs-d the linker blocks I the Activ s;+i -dA 1cudes sbra cte birng as A - t% 964-fold%_LA%' ' LJ L'.% J 1 %''1Xxo LaiJ'. LV..'LI v % 3 1 IL.A IL.. "~J~ LILLLI.L UL1JAA'II& "0 1J L%' __ J - .LL%&

inactivation was achieved. This circular permutation strategy was replicated by Rib6 and

coworkers to create zymogens of the amphibian RNase homolog, Onconase"m, though minimal

inactivation, up to 1.3-fold, was observed.379

Strain has also been employed in zymogen designs. Ozawa and coworkers created circular

variants of firefly luciferase using intein-mediated cis-splicing to connect the termini with a

caspase 3 substrate. 380 The ensuing zymogen is an apoptosis probe with a <10-fold change in signal

in cellulo. Loh and coworkers later pursued strain to inactivate the bacterial RNase, barnase;

however, these proteins were found to be folded and active only at low temperatures. 381 Here, we

extend our original zymogen engineering strategy of sterically occluding the active site with the

imposition of strain to create zymogens with unprecedented inactivation that is relieved by the

action of HIV- 1 protease.

245

7.2 Results

7.2.1 Design of a Circular Zymogen Construct

Circular RNase 1 zymogens were created by intein-mediated cis-splicing with the Nostoc

punctiforme (Npu) DnaE split intein.382 The Npu intein utilizes a "CFN" N-terminal splice

junction.383 We utilized the "CFQ" sequence found starting at residue 58 of human RNase 1. We

omitted the 4-residue C-terminal extension (125-128) of RNase 1 that is not important for

activity.384

7.2.2 Design and Assessment of Glycine (G) Series of Zymogens

Previous RNase A zymogens exhibited modest inactivation suggesting a 14-residue linker would

not effectively block the active site of RNase 1. We designed a series of zymogens to test this

hypothesis by bringing the linker closer to the active site with progressively shorter linkers.

Additionally, we employed a phage display substrate that is among the most efficiently cleaved by

HIV-1 protease (SGIFLETS).207 We flanked this sequence with varying numbers of glycine

residues to create the G series of zymogens (Table 7.1). A plasmid encoding the intein fragments

flanking a circularly permuted RNase 1 connected by the substrate linker directed the expression

of the two soluble proteins and another insoluble one corresponding to the intein fragments and

the circular RNase 1 (Figure 7.S1), indicative of intein-mediated cyclization. 382 These zymogens

all exhibited roughly two orders of magnitude less activity than wild-type RNase 1 and no trend

in inactivation was observed despite a progressive reduction in thermal stability (Figure 7.1).

246

72 . Desicn and A ssisscmnt nf Stmraned (tvr) s c-f 7 mgns-tr-c

We next sought to remove residues from the termini of RNase 1 to bring the linker closer to active

site with the intent of imposing strain. Crystallization optimization of RNase 1 revealed the first 7

N-terminal residues can be removed with only an order of magnitude loss in activity.385 Lys7 is

important for binding the phosphoryl group of the RNA backbone in the P2 subsite.1 94 386 Folding

studies of RNase A demonstrated that residues beyond 122 can be removed without diminishing

activity or stability. 387 We therefore constrained truncation to residues 7 through 122. The terminal

residues of our protease substrate are both serine. Removal of the three N-terminal (AKES) and

two C-terminal (ASV) residues and connecting them with the protease substrate would replace the

truncated serine residues. This construct was selected as a starting point for the strained (Str) series

of zymogens and further truncated by progressively removing residues 4, 5, and 6 (Table 7.1). The

Stri zymogen showed a similar two orders of magnitude reduction in activity similar to that of the

glycine series. Further truncations substantially reduced the activity of the zymogens with a

disproportionately smaller reduction in stability (Figure 7.1).

7.2.4 Assessing Zymogen Activation by HIV-l Protease

The zymogens exhibiting substantial inactivation, i.e., kcat/KM < 104 M-ls-1, were subjected to

activation studies with HIV-1 protease. Proteolysis by the pathogenic protease must be limited to

the inactivating linker of the zymogen. We determined that wild-type RNase 1 is not cleaved by

HIV-1 protease (Figure 7.S2). Next, we digested zymogens with HIV-1 protease and measured the

final activity. Proteolysis restored near wild-type activity to the Str2-4 zymogens (Table 7.2).

247

Finally, we developed a continuous assay to measure the kcat/KM value for HIV-1 protease cleavage

of the zymogens (Table 7.2 and 7.S2, Figure 7.S3).

7.2.5 Modelling the Structural Basis of Str2 Zymogen Inactivation

The zymogen Str2 possessed the best combination of properties: catalytic inactivation, thermal

stability, and proteolytic activation. Accordingly, we modelled the Str2 zymogen with Rosetta to

reveal the structural origins of catalytic inactivation in our zymogen design. We examined the top

ten scoring models of four-hundred and found the N-terminal a-helix was nearly intact while the

C-terminal P-strand was distorted in each case (Figure 7.2A and 7.2B, Figure 7.S4A).

7.2.6 Modelling the Structural Basis of Proteolytic Activation of Str2 by HIV- 1 Protease

We again used modelling with Rosetta to reveal the structural basis of recognition of the

inactivating linker by HIV-1 protease. We examined the top ten scoring models of two-hundred

and found the N-terminal a-helix and C-terminal P-strand must be completely unfolded to

accommodate HIV- 1 protease with its flaps closed upon a substrate installed in the linker (Figure

7.2A and 7.2C, Figure 7.S4B).

248

'7. Discussion

The mechanism of our zymogen inactivation relies on two key strategies: steric occlusion and

strain. Roughly two orders of magnitude in activity are lost by installing the linker across the active

site, as observed with previous circularly permuted RNase A zymogens. We suspect this is the

limit of inactivation imposed by blocking the active site with a flexible linker. Further inactivation

was achieved through local installation of strain, a violation of design criteria in the previous

circularly permuted zymogens enabled here by intein-mediated cyclization.37 6 The key catalytic

residues are located in secondary structural elements at the termini that are not stabilized by

disulfide bonds, unlike the core structure (Figure 7.2). Through installation of strain by progressive

truncation, we were able to perturb active site residues specifically to achieve unprecedented

inactivation of a de novo engineered zymogen without compromising global stability. Our

structural modelling revealed the how steric occlusion and strain are operational in our zymogen

design. The top 10 scoring structures are predicted to be similar in energy, yet conformationally

divergent in the linker. This suggests the zymogen linker is conformationally dynamic but also

unambiguously demonstrates that both termini cannot simultaneously adopt the wild-type fold.

Despite the increased proximity of our linker to the active site compared to previous

zymogens, our designs maintained efficient activation. We observed two-orders of magnitude

reduction of the second order rate constant of HIV- 1 protease toward the zymogens with respect

to a fluorogenic peptide substrate, which is cleaved with a kcat/KM value of 5.0 x 105 M1 s-1.223 Our

chosen substrate is among the most efficiently hydrolyzed by HIV-1 protease and the activity is

reduced to rate constants similar to endogenous substrates when incorporated into zymogens. 2 86

As indicated by our modelling, the loss in catalytic efficiency results from the energetic cost of

249

unfolding the N- and C-terminal structural elements of the zymogen to accommodate the flaps of

the protease. Not all proteases employ flaps and zymogens may be more efficiently activated by

other pathogenic proteases like plasmepsins which have an open active site cleft.

RNases are also a privileged class of enzymes for the creation of zymogens due to their

therapeutically desirable properties: cell permeability and cytotoxic activity. 152 We anticipate

translation of these results will depend on successful evasion of the cytosolic ribonuclease inhibitor

protein and the inactivating linker being sufficiently orthogonal to avoid cleavage by endogenous

proteases while lacking immunogenicity.1 83

250

7A.4 M e-1is and Methods

7.4.1 Protein Expression and Purification

The RNase 1 gene was obtained from a previously reported molecular clone.388 Intein fragments

were obtained from previously described vectors provided by Tom Muir. 3 89 The circular RNase 1

zymogen construct was initially prepared using the linker sequence from a circularly permuted

RNase A zymogen containing the p2/NC cleavage site. PCR was used to prepare five DNA

fragments with terminal homology: the pET32b plasmid, NpuC, a C-terminal RNase fragment, an

N-terminal RNase fragment, and the NpuN fragment. Gibson assembly was used to combine

fragments into the final expression constructs. Modification of the original plasmid was done by

producing RNase fragments with PCR by including the new linkers and truncations and combining

it with a plasmid fragment that contained both intein fragments.

RNase were expressed and purified as previously. 388 All chromatography and assay buffers

were treated with DEPC prior to use, with the exception of Tris which was added from RNase free

stocks (Invitrogen). Zymogens were folded in the presence of 0.5 M arginine-HCl and were

purified with additional chromatography on a MonoS column (GE Healthcare) to ensure purity

and removal of contaminating RNases. HIV-1 protease was expressed and purified as described

previously. 2 23 Protein purity was confirmed by SDS-PAGE and concentrations were determined

using the PierceM BCA assay kit (ThermoFischer Scientific). HIV-1 protease was also treated

with 10 mM DTT at 4' C for 4 h to inactivate contaminating RNases. The ensuing prep was

desalted with a HiTrapo column (GE Healthcare) and did not possess detectable RNase activity.

Analysis of protein purity and other gel-based assays were performed with Any kDTM Mini-

PROTEAN® TGX TM precast protein gels (Bio-Rad).

251

7.4.2 Enzyme Activity Assays

The kinetics of RNA hydrolysis was monitored by the increase in fluorescence intensity of a

doubly labeled fluorogenic substrate, 6-FAM-dArUdAdA-6-TAMRA, upon exposure to

RNases.3 90 Initial (1o) and final (If) intensities along with linear slopes (m = AI/At) or the second

derivative of quadratic fits (2a = A2I/At2) were measured and used to calculate values kcat/KM.

Assays required 10 pM to 1 pM to achieve velocities that reached 10% RNA turnover within

several minutes. Activated zymogens were prepared for kinetic testing by digesting 50 !LM

zymogen with 52 nM HIV-1 protease at 370 C. Continuous activation assays were performed with

2.6 nM HIV-1 protease at room temperature. Fluorescence intensity was measured with a M1000

microplate reader (Tecan) by monitoring emission at 515 nm with excitation at 493 nm. Assays

were performed in quadruplicate in a flat, black 96 well plate (Corning). Assay buffers were DEPC

treated and consisted of 50 mM of either Tris pH 7.4 or Acetate pH 5.0 with 100 mM NaCl.

Kinetics of RNase activity of were determined with equation 1 by assaying initial velocities

under second-order conditions.

kcat __ At (7.1)KM [RNase] (If-Io)

Kinetics of zymogen cleavage by HIV-protease was determined with equation 7.2 by

assaying the increase in RNase activity observed upon the addition of protease.

A2

Jkcat k __ _tz_(7.2)KM HIV Protease K kcat [Zymogen][HIV Protease](If -I,,)

KM Zymogen, Activated

252

V\alues rof ka/K' At H- 7.4 thJ I rpItimna Nf Ras 1 are reportetd n the main tpvt 391T " -7 - 'VLdL' A LIVI Lk J A I ._, LI'W, yJ. IF JL LI I IL4 %J L -L A. I , L4,I, "X% X' If

These values were also determined at pH 5, the optima of HIV- 1 protease, and used as parameters

in fitting equation 7.2 (Table 7.S2). Values of kcat/KM are reported as the mean of quadruplicates

and the standard deviation.

7.4.3 Thermal Stability Assays

Thermal stabilities of RNases were determined by differential scanning fluorometry. G series

zymogen thermal stabilities were measured with a Bio-Rad CFX connect RT-PCR machine.

Samples of 5 [tM protein with 1% v/v sypro orange (Sigma-Aldrich) in 25 uL in PBS were heated

from 25-95 'C at 1 'C/min. Single fluorescent measurements per degree were reported. Change

in fluorescence per degree was calculated by AFt = Ft+i - Ft-1 with Excel software (Microsoft).

The maximal change in fluorescence and the 5 flanking measurements above and below were fit

with the Gaussian function built into Prism 6 (Graphpad). Values of Tm are reported as the mean

and standard deviation of triplicate measurements.

Str zymogens melts were conducted with 0.6% (v/v) Sypro Orange and 30 ptg zymogen in

a 20 pL final volume of PBS. Melts were observed with a ViiA 7 RT PCR machine (Applied

Biosystems) by increasing the temperature from 20 'C to 96 'C at 1 'C/min in steps of 1 'C. The

Tm was determined with the Protein Thermal Shift software (Applied Biosystems) using the

Boltzman model and reported as the mean and standard error.

253

7.4.4 Modelling with Rosetta

Modeling of the Str2 zymogen and its complex with HIV-1 protease were conducted with the

Rosetta.392 RNase 1 from PDB entry lz7x was extracted from chain A. 182 RNase 1 was circularly

permuted at residue 89 to include the linker between the termini. The the 15 N-terminal and 12 C-

terminal residues were removed to be computationally predicted. KIC loop modelling was

performed using fragments picked by the Robetta Server to prepare 400 models of the zymogen

linker and proximal residues of the native termini.

Modelling of the complex was conducted in two steps. RosettaDock was employed to dock

HIV- 1 protease in the closed conformation bound to the "SGIFLETS" from chain A of PDB entry

6bra into the actives site cleft of the N- and C-terminally truncated structure of RNase 1.264, 395

Docking models were manually inspected to identify orientations with proximal termini of RNase

1 and the substrate in the active site of HIV- 1 protease. Five docking models compatible with a

zymogen-protease complex were subjected to further loop modelled. The docking model that

generated the lowest energy complex model by loop modelling was used to prepare an additional

200 models. The top 10 lowest energy models (Table 7.S3) are shown in Figure 7.S4.

254

Table 7.1

Table 7.1. G and Str Zymogen Designs

Zymogen Linker N-truncation C-truncation

3G GGGSGIFLETSGGG None EDST2G GGSGIFLETSGG None EDSTIG GSGIFLETSG None EDSTOG SGIFLETS None EDSTStrI SGIFLETS KES SVEDSTStr2 SGIFLETS KESR SVEDSTStr3 SGIFLETS KESRA SVEDSTStr4 SGIFLETS KESRAK SVEDST

255

Properties of Str

kcat/Km

(activated)

6.7 0.08 x 107

3.2 t 0.02 x 106

1.4 0.02 x 107

Series Zymogens

Relative

Activity

11,000

2,800

24,000

kcat/KM

(HIV- 1 Protease)

3.9 0.3 x 10'

1.3 0.1 x 103

2.5 0.1 X 103

256

Table 7.2

Table 7.2.

Zymogen

Str2

Str3

Str4

Table 7. Si

Table 7.S1. Activity and Stability of RNase

RNase/Zymogen kcat/KM (M-'s-')

RNase Ia 2.1 0.2 x 107

3G 2.8 0.6 x 10'

2G 4.6 0.2 x 104

1G 3.2 0.2 x 10'

OG 2.1 0.2 x 10'

Str1 8.5 0.2 x 104

Str2 5.8 0.05 x 103

Str3 1.1 10.01 x 103

Str4 5.7 0.03 x 102

N.D. = Not determined.aValues are from ref. 183.

1 and Zymogens

Tm (OC)

57 2

50.4 0.4

48.9 0.3

48.1 0.1

46.9 0.2

N.D.

47.5 0.1

42.6 0.1

42.0 0.1

257

Table 7.S2

Table 7.S2. Str Zymogen Kinetic Parameters at pH 5.0

Zymogen kcat/Km (M-'s-') kcat/Km (M-'s- )Zymogen Inactive Activated

Str2 2.9 0.06 x 10 5.2 0.2 x 105

Str3 7.3 0.08 x 102 7.0 0.3 x 105

Str4 9.2 0.3 x 102 2.4 0.4 x 106

258

TabIc 7.S3

Table 7.S3. Total Scores of Models

Str2Model

(REU)

1 -197.697

2 -196.236

3 -193.333

4 -192.159

5 -191.876

6 -191.655

7 -191.556

8 -191.252

9 -191.203

10 -190.923

REU Rosetta Energy Units

Calculated by Rosetta

Complex

(REU)

-522.837

-518.158

-511.494

-466.197

-457.239

-456.408

-454.677

-453.33

-451.303

-451.246

259

108,

107

106

kt/K 105'(M- 1s-1)

104

103

in2

Zymogen

Figure 7.1

-60

-55

T-50 Tm

(*C)

-45

40

260

Figure 7.1. Activity and Stability of Circular Zymogens of RNase 1. Plotting catalytic efficiency

(kcat/KM) and thermal stability (Tm) of wild-type RNase 1 and zymogen reveals an exponential loss

in activity is associated with a linear loss of stability. Wild-type values are taken from Reference

183. Errors bars for report standard deviation, with the exception of Str zymogen stabilities which

report standard error.

261

C

:4

C

262

Figure 7.2

A B

Figure 7.2. Modelling of Str2 Zymogen and Complex with HIV-1 Protease. The active site

residues H12, K41, and H118 and disulfide-bonded cysteine sidechains are shown as sticks.

Connecting the termini of A. wild-type RNase 1 with a flexible linker and truncating 4 residues

from the N-terminus and 6 residues from the C-terminus prevents both terminal secondary

structure elements from folding. B. The lowest energy structures generated by Rosetta are

predicted to have an unfolded C-terminal P-strand. C. Unfolding of both terminal secondary

structural elements are required for HIV- 1 protease to recognize the substrate containing linker as

modelled by Rosetta.

263

SpectraTMBR

LadderInsoluble

TotalSoluble P dtl

Pre-induction

25 kDa

15 kDa

10 kDa

264

Figure 7.S1

Figure 7.S1. Expression of 3G Zymogen. Induction of expression of the single open-reading-

frame zymogen construct results in a single band in the insoluble fraction (the circular zymogen)

and two bands in the soluble fraction (intein fragments), neither of which are observed pre-

induction, as analyzed by SDS-PAGE.

265

Figure 7.S2

SpectraTM + + RNase 1BR + - HIV-1 Protease

Ladder

25 kDa

15 kDa

10 kDa

266

Figure 7.S2. Proteolytic Treatment of RNase 1 with HIV-1 Protease. After 3 hours of treatment at

370 C, no detectable cleavage of wild-type RNase 1 was observed by SDS-PAGE.

267

Figure 7.S3

-- Str2 zymogenStr2 zymogen +HIV-1 protease

4000

5000-

4000-

3000-

2000-

1000-

U-

00 1000 2000

time (s)

.. 3..3000

268

'

Figure 7.S3. Activation Kinetics of Str2. Hydrolysis of 20 nM of fluorogenic RNase substrate at

pH 5.0 by 10 nM Str2 zymogen lead to a linear increase in product formation (as reported by RFU)

at substrate turnover below 10%. Addition of 2.6 nM HIV-1 protease leads to a time dependent

increase in activity, which is fit by a second-order polynomial.

269

Figure 7.S4

A B

270

Figure 7.S4. Top 10 Scoring Rosetta Models of Str2 and Complex with HIV- 1 Protease. A. The

linker inactivated the zymogen is predicted to occupy multiple conformations which indicates this

region may be conformationally dynamic. B. HIV-1 protease requires partial unfolding of RNase

1 in order to close its flaps on the substrate containing linker.

271

Appendix A

Stilbene Boronic Acids Form a Covalent Bond with

Human Transthyretin and Inhibit its Aggregation

Contribution:

I prepared proteins, established biochemical assays, and performed crystallization and diffraction

analysis. I contributed to ligand design, preparation of final crystallographic models, preparation

of the initial manuscript, and revision of the final manuscript.

This appendix has been published in part, under the same title. Reference:

Smith, T.P.*; Windsor, I.W.*; Forest, K.T.; Raines, R.T., Stilbene boronic acids form a covalent bond

with human transthyretin and inhibit its aggregation. J. Med Chem. 2017, 60, 7820-7834.

*Denotes equal contributions

272

A

Transthyretin (TTR) is a homotetrameric protein. Its dissociation into monomers leads to the

formation of fibrils that underlie human amyloidogenic diseases. The binding of small molecules

to the thyroxin-binding sites in TTR stabilizes the homotetramer and attenuates TTR amyloidosis.

Herein, we report on boronic acid-substituted stilbenes that limit TTR amyloidosis in vitro. Assays

of affinity for TTR and inhibition of its tendency to form fibrils were coupled with X-ray

crystallographic analysis of nine TTR ligand complexes. The ensuing structure-function data led

to a symmetrical diboronic acid that forms a boronic ester reversibly with serine 117. This

diboronic acid inhibits fibril formation by both wild-type TTR and a common disease-related

variant, V30M TTR, as effectively as does tafamidis, a small-molecule drug used to treat TTR-

related amyloidosis in the clinic. These findings establish a new modality for covalent inhibition

of fibril formation and illuminate a path for future optimization.

273

A.1 Introduction

Amyloidosis is a disease caused by the aggregation of a normally soluble protein.396-397

Endogenous proteins can be causal for these diseases, which include Alzheimer's, Huntington's,

and Parkinson's.398 One such protein, transthyretin (TTR),399 is a homotetrameric protein

comprised of four identical monomer units, each consisting of 127 amino-acid residues that fold

into a P-sandwich (Figure A. 1).400~401 The dissociation of the TTR tetramer and aggregation of the

ensuing monomers underlies familial amyloid polyneuropathy, familiar cardiomyopathy, and

senile systemic amyloidosis.3 98, 4 02

TTR is present in both blood (0.3 g/L = 4 pM) and cerebrospinal fluid (0.1-0.4 'M). 399,403

A primary role of TTR in vivo is to transport thyroxin (T4) and retinol, a hydrophobic hormone

and fat-soluble vitamin Al, respectively. Due to the abundance of other lipid-binding proteins (e.g.,

thyroid-binding globulin and albumin), most of the T4-binding sites of TTR are empty in blood. In

cerebrospinal fluid, TTR also binds to $-amyloid, attenuating the neurotoxicity that underlies

Alzheimer's disease.4 03-4 0 5

The binding of a ligand can stabilize the folded state of a protein. 406-409 Evidence for the

coupling of binding and stability appeared as early as 1890, when O'Sullivan and Thompson

demonstrated that cane sugar increases markedly the thermostability of invertase, which is an

enzyme that catalyzes the hydrolysis of sucrose. 4 10 Since then, ligands have been used to enhance

the conformational stability of countless proteins, including TTR. Many small molecules have

been synthesized and tested as putative TTR ligands, and several have demonstrated efficacy in

attenuating amyloidosis. 4 114 12 Most efforts have focused on ligands that bind to the two identical

T4-binding sites at a dimer-dimer interface (Figure A. 1), as such ligands discourage dissociation

274

to the monomeric state. 413 A few of these compounds have become viable treatment options,

including diflunisal, which is an FDA-approved non-steroidal anti-inflammatory drug that has had

limited adoption due to long-term gastrointestinal side-effects associated with cyclooxygenase

inhibition, 414-4 and tafamidis, which is used in the clinic to treat TTR-related amyloidosis.416418

An attractive approach to increase the potency and pharmacokinetics of a ligand is to evoke the

formation of a covalent bond. 4 19-42' This strategy is well suited for TTR amyloidosis, not only

because with an optimized dosage there might be no appreciable competition in serum with the

natural ligand, T4, but also because sustained stabilization of the TTR tetramer deters the

accumulation of monomers that leads to a cascade of aggregation. 422 Previous work by Kelly and

coworkers has shown that small molecules can modify TTR chemoselectively by targeting the c

amino group of Lys 15/Lys 15' at the entry to the T4-binding binding site. This work employed

irreversible reactions, including conjugate addition with activated esters and thioesters 4 23-4 24 and

vinyl sulfonamides, 42 5 and sulfation with aryl fluorosulfates. 42 64 27 Such ligands can, however,

react irreversibly with other plasma proteins,42 5 leading to potential immunogenic responses to the

protein-ligand adduct419 and the generation of potential toxic byproducts. 424

To accrue the benefits of covalent binding without the liabilities, we sought ligands for

TTR that bind in a covalent but reversible manner. Boronic acids interact with Lewis bases in

aqueous media.1 28,428 Boronic acids (including the FDA-approved drug Bortezomib 429 ) are well

known as serine/threonine protease inhibitors, 12 6, 139, 430-431 anti-microbial and anti-cancer

agents, 3 3 8 , 341 and delivery vehicles. 3 68 , 432-434 Boronic acid-based fluorogenic probes have been

developed for sensing both saccharides 435 and reactive oxygen species, 348-349 as well as for

molecular recognition 4 3 6 and protein conjugation. 4 374 38 These applications arise from the ability

275

of boronic acids to form a covalent bond with a Lewis base that is reversible under physiological

conditions.309,439 Notably, boronic acids are benign, as their metabolic byproduct, boric acid, is

present in a normal diet.440

Here, we report on the development of boronic acid-based ligands for the T4-binding site

of the TTR tetramer. An iterative strategy involving chemical synthesis and structure-function

analysis led us to covalent inhibitors of TTR aggregation. This strategy serves as a model for a

new class of amyloidosis inhibitors.

276

A.2 Results

We chose stilbene as a scaffold for the design of an initial series of boronic acid-containing TTR

ligands (Chart A. 1). This scaffold is present in the natural product resveratrol (1), and has been

employed in other TTR ligands. 400 , 412, 424, 441443 Moreover, stilbenes are readily accessible by a

convergent synthetic route, as the two rings can be functionalized separately and then joined with

a Wittig reaction (Schemes A. 1 and A.2).

Resveratrol occupies the T4-binding site with moderate affinity (Table A. 1 and Figure

A. 1).400 We began by replacing the phenolic hydroxyl group, which is known to form a hydrogen

bond with Ser 17/117', with a boronic acid group to generate stilbene 2 (Chart A. 1). Halogen

substitution is known to provide additional van der Waals interactions within the inner pocket of

the T4-binding site, enhancing the affinity of TTR ligands.444 Accordingly, we added a chloro

group meta to the boronic acid moiety to generate stilbenes 3 and 4.

We performed competitive binding assays to compare affinities among the diphenol series

of ligands (Table A.1 and Figure A.SlA). We observed no change in values of Kd,2 between

stilbene 1 and 2. Interestingly, chlorinated stilbene 3 showed a higher value of Kd,2 relative to its

non-halogenated counterpart, stilbene 1. This decrease in affinity contrasts with stilbene 4, which

exhibited increased affinity as a result of chlorination and showed the strongest affinity for TTR

of stilbenes 1-4, having a Kd,2 value of 441 nM.

Next, we assessed the ability of these molecules to inhibit fibril formation by both wild-

type TTR and the common V30M variant, which is associated with familial amyloid

polyneuropathy (Table A. 1 and Figure A.S2A). We found that all of the stilbenes in this series

inhibited aggregation at 7.2 pM and at a 2:1 ligand/protein ratio. Stilbenes 1 and 2 showed 25%

277

and 23% fibril formation for V30M TTR, whereas stilbenes 3 and 4 showed 14% and 11%,

respectively. Herein, we consider compounds that limit aggregation to <1 0% over 96 h as potent

inhibitors, and stilbenes 1-4 did not achieve this threshold.

Next, we solved co-crystal structures of TTR and resveratrol analogs 2-4 to discern if the

boronated stilbenes formed a covalent bond with TTR. To our surprise, each phenylboronic acid

moiety was observed in the "reverse" binding mode (Figure A.2A and 2B) relative to its parent

phenol, resveratrol (Figure A. 1 and 3A). In other words, the boronic acid group resided in the outer

pocket of the T4-binding site No indication of boronic ester formation with amino acid residues

was apparent (Figure A.S4 and S6).

In an attempt to reconfigure this unexpected orientation, we designed a second series of

stilbenes in which a carboxylic acid group was installed at the meta position of the ring not

modified in the first series (Chart A.2). Previous work had shown that incorporating an anionic

substituent into ligands could introduce advantageous electrostatic interactions with Lys15/15' of

TTR.417 445 We hypothesized that this interaction would orient the boronic acid to the inner

pocket of the T4-binding site, and perhaps promote boronate-ester formation.

The first pair, stilbenes 5 and 6, exhibited Kd,2 values in the low micromolar range, 3- and

2-fold higher than those of stilbenes 1 and 2, respectively (Table A.2 and Figure A.S1B). The

installation of a chloro group in stilbenes 7 and 8 restored values of Kd,2 to the high nanomolar

range. Consistent with a decreased affinity apparent in the value of Kd,2, the non-halogenated

stilbenes 5 and 6 also showed a diminished ability to prevent fibril formation under acidic fibril-

forming conditions (Table A.2 and Figure A.S2B). Incubation of wild-type TTR with phenol 5

showed 77% fibril formation and 112% at 7.2 [tM for the V30M variant, respectively. Boronic

278

acid 6 nerformed better as an inhibitor than did phenol 5 for both wild-type TTR and the V3OM

variant (11% and 28%, respectively). The chlorinated pair 7 and 8 showed potent fibril inhibition

(<10%) for both TTRs. These differences were, however, within experimental error. Again, co-

crystallography revealed that boronic acids 6 and 8 were bound in the reverse mode, relative to

their paired phenols. In other words, the carboxylic acid group was in the inner pocket, near

Ser 17/117', and the boronic acid group was in the outer pocket, near Lys15/15' (Figures A.2C

and A.2D, and Table A.S 11). As with boronic acids 2 and 4, boronic acids 6 and 8 did not exhibit

covalent interactions with binding-pocket residues. The structure of 5 in complex with TTR may

include alternative conformations for the phenolic group occupying the inner pocket but we were

not confident placing those conformations (Figures A.S7A and A.S7B).

The final series of stilbenes contained a boronic acid moiety on each ring (Chart A.3). We

synthesized C2 symmetrical diboronic acid 10, which has two meta-chloro and para-boronic acid

groups (relative to the stilbene olefin), as well as the analogous C2 symmetrical diphenol 9.

Additionally, we synthesized stilbene 11, which has one boronic acid group meta to the linker, to

investigate whether this position of the boronic acid enhances interactions with Lys 15/15' in the

outer pocket. We also included tafamidis (12) in this series as a benchmark for our assays.

In the competitive binding assay, diphenol 9 had a Kd,2 value of 819 nM (Table A.3 and

Figure A.SlC). The analogous diboronic acid, 10, had a Kd,2 value of 469 nM, which was the

largest decrease in Kd,2 value that we observed between a boronic acid and its paired phenol. These

two stilbenes comprise the only pair that can be compared directly, as their C2 symmetry precludes

alternative binding orientations. The difference in affinity for TTR was amplified in the fibril

formation assay, where diboronic acid 10 exhibited more potent inhibition of fibril formation than

279

did diphenol 9, both for wild-type TTR (3% versus 12% at 7.2 pM) and for the V30M variant (8%

versus 27% at 7.2 ptM) (Table A. 1 and Figure A.S2C). The binding of diboronic acid 10 with TTR

was reversible, as assessed with mass spectrometry (Figure A.S3). Asymmetric diboronic acid 11

had a value of Kd,2 similar to that of diphenol 9 (864 nM) as well as a similar ability to inhibit fibril

formation, suggesting that the location of the boronic acid plays a role in optimizing interactions

within the binding pocket. Gratifyingly, co-crystallographic data of TTR with either diboronic acid

10 or diboronic acid 11 showed the formation of a boronic ester with Ser 17/117' (Figures A.2E

and A.2F).

280

A.3 Diselsinn

We sought a new class of small-molecule ligands for TTR, which is a validated target for

pharmacological intervention. 4 02 , 416-418 Towards that goal, we investigated the effects of

incorporating a boronic acid substituent on the well-known stilbene scaffold, embodied in

resveratrol (1). The results enabled us to reach two conclusions. First, a boronic acid group can

enhance the potency of a TTR ligand. Second, a boronate ester can form with a weakly nucleophilic

amino-acid residue.

The installation of a boronic acid group tends to increase the affinity of a stilbene for TTR.

The value of Kd,2 for each boronic acid ligand for TTR is lower than (or equivalent to) that of its

analogous phenol (Tables A. 1-3). Moreover, a boronic acid group enhances the ability of a stilbene

to deter the formation of TTR fibrils (Tables A. 1-3). Nonetheless, our structural studies revealed

that in each complex between TTR and a stilbene containing a single boronic acid group, that

group was in the outer pocket of the T4-binding site, exposed to solvent (Figures A.2A-A.2D,

A.S4, A.S6, A.S8, and A.S10).

The modest increase in affinity incurred by adding a single boronic acid group could be

due to a weak noncovalent interaction between the 6-amino group of Lys15/15' and the vacant

p-orbital of the boron. Kelly and coworkers demonstrated that this amino group, which likely has

a low pKa, can act as a nucleophile. 423 In our complexes, the relevant B N distances are 3.3-

3.5 A for stilbenes 2, 4, 6, 8, and 10 (Table A.S1 1). We did not, however, observe electron-

densities or atomic geometries consistent with the formation of a dative N-*B bond between these

two functionalities, nor were any additional hydrogen bonds apparent between TTR residues and

the boronic acid group (Figures A.2A-A.2D, A.S4, A.S6, A.S8, and A.S 10). Additional hydrogen

281

bonds did, however, arise elsewhere in the complexes with a single boronic acid. For example, the

two meta hydroxyl groups in stilbenes 2 and 4 (but not 1 and 3) interact closely (2.0-2.3 A) with

Ser 17/117' (Table A.S1 1).

Stilbenes and similar compounds bind to TTR in one of two modes.4 14 , 422, 427, 446-449

Although a consensus explanation is not apparent, the polarity of pendant functional groups can

play a role in ligand orientation.443 450 In our stilbenes, however, the relevant logP values of phenol

(1.46), benzoic acid (1.87), and phenylboronic acid (1.59) are similar,4 51 suggesting that

hydrophilicity alone contributes little to their orientation in the T4-binding site. The installation of

a chloro group on one ring, as in stilbenes 4 and 8, led to increased affinity and enhanced efficacy

(Tables A. 1 and A.2), but did not affect binding orientation (Figures A.2B and A.2D).

To negate binding orientation as a factor, we designed a class of molecules containing a

boronic acid substituent on each stilbene ring. If such a ligand were to bind to TTR, then a boronic

acid group would necessarily be in the inner pocket of the T4-binding site. We were gratified that

this strategy was successful, as the boronic acid group of both stilbenes 10 and 11 that bound in

the inner pocket formed an ester with Ser 17/117'. Boronic esters have demonstrable utility as

mimics of the high-energy tetrahedral intermediate in reactions catalyzed by serine/threonine

proteases.126, 139,430-431 In those covalent complexes, an active-site serine or threonine residue forms

a tetrahedral, sp3-hybridized boronate ester with a boronic acid group.45 2

Despite the hydration of boronic acid groups in aqueous solution 428 and in marked contrast

to other known boronate esters with proteins, we observe planar, sp2-hybridized boronate esters

with Ser 17/117' of TTR (Figures A.2E and A.2F). We are aware of only one other structure in

which a planar boronate ester is formed with a hydroxyl residue of a protein (PDB entry 1p06).453

282

That other structure is, however, distinct because alone pair of electrons from a proximal histidine

residue appears to participate in a dative bond with the vacant p-orbital of the boron. The presence

of a planar ester could indicate that formation of a tetrahedral adduct is hindered sterically, unlike

in the active site of a serine/threonine protease that has evolved to bind tightly to a tetrahedral

intermediate. Hence, our results could demarcate the lower limit of affinity enhancement that can

be realized from boronic acid-based inhibitors.

Chloro groups have a variable contribution to the affinity of stilbenes for TTR. A chloro

group can fill unoccupied cavities, which would otherwise compromise affinity.4 544 55 Second, the

position of the chloro group in both of the ester-forming boronates enables the formation of a

halogen bond.4 56 The relevant ... Cl distance is close to the sum of the van der Waals radii (ro +

rci = 3.27 A; Figure A.4).444 The geometries observed in the TTR complexes with stilbenes 10 and

11 (Table A.S12) suggest a halogen-bond energy of 0.7-0.9 kcal/mol.457 Still, the consequences

of installing a chloro group on the stilbene scaffold are unlike those of installing a boronic acid

group, which consistently increases affinity for TTR (vide supra). For example, the addition of a

chloro group to stilbene 1 to form stilbene 3 decreases affinity, whereas the addition of a chloro

group to stilbene 5 to form stilbene 7, increases affinity. Notably, the phenolic ring of stilbene 5

was found to occupy two conformations in the inner pocket of the T4-binding site, which could

explain, in part, the lower affinity of stilbene 5 relative to stilbene 7 (Figures A.3B, A.S7, and

A.S9).

The judicious use of halogen substituents in boronated stilbenes merits further

investigation. For example, a chloro group positioned meta to the boron atom has been found to

decrease the pKa of phenyl boronic acid from 8.8 to 8.2.362 Such a more acidic boronic acid can

283

form more stable boronate esters.458 An analogous difference in Lewis acidity could be responsible

for some of the differences observed in the affinity of boronic acids for TTR.

Likewise, the esterification of TTR by boronic acid ligands warrants additional

optimization. In particular, a mono-boronate analog of compound 10 that binds with its lone

boronic acid group in the inner pocket could be used to reveal the precise contribution of a boronate

ester to the thermodynamics and kinetics 459 of binding. For proper orientation, such a ligand would

likely require installation of a highly polar functional group that demands the solvation attainable

in the outer pocket.

284

A.4 Materials and Methods

A.4.1 Materials

Resveratrol (1), 8-anilino- 1 -naphthalenesulfonic acid (ANS), and other reagents for biochemical

assays were from Sigma-Aldrich (St Louis, MO). Tafamidis (2-(3,5-dichlorophenyl)-6-

benzoxazolecarboxylic acid) (12) was from Carbosynth Limited (Berkshire, UK). DNA

oligonucleotides were from Integrated DNA Technologies (Coralville, IA).

A.4.2 Chemical Synthesis

Stilbenes 2-11 were synthesized by the routes shown in Schemes A. 1 and A.2.

A. 4.2.1 Materials

Reagents and solvents were from Sigma-Aldrich (Milwaukee, WI) and were used without further

purification. All glassware was flame-dried, and all reactions were performed under an atmosphere

of N2(g). Reagent-grade solvents: dichloromethane (DCM), tetrahydrofuran (THF), triethylamine

(TEA), and dimethylformamide (DMF) were dried over a column of alumina and were removed

from a dry still under an inert atmosphere. Flash column chromatography was performed with

Silicycle 40-63 A silica (230-400 mesh), and thin-layer chromatography (TLC) was performed

with EMD 250-pm silica gel 60-F254 plates.

A. 4.2.2 Conditions

All procedures were performed in air at ambient temperature (~22 'C) and pressure (1.0 atm)

unless indicated otherwise.

285

A. 4.2.3 Solvent Removal

The phrase "concentrated under reduced pressure" refers to the removal of solvents and other

volatile materials using a rotary evaporator at water aspirator pressure (<20 torr) while maintaining

a water bath below 40 'C. Residual solvent was removed from samples at high vacuum (<0.1 torr).

A. 4.2.4 NMR Spectroscopy

'H and 13C NMR spectra were acquired with a Bruker Avance III 500i spectrometer at the National

Magnetic Resonance Facility at Madison (NMRFAM). Chemical shift data are reported in units of

6 (ppm) relative to residual solvent.

A. 4.2.5 Mass Spectrometry

Mass spectra of small molecules were acquired with an LCT electrospray ionization (ESI)

instrument from Waters. Mass spectra of proteins were acquired with a microflex LRF desorption

ionization-time-of-flight (MALDI-TOF) instrument from Bruker (Billerica, MA). Both

instruments were in the Paul Bender Chemical Instrumentation Center at the University of

Wisconsin-Madison.

A. 4.2.6 Melting Points

Melting points were determined with an OptiMelt MPA100 instrument from Stanford Research

Systems (Sunnyvale, CA) over a range of 100-400 'C with a 0.5 'C/min heating rate. Melting

286

points values are given as the sngLe Mnis-Ics -nt Clponds 2, 3, andA 5 had mlting pIints

>400 OC.

A. 4.2. 7 Compound Purity

The purity of all compounds was judged to be >95%, as assessed by 'H and 13 C NMR

spectroscopy, mass spectrometry, melting-point determination, and reversed-phase high-

performance liquid chromatography (HPLC) using a C18 column and 515/717/996 analytical

instrument from Waters (Milford, MA) with a gradient of 10-80% v/v acetonitrile in water over

40 min.

A.4.2.8 Synthesis

5-[(1E)-2-(4-Bromo)ethenyl]-1,3-dimethoxybenzene (2a). 3,5-Dimethoxybenzyl bromide (1.2 g,

5.5 mmol) was dissolved in neat triethylphosphite (1.2 mL, 6.6 mmol) and heated to 150 'C for 4

h. The reaction mixture was cooled to 0 'C and diluted with DMF (40 mL). NaH (60% w/v in

mineral oil, 0.28 g, 7.12 mmol) was added to the resulting solution, and the reaction mixture was

stirred at 0 'C for 20 min. A solution of 4-bromo-benzaldehyde (1.0 g, 5.5 mmol) in DMF (15 mL)

was then added dropwise. The reaction mixture was allowed to warm to room temperature and

stirred overnight. The reaction mixture was then diluted with EtOAc (20 mL), and washed with

10% w/v citric acid (30 mL), followed by brine (30 mL). The organic layer was separated, dried

with Na2SO4(s), and filtered. The solvent was removed under reduced pressure, and the crude

product was purified by flash column chromatography (10% v/v EtOAc in hexanes) to afford

compound 2a as a white solid (1.56 g, 89%). 'H NMR (500 MHz, CDCL 3, 6): 3.83 (s, 6H), 6.41 (s,

287

1H), 6.65 (s, 2H), 6.98-7.01 (d, J= 16.28 Hz, 1H), 7.01-7.04 (d, J= 16.34 Hz, 1H), 7.35-7.37 (d,

J= 8.48 Hz, 2H), 7.46-7.48 (d, J= 8.51 Hz, 2H); '3C NMR (100 MHz, CDCl3, 6): 55.42, 100.31,

104.74, 121.57, 128.03, 128.16, 129.50, 131.92, 136.19, 139.07, 161.12; ASAP-MS (m/z): [M +

H] caled for C16HI 5BrO2, 319.0329; found, 319.0331.

5-[(JE)-2-(4-Bromo)ethenyl]-1,3-benzenediol (2b). Compound 2a (0.5 g, 1.6 mmol) was

dissolved in DCM (7 mL), and the resulting solution was cooled to 0 'C. A solution of 1.0 M BBr3

in DCM (7.8 mL) was added dropwise at 0 'C. The reaction mixture was allowed to warm to room

temperature and stirred for 4 h. The reaction mixture was then poured carefully into a separation

funnel containing ice water (-15 mL). The mixture was extracted with DCM (3 x 15 mL). The

organic layers were combined and washed with brine (15 mL), dried with Na2SO4(s), and filtered.

The solvent was removed under reduced pressure, and the crude product was suspended in ice-

cold DCM. The resulting precipitate was isolated by filtration to afford compound 2b as a white

solid (0.326 g, 70%). 'H NMR (500 MHz, CD30D, 6): 6.22 (s, 1H), 6.51 (s, 2H), 7.00-7.03 (d, J

16.33 Hz, 1H), 7.04-7.07 (d, J= 16.33 Hz, 1H), 7.45-7.47 (d, J= 8.5 Hz, 2H), 7.50-7.51 (d, J

8.5 Hz, 2H); 13C NMR (125 MHz, CD30D, 6): 103.43, 106.15, 121.97, 128.05, 129.17, 130.97,

132.78, 138.04, 140.42, 159.80; HRMS-ESI (m/z): [M - H]- calcd for C14HilBrO 2, 288.9870;

found, 288.9869.

5-[(1E)-2-(4-Boronic Acid Pinacol Ester) ethenylU-1,3-benzenediol (2c). Compound 2b (0.250 g,

0.858 mmol), KOAc (0.245 g, 2.57 mmol), bis(pinacolato)diboron (0.652 g, 2.57 mmol), and

Pd(dppf)C12 (0.062 g, 0.0858 mmol) were added to a Schlenk flask, which was then evacuated and

backfilled with N2(g). Dioxane (8.5 mL) was deoxygenated by sonication under high vacuum and

backfilled with N2(g). The deoxygenated dioxane was then added by cannula into the reaction

288

flask, and the reaction mixture was heated to 80 0C and stirred overnight. The reaction mixture

was then filtered, and the solvent was removed under reduced pressure. The crude product was

purified by flash column chromatography (20% v/v EtOAc in hexanes) to afford compound 2c as

a white solid (0.245 g, 85%). 'H NMR (500 MHz, CD 30D, 6): 1.34 (s, 12H), 6.22 (s, 1H), 6.51 (s,

2H), 7.02-7.06 (d, J== 16.27 Hz, 1H), 7.06-7.10 (d, J= 16.36 Hz, 1H), 7.50-7.52 (d, J= 7.93 Hz,

2H), 7.71-7.72 (d, J= 7.79 Hz, 2H); 13C NMR (125 MHz, CD30D, 6): 25.19, 85.04, 103.42,

160.21, 126.77, 129.23, 131.16, 136.08, 140.54, 141.63, 159.75; HRMS-ESI (m/z): [M - H]' calcd

for C20H23BO4 , 336.1653; found, 336.1653.

5-[(]E)-2-(4-Boronic acid)ethenyl]-1,3-benzenediol (2). Compound 2c (0.050 g, 0.147 mmol)

was dissolved in 4:1 THF/H 20 (1.5 mL). NaIO4 (0.157 g, 0.739 mmol) was added to the resulting

solution, followed by 1.0 M HCl (36 ptL, 0.036 mmol). The reaction mixture was allowed to stir

overnight. The reaction mixture was then diluted with H20 (2 mL) and extracted with EtOAc (3 x

4 mL). The organic layers were combined and washed with brine (10 mL), dried with Na2SO4(s),

and filtered. The solvent was removed under reduced pressure, and the crude product was purified

by flash column chromatography (3% v/v CH30H in DCM) to afford compound 2 as a white

crystalline solid (0.027 g, 67%) with mp >400 'C. 1H NMR (500 MHz, CD30D, 6): 6.20 (s, 1H),

6.50 (s, 2H), 7.95 (bs, 2H), 7.51-7.52 (d, J= 8.17 Hz, 2H), 7.60-7.62 (d, J= 8.13 Hz, 2H); 13C

NMR (125 MHz, CD30D, 6): 103.32, 106.13, 126.69, 129.26, 130.67, 135.09, 140.06, 140.64,

159.78; HRMS-ESI (m/z): [M - H]- calcd for the single methyl boronic ester C, 5 Hi5 BO4 ,

268.1027; found, 268.1027.

5-[(]E)-2-(2-Chloro-4-bromo)ethenyl]-1, 3-dimethoxybenzene (3a). 3,5-Dimethoxybenzyl

bromide (0.74 g, 3.4 mmol) was dissolved in neat triethylphosphite (0.7 mL, 4.0 mmol), and the

289

resulting solution was heated to 150 'C for 4 h. The reaction mixture was cooled to 0 'C and added

to DMF (20 mL). NaH (60% w/v in mineral oil, 0.17 g, 4.36 mmol) was added to the resulting

solution, and the reaction mixture was stirred at 0 'C for 20 min. A solution of 4-bromo-2-chloro

benzaldehyde (0.74 g, 3.36 mmol) in DMF (10 mL) was then added dropwise. The reaction

mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was

then diluted with EtOAc (15 mL), and washed with 10% w/v citric acid (20 mL), followed by brine

(20 mL). The organic layer was separated, dried with Na2SO4(s), and filtered. The solvent was

removed under reduced pressure, and the crude product was purified by flash column

chromatography (5% v/v EtOAc in hexanes) to afford compound 3a as a white solid (1.10 g, 91%

over 2 steps). 'H NMR (400 MHz, CDCl3, 6): 3.84 (s, 6H), 6.44 (s, 1H), 6.69 (s, 2H), 6.97-7.01

(d, J= 16.24 Hz, 1H), 7.36-7.40 (m, 2H), 7.50-7.52 (d, J= 8.42 Hz, 1H), 7.55 (s, 1H); 13C NMR

(100 MHz, CDCl 3, 5): 55.51, 100.60, 105.11, 121.37, 124.27, 127.60, 130.27, 131.89, 132.43,

134.20, 134.41, 138.81, 161.12; ASAP-MS (m/z): [M + H]' calcd for C1 6HI4BrClO2, 352.9939;

found, 352.9939.

5-[(1E)-2-(2-Chloro-4-bromo)ethenyl]-1,3-benzenediol (3b). Compound 3a (0.500 g, 1.42

mmol) was dissolved in DCM (14 mL), and the reaction mixture was cooled to 0 'C. A solution

of 1.0 M BBr3 (7.10 mmol) in DCM (7.1 mL) was then added dropwise at 0 'C. The reaction

mixture was allowed to warm to room temperature and stirred for 4 h. The reaction mixture was

then poured carefully into a separation funnel containing ice water (~10 mL) and extracted with

DCM (3 x 10 mL). The organic layers were combined and washed with brine (15 mL), dried with

Na2SO4(s), and filtered. The solvent was removed under reduced pressure, and the crude product

was suspended in cold DCM (5 mL). The resulting precipitate was filtered to afford compound 3b

290

as a white solid (0.335 g, 72%). IH NMMR (500 '-"z, CD3OD, (5): 6.23, (s, 1ITh) 6.51 (s, '),' 7.03-

7.07 (d, J= 16.23 Hz, 1H), 7.31-7.35 (d, J= 16.23 Hz, 1H), 7.45-7.47 (d, J= 8.31 Hz, 1H), 7.60

(s, 1H), 7.67-7.69 (d, J= 8.47 Hz, 1H); 13 C NMR (125 MHz, CD30D, 6): 103.79, 106.34, 124.89,

126.71, 133.26, 133.34, 133.90, 135.91,137.59,140.29, 159.89; HRMS-ESI (m/z): [M - H]- calcd

for C14HIoBrClO 2, 322.9480; found, 322.9480.

5-[(]E)-2-(2-Chloro-4-boronic Acid Pinacol Ester)ethenyl]-1,3-benzenediol (3c). Compound 3b

(0.100 g, 0.308 mmol), KOAc (0.088 g, 0.926 mmol), bis(pinacolato)diboron (0.235 g, 0.926

mmol), and Pd(dppf )C1 2 (0.0225 g, 0.0308 mmol) were added to a flame-dried Schlenk flask,

which was then evacuated and backfilled with N2(g). Dioxane (3.5 mL) was deoxygenated by

sonication under high vacuum and backfilled with N2(g). The deoxygenated dioxane was then

added by cannula into the reaction flask, and the reaction mixture was heated to 80 'C and stirred

overnight. The reaction mixture was filtered and the solvent was removed under reduced pressure.

The crude product was purified by flash column chromatography (2% v/v CH30H in DCM) to

afford compound 3c as a white solid (0.080 g, 69%). 'H NMR (500 MHz, CD30D, 6): 1.33 (s,

12H), 6.23 (s, 1H), 6.53 (s, 2H), 7.05-7.09 (d, J= 16.29 Hz, lH), 7.40-7.44 (d, J= 16.28 Hz, 1H),

7.60-7.62 (d, J= 7.98 Hz, 1H), 7.70 (s, 1H), 7.72-7.74 (d, J= 7.83 Hz, 1H); 13C NMR (125 MHz,

CD 30D, 6): 25.18, 75.82, 85.42, 103.90, 106.42, 124.82, 126.88, 133.83, 133.90, 134.11, 136.74,

139.11, 140.17, 159.84; HRMS-ESI (m/z): [M - H]- caled for C 2 0H2 2BC10 4, 370.1263; found,

370.1263.

5-[(E)-2-(2-chloro-4-hydroxyphenyl)ethenyl]-1,3-benzenediol (3). Compound 3 was derived

from the oxidation of compound 3c by an aryl N-oxide in one step as described previously. 460 Here,

compound 3c (0.020 g, 0.053 mmol) was dissolved in DCM (0.6 mL). NN-Dimethyl-p-toluidine-

291

N-oxide (0.012 g, 0.081 mmol) was added to the resulting solution, and the reaction mixture was

stirred for 1 h. The solvent was removed under reduced pressure, and the crude product was

purified by flash column chromatography (15% v/v EtOAc in hexanes) to afford compound 3 as a

pale yellow solid (0.010 g, 70%) with mp >400 'C. 1 H NMR (500 MHz, CD30D, 6): 6.18-6.19 (t,

J= 2.15 Hz, 1H), 6.47 (s, 2H), 6.74-6.76 (d, J= 8.66 Hz, 1H), 6.82 (s, 1H), 6.82-6.85 (d, J=

14.57 Hz, 1H), 7.31-7.34 (d, J= 16.16 Hz, 1H), 7.58-7.59 (d, J= 8.64 Hz, 1H); 13C NMR (125

MHz, CD30D, 6): 103.10, 105.96, 115.92, 117.03, 124.95, 127.75, 128.32, 129.78, 134.77,

140.91, 159.08, 159.77; HRMS-ESI (m/z): [M - H]- calcd for C 14Hi 1C10 3, 261.0324; found,

261.0325.

5-[(]E)-2-(2-Chloro-4-boronic Acid)ethenyl]-1, 3-benzenediol (4). Compound 3c (0.050 g,

0.134 mmol) was dissolved in 4:1 THF/H20 (1.3 mL). NaIO4 (0.143 g, 0.670 mmol) was added to

the resulting solution, followed by 1.0 M HCl (33 tL, 0.033 mmol). The reaction mixture was then

stirred overnight. The reaction mixture was diluted with H20 (1.0 mL) and extracted with EtOAc

(3 x 2 mL). The organic layers were combined and washed with brine (6 mL), dried with

Na2SO4(s), and filtered. The solvent was removed under reduced pressure, and the crude mixture

was purified by flash column chromatography (4% v/v CH30H in DCM) to afford compound 4 as

a white crystalline solid (0.022 g, 88%) with mp 186.0 'C. 1H NMR (500 MHz, CD30D, 6): 6.23

(s, 1H), 6.52 (s, 2H), 7.05-7.09 (d, J= 16.20 Hz, 1H), 7.41-7.45 (d, J= 16.23 Hz, 1H), 7.54-7.56

(d, J= 7.85 Hz, 1H), 7.63 (s, 1H), 7.75-7.76 (d, J= 7.81 Hz, 1H); 13C NMR (125 MHz, CD 30D,

6): 103.77, 106.32, 124.88, 126.71, 133.26, 133.34, 133.90, 135.91, 137.59, 140.29, 159.89;

HRMS-ESI (m/z): [M - H]- calcd for C14HI 2BClO 4, 288.0481; found, 288.0482.

292

3-[(!E )-2-4Boohnlehn!-ezi AA//d Mthy Es 7 "4tr' (I. ) E/Cy I3fI-

(bromomethyl)benzoate (2.0 g, 8.7 mmol) was dissolved in neat triethylphosphite (1.78 mL,

10.4 mmol) and heated to 150 'C for 4 h. The reaction mixture was cooled to 0 'C and diluted with

DMF (87 mL). NaH (60% w/v in mineral oil, 0.69 g, 17 mmol) was added to the resulting solution,

and the reaction mixture stirred at 0 'C for 20 min. A solution of 4-bromobenzaldehyde (1.62 g,

8.73 mmol) in DMF (87 mL) was then added dropwise. The reaction mixture was allowed to warm

to room temperature and stirred overnight. The reaction mixture was then diluted with EtOAc (50

mL), and washed with 10% w/v citric acid (20 mL), followed by brine (20 mL). The organic layer

was separated, dried with Na2SO4(s), and filtered. The solvent was removed under reduced

pressure, and the crude product was purified by flash column chromatography (10 % v/v EtOAc

in hexanes) to afford the 5a as a white solid (1.75 g, 63% over 2 steps). 'H NMR (500 MHz,

CD30D, 6): 3.84 (s, 3H), 6.94-6.96 (d, J= 8.70 Hz, 2H), 7.09-7.12 (d, J= 16.28, 1H), 7.20-7.25

(d, J= 16.41 Hz, 1H), 7.45-7.48 (t, J= 7.67, 1H), 7.53-7.55 (d, J= 8.62 Hz, 2H), 7.76-7.78 (d, J

= 7.85 Hz, 1H), 7.88-7.90 (d, J= 7.72, 1H), 8.18 (s, 1H); 13C NMR (125 MHz, CD30D, 6): 54.31,

113.73, 124.97, 126.93, 127.58, 127.80, 128.40, 129.13, 130.01, 138.21, 159.72, 168.62; HRMS-

ESI (m/z): M calcd for C16H13BrO 2 , 316.0094; found, 316.0081.

3-[(JE)-2-(4-Bromophenyl)ethenyl]-benzoic Acid (5b). Compound 5a (1.5 g, 4.7 mmol) was

dissolved in 3:1 THF/EtOH (47 mL). A solution of 2 M NaOH (4.7 mL, 9.4 mmol) was added to

the resulting solution, and the reaction mixture was stirred overnight. The reaction mixture was

then diluted with EtOAc (20 mL), and washed with 10% w/v citric acid (20 mL), followed by brine

(20 mL). The organic layer was separated, dried with Na2SO4(s), and filtered. The solvent was

removed under reduced pressure, and the crude product was purified by flash column

293

chromatography (20% v/v EtOAc in hexanes) to afford compound 5b as a white solid (1.36 g,

96%). 'H NMR (500 MHz, DMSO, 6): 7.33-7.36 (d, J= 16.52 Hz, 1H), 7.40-7.43 (d, J= 16.44

Hz, 1H), 7.50-7.54 (t, J = 7.68 Hz, 1H), 7.58-7.60 (d, J= 8.64 Hz, 2H), 7.63-7.61 (d, J= 8.66

Hz, 2H), 7.84-7.88 (t, J= 8.63 Hz, 2H), 8.16 (s, 1H), 13.10 (s, 1H); 13 C NMR (125 MHz, DMSO,

6): 121.27, 127.81, 128.68, 128.92, 129.00, 129.10, 129.51, 131.04, 132.09, 136.61, 137.66,

167.69; HRMS-ESI (m/z): M caled for CI 5Hl1BrO2, 301.9937; found, 301.9944.

3-[(]E)-2-(4-Boronic acid Pinacol Ester) ethenyl]-benzoic Acid (Sc). Compound 5b (1.0 g, 3.3

mmol), KOAc (0.971 g, 9.9 mmol), bis(pinacolato)diboron (2.5 g, 9.9 mmol), and Pd(dppf)C12

(0.24 g, 0.33 mmol) were added to a flame-dried Schlenk flask, which was then evacuated and

backfilled with N2(g). Dioxane (33 mL) was deoxygenated by sonication under high vacuum and

backfilled with N2(g). The deoxygenated dioxane was then added by cannula into the reaction

flask, and the reaction mixture was stirred overnight at 80 'C. The reaction mixture was filtered,

and the solvent was removed under reduced pressure. The crude product was purified by flash

column chromatography (20% v/v EtOAc in hexanes) to afford compound 5c as a white solid

(0.850 g, 73%). 'H NMR (500 MHz, CDC13, a): 1.36, (s, 12H), 7.22 (s, 2H), 7.46-7.50 (t, J= 7.44

Hz, 1H), 7.54-7.55 (d, J= 7.72 Hz, 1H), 7.76-7.77 (d, J= 8.12 Hz, 2H), 7.81-7.83 (d, J= 8.10

Hz, 2H), 8.00-8.01 (d, J= 8.11 Hz, 1H), 8.27-8.28 (d, J= 7.72 Hz, 1H), 8.28 (s, 1H); 13 C NMR

(125 MHz, CDCl 3, 6): 24.90, 83.87, 125.97, 128.19, 128.32, 128.94, 129.28, 129.70, 130.06,

131.67, 135.23, 137.70, 139.50; HRMS-ESI (m/z): [M + NH4] calcd for C2 1H2 3BO4, 367.2064;

found, 367.2062.

3-[(]E)-2-(4-Hydroxyphenyl)ethenyl]-benzoic Acid (5). Compound 5c (0.050 g, 0.142 mmol)

was dissolved in DCM (1.4 mL). NN-Dimethyl-p-toluidine-N-oxide (0.032 g, 0.213 mmol) was

294

added to the resulting solution, and the reaction mixture was allowed to stir for 1 h. The

mixture was filtered, and the solvent was removed under reduced pressure. The crude product was

purified by flash column chromatography (2% v/v MeOH in DCM) to afford compound 5 as a

white solid (0.030 g, 88%) with mp >400 0C. 'H NMR (500 MHz, CD30D, 5): 6.78-6.80 (d, J=

8.61 Hz, 2H), 7.02-7.05 (d, J= 16.32 Hz, 1H), 7.15-7.19 (d, J= 16.34 Hz, 1H), 7.42-7.44 (d, J=

8.26 Hz, 2H), 7.42-7.45 (t, J= 7.28 Hz, 1H), 7.73-7.75 (d, J= 7.82 Hz, 1H), 7.85-7.87 (d, J=

7.79 Hz, 1H), 8.15 (s, 1H); 13C NMR (125 MHz, CD 30D, 6): 116.52, 125.56, 128.25, 129.04,

129.10, 129.80, 130.04, 130.90, 131.42, 132.30, 139.80; HRMS-ESI (m/z): [M - H]- calcd for

Ci5 H,20 3, 239.0714; found, 239.0716.

3-[(]E)-2-(4-Boronic acid)ethenyl]-benzoic Acid (6). Compound 5c (0.5 g, 1.48 mmol) was

dissolved in 4:1 THF/H20 (15 mL). NaIO4 (0.405 g, 1.89 mmol) was added to the resulting

solution, followed by 1.0 M HCl (0.15 mL, 0.158 mmol). The reaction mixture was then stirred

overnight. The reaction mixture was diluted with H2 0 (10 mL) and extracted with EtOAc (3 x 15

mL). The organic layers were combined and washed with brine (20 mL), dried with Na2SO4(s),

and filtered. The solvent was removed under reduced pressure, and the crude product was purified

by flash column chromatography (10% v/v CH30H in DCM) to afford compound 6 as a white

crystalline solid (0.345 g, 90%) with mp 174.2 'C. 'H NMR (500 MHz, CDCl 3, 6): 7.24-7.27 (d,

J= 16.43 Hz, 1H), 7.28-7.32 (d, J= 16.45 Hz, 1H), 7.49-7.76 (t, J= 7.70 Hz, 1H), 7.58-7.59 (d,

J= 7.94 Hz, 2H), 7.62-7.64 (d, J= 7.92 Hz, 2H), 7.80-7.82 (d, J= 7.87 Hz, 1H), 7.90-7.92 (d, J

= 7.81 Hz, 1H), 8.21 (s, 1H); 13C NMR (125 MHz, CDCl 3, 6): 125.51, 127.32, 127.86, 128.37,

128.51, 129.41, 130.43, 131.07, 133.70, 137.77, 138.27, 168.32; HRMS-ESI (m/z): [M - H]- calcd

for single methyl boronic acid C1 6H1 5BO4, 280.1026; found, 280.1033.

295

Methyl 4-Bromo-2-chlorobenzoate (7a). 4-Bromo-2-chlorotoluene (1.0 g, 4.8 mmol) was

dissolved in 1:1 water/tert-butanol (20 mL). KMnO4 (1.53 g, 9.7 mmol) was added to the resulting

solution, and the reaction mixture was heated to 70 'C with a reflux condenser for 2 h. The reaction

mixture was then allowed to cool to room temperature, and more KMnO4 (1.5 g, 9.7 mmol) was

added. The reaction mixture was then reheated to 70 'C in a flask with a reflux condenser and

stirred overnight at 70 'C. The warm reaction mixture was filtered, and the resulting KMnO4 cake

was rinsed with water (~10 mL). The filtrate was acidified to pH 3 with concentrated HCl and

extracted with EtOAc (3 x 20 mL). The organic layers were combined, dried with NaSO 4 (s), and

filtered. The solvent was removed under reduced pressure to afford the carboxylic acid precursor

as a white solid. This precursor was dissolved in 3 M HCl in MeOH (15 mL) and heated to reflux

for 12 h. The reaction mixture was allowed to cool to room temperature, and N2(g) was bubbled

through the solution for 20 min to remove excess HCl(g). The solvent was removed under reduced

pressure, and the crude product was purified by flash column chromatography (20% v/v EtOAc in

hexanes) to afford compound 7a as a colorless oil (1.15 g, 96% yield over 2 steps). IH NMR (500

MHz, CD30D, 6): 3.93 (s, 3H), 7.59-7.61 (d, J= 8.47 Hz, 1H), 7.76-7.77 (m, 2H); 13 C NMR (125

MHz, CD30D, 6): 53.07, 127.34, 130.51, 131.41, 133.71, 134.63, 135.53, 166.72; HRMS-ESI

(m/z): M calcd for C8H6BrClO 2, 247.9235; found, 247.9237.

4-Bromo-2-chlorobenzyl Alcohol (7b). Compound 7a (1.00 g, 4.01 mmol) was dissolved in

THF (47 mL), and the resulting solution was cooled to 0 'C. 2 M LiBH4 in THF (12 mL, 23.5

mmol) was then added dropwise, followed by methanol (4 mL). The reaction mixture was allowed

to warm to room temperature and stirred overnight. The reaction mixture was quenched by adding

EtOAc (20 mL) dropwise, followed by water (15 mL), and then acidification to pH 5 with 1.0 M

296

HCl. The resulting lithium salts wTere removed by filtration, and the filtrate iwas extracted with

EtOAc (3 x 15 mL). The organic layers were combined, dried with NaSO4(s), and filtered. The

solvent was removed under reduced pressure, and the crude product was purified by flash column

chromatography (20% v/v EtOAc in hexanes) to afford compound 7b as a white solid (0.843 g,

95%). 'H NMR (500 MHz, CDCl 3, 6): 1.91 (bs, 1H), 4.74 (s, 2H), 7.38-7.39 (d, J= 8.22 Hz, 1H),

7.42-7.44 (d, J= 8.23, 1H) 7.53 (s, 1H); 13 C NMR (500 MHz, CDCl 3, 6): 62.48, 121.68, 129.93,

130.42, 132.05, 133.54, 137.43; HRMS-ESI (m/z): M calcd for C7H6OBrCl, 219.9286; found,

219.9282.

4-Bromo-2-chlorobenzaldehyde (7c). Compound 7b (0.5 g, 2 mmol) was dissolved in DCM

(22 mL). Pyridinium dichromate (PDC, 2.57 g, 6.84 mmol) was added to the resulting solution,

and the reaction mixture was allowed to stir ovemight. The reaction mixture was then filtered

through a pad of Celite*, and the solvent was removed under reduced pressure. The crude product

was purified by flash column chromatography (10% v/v EtOAc in hexanes) to afford compound

7c as a white solid (0.440 g, 88%). 'H NMR (500MHz, CDCl 3, a): 7.54-7.5 5 (d, J= 8.25 Hz, 1H),

7.66 (s, 1H), 7.78-7.80 (d, J=8.32 Hz, IH), 10.42 (s, 1H); 13C NMR (125 MHz, CDCl3,A) 129.78,

130.55, 131.09, 131.45, 133.49, 138.71, 188.95; ASAP-MS (m/z): [M+H]* calcd for C7H4BrClO,

218.9207; found, 218.9216.

3-[(JE)-2-(2-Chloro-4-bromophenyl)ethenyl]-benzoic Acid Methyl Ester (7d). Ethyl 3-

(bromomethyl)benzoate (1.04 g, 4.55 mmol) was dissolved in neat triethylphosphite (0.9 mL, 5.46

mmol), and the resulting solution was heated to 150 'C for 4 h. The reaction mixture was cooled

to 0 'C and diluted with DMF (40 mL). NaH (60% w/v in mineral oil, 0.23 g, 5.91 mmol) was

added to the resulting solution, and the reaction mixture was stirred at 0 0C for 20 min. A solution

297

of 4-bromo-2-chlorobenzaldehyde (7c; 1.0 g, 4.6 mmol) in DMF (5 mL) was added dropwise. The

reaction mixture was then allowed to warm to room temperature and stirred overnight. The reaction

mixture was diluted with EtOAc (5 mL), and washed with 10% w/v citric acid (5 mL), followed

by brine (5 mL). The organic layer was separated, dried with Na2SO4(s), and filtered. The solvent

was removed under reduced pressure, and the crude product was purified by flash column

chromatography (10% v/v EtOAc in hexanes) to afford compound 7d as a white solid (1.3 g, 83%

over 2 steps). 1H NMR (500MHz, CDCl 3, 6): 3.95 (s, 3H), 7.08-7.11 (d, J= 16.31 Hz, 1H), 7.39-

7.41 (d, J= 8.53 Hz, 1H), 7.44-7.47 (t, J= 8.02 Hz, 1H), 7.45-7.49 (d, J= 16.56 Hz, 1H), 7.53-

7.55 (d, J= 8.45 Hz, 1H), 7.56 (s, 1H), 7.71-7.73 (d, J= 7.73 Hz, 1H), 7.96-7.97 (d, J = 7.70 Hz,

lH), 8.19 (s, 1H); 13C NMR (125 MHz, CDCl 3, 6): 52.43, 121.70, 125.02, 127.66, 128.15, 129.02,

129.35, 130.39, 130.88, 131.12, 132.55, 134.26, 134.35, 137.19, 167.02; HRMS-ESI (m/z): [M +

NH4] calcd for C16HI 2BrClO2, 368.0048; found, 368.0053.

3-[(]E)-2-(2-Chloro-4-bromophenyl)ethenyl]-benzoic Acid (7e). Compound 7d (1.34 g, 3.83

mmol) was dissolved in 3:1 THF/EtOH (40 mL). 2.0 M NaOH (3.3 mL, 7.7 mmol) was added to

the resulting solution, and the reaction mixture was stirred overnight. The reaction mixture was

diluted with EtOAc (20 mL), and washed with 10% w/v citric acid (30 mL), followed by brine (30

mL). The organic layer was separated, dried with Na2SO4(s), and filtered. The solvent was removed

under reduced pressure, and the crude product was purified by flash column chromatography (20%

v/v EtOAc in hexanes) to afford compound 7e as a white solid (1.22 g, 95%). 'H NMR (500 MHz,

DMSO, 6): 7.42-7.45 (d, J= 16.39 Hz, 1H), 7.47-7.50 (d, J= 16.41 Hz, 1H), 7.53-7.56 (t, J=

7.71 Hz, 1H), 7.60-7.62 (d, J= 8.54, 1H), 7.79 (s, 1H), 7.86-7.88 (d, J= 8.63 Hz, 1H), 7.88-7.92

(t, J= 7.86, 2H), 8.15 (s, 1H), 13.01 (bs, IH); 13 C NMR (125 MHz, DMSO, 6): 121.99, 124.85,

298

128.89, 129.10, 129.94 130.17, 131.41, 131 73, 12),Q 132.3Q 13Q7, 13A61, 1322 1Q()7,

167.31; HRMS-ESI (m/z): [M - H]- caled for Ci5H oBrClO 2, 334.9479; found, 334.9476.

3-[(]E)-2-(2-Chloro-4-boronic Acid Pinacol Ester)ethenyl]-benzoic Acid (7f). Compound 7e

(0.200 g, 0.580 mmol), KOAc (0.171 g, 1.791 mmol), bis(pinacolato)diboron (0.45 g, 1.79 mmol),

and Pd(dppf)C1 2 (0.043 g, 0.059 mmol) were added to a flame-dried Schlenk flask, which was then

evacuated and backfilled with N2(g). Dioxane (6 mL) was deoxygenated by sonication under high

vacuum and backfilled with N2(g). The deoxygenated dioxane was then added by cannula into the

reaction flask, and the reaction mixture was stirred overnight at 80 'C. The reaction mixture was

filtered, and the solvent was removed under reduced pressure. The crude product was purified by

flash column chromatography (1% v/v CH30H in DCM) to afford compound 7f as a white solid

(0.183 g, 82%). 'H NMR (500 MHz, CD30D, &): 1.36 (s, 12H), 7.33-7.36 (d, J= 16.30 Hz, 1H),

7.49-7.52 (t, J= 8.41 Hz, 1H), 7.61-7.64 (d, J= 16.47 Hz, 1H), 7.66-7.67 (d, J= 7.46, 1H), 7.73

(s, 1H), 7.83-7.85 (d, J= 7.75 Hz, 2H), 7.95-7.97 (d, J= 7.73, 1H), 8.24 (s, 1H); 13C NMR (125

MHz, CD30D, 6): 25.19, 85.51, 126.33, 127.18, 129.00, 130.07, 130.41, 132.16, 132.61, 134.18,

136.79, 138.74, 138.86, 169.56; HRMS-ESI (m/z): [M + NH4]'calcd for C21H22BClO4, 401.1675;

found, 401.1666.

3-[(JE)-2-(2-Chloro-4-hydroxyphenyl)ethenyl]-benzoic Acid (7). Compound 7f (0.100 g,

0.259 mmol) was dissolved in DCM (2.6 mL). NN-Dimethyl-p-toluidine-N-oxide (0.060 g, 0.389

mmol) was added to the resulting solution, and the reaction mixture was then stirred overnight.

The solvent was removed under reduced pressure, and the crude product was purified by flash

column chromatography (2% v/v MeOH in DCM) to afford compound 7 as a white solid (0.051

g, 72%) with mp 213.6 0C. 'H NMR (500 MHz, CD30D, 6): 6.77-6.68 (d, J= 8.63 Hz, 1H), 6.84

299

(s, 1H), 7.06-7.10 (d, J= 16.34 Hz, 1H), 7.45-7.48 (t, J= 7.72 Hz, 1H), 7.49-7.52 (d, J= 16.31

Hz, 1H), 7.65-7.67 (d, J= 8.63 Hz, 1H), 7.75-7.77 (d, J= 7.91 Hz, 1H), 7.89-7.91 (d, J= 7.78

Hz, 1H), 8.17 (s, 1H); 13C NMR (125 MHz, CD30D, 6): 115.98, 117.09, 126.42, 127.43, 128.47,

128.48, 128.58, 129.60, 129.92, 131.70, 132.43, 135.06, 139.41, 159.45, 169.74; HRMS-ESI

(m/z): [M - H]- caled for C15 HIClO3, 273.0324; found, 273.0327.

3-[(JE)-2-(2-Chloro-4-boronic Acid)ethenyl]-benzoic (8). Compound 7f (0.100 g, 0.260 mmol)

was dissolved in 4:1 THF/H20 (2.6 mL). NaIO4 (0.28 g, 1.30 mmol) was added to the resulting

solution, followed by 0.02 mL of 1.0 M HCl (0.02 mmol). The reaction mixture was then stirred

overnight. The reaction mixture was diluted with H20 (2 mL) and extracted with EtOAc (3 x 3

mL). The organic layers were combined and washed with brine (5 mL), dried with Na2SO4(s), and

filtered. The solvent was removed under reduced pressure, and the crude product was purified by

flash column chromatography (1-3% v/v CH30H in DCM) to afford compound 8 as a white

crystalline solid (0.058 g, 75%) with mp 161.0 'C. 'H NMR (500 MHz, CD30D, 6): 7.32-7.35 (d,

J= 16.37 Hz, 1H), 7.51-7.54 (t, J= 7.73 Hz, 1H), 7.51-7.67 (in, 3H), 7.84-7.85 (d, J= 7.29 Hz,

2H), 7.97-7.98 (d, J= 7.76 Hz, 1H), 8.25 (s, 1H); 13 C NMR (125 MHz, CD30D, 6): 126.41,

126.94, 128.93, 130.04, 130.29, 132.07, 132.63, 133.31, 134.12, 135.95, 137.29, 138.82, 169.61;

HRMS-ESI (m/z): [M - H]- calcd for the single methyl boronic ester C1 6H 14BC10 4 , 314.0637;

found, 314.0635.

1,1'-(]E)-(1,2-Ethenediyl)bis[2-chloro-4-bromo]-benzene (9a). 4-Bromo-1-(bromomethyl)-2-

chloro-benzene (0.300 g, 1.05 mmol) was dissolved in neat triethylphosphite (0.217 mL, 1.26

mmol) and heated to 150 'C for 4 h. The reaction mixture was then cooled to 0 'C and diluted with

DMF (8 mL). NaH (60% w/v in mineral oil, 0.054 g, 1.36 mmol) was added to the resulting

300

schltinn and the reactinn Miviir Aw7as ctrreA at 0 C 4fror '2 m;i. A -lft f_4-rrr'o m -chrf--on, -t e r~qC xx x XXLIx% VV O L 'J L11I% %.L ".L WJ '.- lk L Z1.j .J 111I. 1 1. 13%JL4LI.J11 JL

benzaldehyde (7c; 0.230 g, 1.05 mmol) in DMF (2.5 mL) was then added dropwise. The reaction

mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was

diluted with EtOAc (8 mL), and washed with 10% w/v citric acid (10 mL), followed by brine (10

mL). The organic layer was then separated, dried with Na2SO 4 (s), and filtered. The solvent was

removed under reduced pressure, and the crude product was suspended in cold DCM (10 mL). The

resulting precipitate was collected by filtration to afford compound 9a as a white solid (0.306 g,

72% yield over 2 steps). 'H NMR (500 MHz, CDCl 3, 6): 7.39 (s, 2H), 7.41-7.43 (d, J= 8.42 Hz,

2H), 7.58 (s, 2H), 7.58-7.59 (d, J= 7.12 Hz, 2H); 3C NMR (125 MHz, CDCl 3, 6): 121.99, 126.70,

127.83, 130.35, 132.45, 133.89, 134.30; ASAP-MS (m/z): M' calcd for C14H8Br2C2, 403.8365;

found, 403.8367.

1,] '-(JE)-(1,2-Ethenediyl)bis[2-chloro-4-boronic Acid Pinacol Ester]-benzene (9b). Compound

9a (0.050 g, 0.123 mmol), KOAc (0.071 g, 0.742 mmol), bis(pinacolato)diboron (0.187 g,

0.742 mmol), and Pd(dppf)C12 (9 mg, 0.012 mmol) were added to a flame-dried Schlenk flask,

which was then evacuated and backfilled with N2(g). Dioxane (2 mL) was deoxygenated by

sonication under high vacuum and backfilled with N2(g). The deoxygenated dioxane was then

added by cannula into the reaction flask, and the reaction mixture was heated to 80 'C and stirred

overnight (Note: higher yields of the diboronated product were found at more dilute reaction

concentrations). The reaction mixture was filtered, and the solvent was removed under reduced

pressure. The crude product was purified by flash column chromatography (50% v/v DCM in

hexanes) to afford 9b as a white solid (0.056 g, 92%). 'H NMR (500 MHz, CDCl3, a): 1.35 (s,

24H), 7.56 (s, 2H), 7.67-7.69 (d, J= 7.76 Hz, 2H), 7.73-7.75 (d, J= 7.85 Hz, 2H), 7.83 (s, 2H);

301

"C NMR (125 MHz, CDCl 3, 6): 24.88, 84.18, 126.17, 127.94, 133.02, 133.45, 136.06, 137.45;

ASAP-MS (m/z): [M + H]f calcd for C26H32B2C1204, 499.2009; found, 499.2001.

1,1 '-(JE)-(1,2-Ethenediyl)bis[2-chloro-4-hydroxy]-benzene (9). Compound 9b (0.020 g,

0.040 mmol) was dissolved in DCM (0.5 mL). NN-Dimethyl-p-toluidine-N-oxide (0.0 18 g, 0.120

mmol) was added to the resulting solution, and the reaction mixture was stirred for 1 h. The solvent

was removed under reduced pressure, and the crude product was purified by flash column

chromatography (2% v/v MeOH in DCM) to afford compound 9 as a white solid (9 mg, 80%) with

mp 203.9 'C. 'H NMR (500 MHz, CD 30D, 6): 6.75-6.77 (d, J= 8.61 Hz, 2H), 6.83 (s, 2H), 7.24

(s, 2H), 7.55-7.57 (d, J= 8.59 Hz, 2H); 13 C NMR (125 MHz, CD 30D, a): 115.93, 117.07, 125.22,

128.00, 128.36, 134.73, 159.09; HRMS-ESI (m/z): [M - H]- calcd for C1 4H1 0C1202 , 278.9985;

found, 278.9985.

1,1'-(JE)-(1,2-Ethenediyl)bis[2-chloro-4-boronic acid]-benzene (10). Compound 9b (0.020 g,

0.040 mmol) was dissolved in 4:1 THF/H20 (0.6 mL). NaIO4 (0.042 g, 0.200 mmol) was added to

the resulting solution, followed by a few drops of 1.0 M HCl. The reaction mixture was then stirred

overnight. The reaction mixture was then diluted with H20 (1 mL) and extracted with EtOAc (3 x

2 mL). The organic layers were combined and washed with saturated brine (3 mL), dried with

Na2SO4(s), and filtered. The solvent was removed under reduced pressure, and the crude product

was purified by flash column chromatography (3% v/v CH30H in DCM) to afford compound 10

as a white crystalline solid (0.010 g, 74%) with mp 234.2 0 C. 'H NMR (500 MHz, CD 30D, a):

7.59 (s, 1H), 7.59-7.60 (d, J= 8.42 Hz, 2H), 7.66 (s, 1H), 7.78-7.80 (d, J= 7.78 Hz, 2H); '3 C

NMR (125 MHz, CD30D, 6): 127.14, 128.69, 133.38, 134.25, 135.98; MALDI-MS (m/z): M

calcd for C14H12 B2C12 0 4 , 336.03; found, 336.00.

302

2-Bromo-1-[1-72-(3-bromophenyflethenyl]-4ch/orobenzene (1a). 3 -Bromobenzyl bromide

(0.500 g, 2.00 mmol) was dissolved in neat triethylphosphite (0.411 mL, 2.4 mmol), and the

resulting solution was heated to 150 'C for 4 h. The reaction mixture was cooled to 0 'C and then

diluted with DMF (10 mL). NaH (60% w/v in mineral oil, 0.096 g, 2.4 mmol) was added to the

resulting solution, and the reaction mixture stirred at 0 'C for 20 min. A solution of 4-bromo-2-

chloro-benzaldehyde (7c; 0.447 g, 2.04 mmol) in DMF (10 mL) was then added drop-wise. The

reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction

mixture was diluted with EtOAc (20 mL), and washed with 10% w/v citric acid (30 mL) followed

by brine (30 mL). The organic layer was separated, dried with Na2SO4(s), and filtered. The solvent

was removed under reduced pressure, and the crude product was suspended in ice-cold DCM (5

mL). The resulting precipitate was collected by filtration to afford compound IIa as a white solid

(0.514 g, 69% over 2 steps). 'H NMR (500 MHz, CD30D, 6): 7.09-7.13 (d, J= 16.3 Hz, 1H),

7.26-7.29 (d, J= 12.53 Hz, 2H), 7.35-7.39 (t, J= 7.61 Hz, 1H), 7.53 (s, 3H), 7.61-7.63 (d, J=

7.74 Hz, 1H), 7.79 (s, 1H); 13C NMR (125 MHz, CD30D, 6): 124.42, 125.32, 126.40, 127.54,

127.67, 131.61, 131.80, 131.80, 132.97, 134.94, 135.71, 141.09; ASAP-MS (m/z): M calcd for

C14H9Br2Cl, 369.8754; found, 369.8740.

2-Boronic Acid Pinacol Ester-1-[(1E)-2-(3-Boronic acid pinacol ester)ethenylU-4-chloro-benzene

(11b). Compound 1 la (0.100 g, 0.270 mmol), KOAc (0.152 g, 1.62 mmol), bis(pinacolato)diboron

(0.410 g, 1.62 mmol), and Pd(dppf)C12 (0.0270 g, 0.020 mmol) were added to a flame-dried

Schlenk flask, which was then evacuated and backfilled with N2(g). Dioxane (3.6 mL) was

deoxygenated by sonication under high vacuum and backfilled with N2(g). The deoxygenated

dioxane was then added by cannula into the reaction flask, and the reaction mixture was heated to

303

80 'C and stirred overnight. The reaction mixture was filtered, and the solvent was removed under

reduced pressure. The crude product was purified by flash column chromatography (2% v/v DCM

in hexanes) to afford 11b as a white solid (0.111 g, 87%). 'H NMR (500 MHz, CD30D, 6): 1.36

(s, 12H), 1.38 (s, 12H), 7.27-7.30 (d, J= 16.29 Hz, 1H), 7.38-7.41 (t, J= 7.47 Hz, lH), 7.55-7.58

(d, J= 16.30 Hz, 1H), 7.64-7.66 (d, J= 7.73 Hz, lH), 7.67-7.70 (t, J= 7.88 Hz, 2H), 7.72 (s, lH),

7.81-7.82 (d, J= 7.75 Hz, 1H), 7.96 (s, 1H); 13C NMR (125 MHz, CD30D, 6): 26.45, 26.48, 50.43,

50.60, 86.55, 86.74, 126.43, 128.29, 130.59, 132.18, 134.79, 135.29, 135.31, 135.40, 136.90,

138.03, 138.91, 140.44; HRMS-ESI (m/z): [M + NH4]* calcd for C2 6 H33B2C1O4, 484.2607; found,

484.2600.

2-Boronic Acid-1-[(JE)-2-(3-boronic Acid)ethenyl]-4-chloro-benzene (11). Compound 11b

(0.100 g, 0.214 mmol) was dissolved in 4:1 THF/H20 (2.1 mL). NaIO4 (0.227 g, 1.07 mmol) was

added to the resulting solution, followed by a few drops of 1.0 M HCl. The reaction mixture was

then stirred overnight. The reaction mixture was diluted with H20 (2 mL) and extracted with

EtOAc (3 x 2 mL). The organic layers were combined and washed with brine (4 mL), dried with

Na2SO4(s), and filtered. The solvent was removed under reduced pressure, and the crude product

was purified by flash column chromatography (2% v/v CH30H in DCM) to afford compound 11

as a white crystalline solid (0.040 g, 63%) with mp 146.3 0C. 'H NMR (500 MHz, CD 30D, 6):

7.24-7.27 (d, J= 16.3 Hz, 1H), 7.37-7.40 (t, J= 7.57 Hz, 1H), 7.52-7.57 (m, 3H), 7.63-7.64 (d,

J= 4.84 Hz, 2H), 7.79 (s, 2H); 13C NMR (125 MHz, CD 30D, 6): 125.12, 126.78, 128.85, 129.02,

129.16, 129.80, 133.24, 133.95, 134.31, 135.93, 137.64; HRMS-ESI (m/z): [M - H]- caled forthe

single methyl boronate ester C15H1 4B2CIO4, 315.0772; found, 315.0772.

304

A.4.3 Protein Expression and Purification

Plasmids that direct the expression of wild-type TTR and its V30M variant were prepared in the

pET32b vector from Merck KGaA (Darmstadt, Germany) by standard methods. 223 To create the

plasmid encoding V30M TTR, two double-stranded DNA fragments were prepared by PCR using

complementary primers containing V30M-generating substitutions and gene-specific primers

targeting opposite termini for assembly with the plasmid fragment. Wild-type TTR and its V30M

variant were produced in Escherichia coli strain BL-21 from Merck KGaA cultured in Luria-

Bertani medium containing ampicillin (200 iM) at 37 'C. Gene expression was induced when

OD6oo nm reached ~2.0, and cells were then grown for an additional 4 h at 37 'C. Cell pellets were

resuspended in 20 mM Tris-HCl buffer, pH 7.4, containing EDTA (1.0 mM) and lysed with a

high-pressure cell disruptor from Constant Systems (Kennesaw, GA). The soluble fraction was

isolated by centrifugation for 10 min at 10,500g and for 1 h at 30,000g.

Wild-type TTR and its V30M variant were purified as described previously, 461 with minor

modifications. The lysate was fractionated with aqueous ammonium sulfate at 60-85% saturation.

The precipitate was dissolved in 20 mM Tris-HCl buffer, pH 7.8, containing EDTA (1.0 mM) and

dialyzed overnight against this same buffer. The isolate was clarified at 30,000g for 30 min,

filtered, and applied to a Hitrap Q HP column from GE Healthcare Life Sciences (Pittsburgh, PA)

that had been equilibrated with the dialysis buffer. TTR was eluted with the same buffer containing

NaCl (1.0 M) and was subjected to gel-filtration chromatography on a Superdex 75 column from

GE Healthcare Life Sciences (Pittsburgh, PA) that had been pre-equilibrated with 10 mM sodium

phosphate buffer, pH 7.6, containing KCl (100 mM). Pure tetrameric TTR eluted at -0.6 column

volumes as monitored by SDS-PAGE. The concentration of wild-type TTR and its V30M variant

305

was determined from the A280 .m by using e = 18.5 x 10' M'cm' and confirmed with a

bicinchoninic acid (BCA) assay using a kit from Pierce Biotechnology (Rockford, IL).

A.4.4 Competitive Fluorescence Assay

Fluorescence measurements were performed with a Photon Technology International

Quantamaster spectrofluorometer (Edison, NJ). Wild-type TTR was found to form a complex with

ANS that has a Kd value of 3.2 pM (data not shown).462 To determine the affinity for ligands, wild-

type TTR tetramers (500 nM) were incubated in 2 mL of 10 mM sodium phosphate buffer, pH 7.6,

containing KCl (100 mM) and ANS (5.0 pM) until the fluorescence signal (excitation: 410 nm;

emission: 460 nm) was stable (~30 min). A ligand (1 nM-10 pM) was then added in aliquots (5

[tL) from a stock solution in dimethyl formamide (DMF). The fluorescence intensity at each ligand

concentration was recorded before adding the next dose. Control experiments with only the solvent

(DMF) revealed that the fluorescence intensity dropped proporationally with the dilution factor.

Accordingly, average intensities were adjusted for the dilution incurred upon adding ligand and

were expressed as a percent change from the initial measurement. The final DMF concentration

was 6.3% by volume after 27 additions of ligand.

The data did not fit well to the one- or two-site competitive binding models used previously

to describe other systems. 462 The asymmetric behavior is likely a consequence of two distinct

binding events and a modest decrease in fluorescence intensity resulting from the first ANS

dissociation event. The steepest inflection point at higher ligand concentrations can be attributed

to the half-maximal concentration required to compete ANS from the second binding site. The

Prusoff-Cheng relation was used to account for this competition, resulting in a logistic equation: 463

306

% I = %O + %sf S (1)

1 '2 1+ ANS - [ligand])

where Kd,2 is the equilibrium dissociation constant of the second site, n is the Hill coefficient, and

S is the symmetry parameter. Values of Kd,2 were determined with Prism 6 software from Graphpad

(La Jolla, CA) by holding constant the ligand concentration, ANS concentration, and Kd,ANS

3.2 ptM and varying other parameters to maximize the value of R2, which was >0.99 for all datasets.

The value of Kd,2= (373 10) nM for the TTR-tafamidis complex determined with this method is

similar to the value of Kd,2 = 278 nM determined with isothermal titration calorimetry. 41" Values

of Kd,1 were not obtainable by this method, though modest inflection points were observed in the

low nanomolar range (Figure A.S 1). Reported values of K,1 for other ligands fall within this range,

however, our efforts to improve kinetic stabilization properties focus on reducing the Kd,2 value.

A.4.5 Fibril-Formation Assay

Light-scattering at 400 nm was used to assess the formation of fibrils under acidic conditions, as

described previously. 464 In this assay, turbidity is assumed to be proportional to the mass of the

protein converted to fibrils. Ligands (7.2 or 14.4 pM) were incubated with TTR tetramers (7.2

pM) for 30 min prior to twofold dilution with 50 mM sodium acetate buffer, pH 4.4, containing

KCl (100 mM). Absorbance at 400 nm was measured ten times with four replicates in clear, flat-

bottomed, 96-well plates at 0 and 96 h using an M1000 plate reader from Tecan (Maennedorf,

Switzerland). Percent fibril formation was calculated from the difference in the light scattering that

307

accumulated after 96 h in ligand-containing wells versus wells containing only vehicle (DMF),

using the equation:

% fibril formation = A 4 0 0 nm,ligand,96 h- A 4 0 0 nmigand,o h X 100% (2)A 4 0 0 nm,DMF,96 h- A 4 0 0 nm,DMF,0 h

Standard deviations from the four replicates were propagated through this calculation.

A.4.6 Protein Crystallization and X-ray Structure Determination

Crystals were grown by vapor diffusion using a solution of wild-type TTR (-6.0 mg/mL in 10 mM

sodium phosphate buffer, pH 7.6, containing 100 mM KCl) and stilbenes 2-8, 10, or 11 (7.2 mM,

added from a stock solution in DMF). Hanging drops (2 !IL of solution containing protein and

ligand plus 2 ptL of mother liquor) were equilibrated with a reservoir of 1.0-1.3 M sodium citrate

buffer, pH 5.5, containing glycerol (1-3% v/v). 4 00 Crystals appeared after 1-3 days. TTR

precipitated in the presence of stilbene 9 (7.2 mM). Crystals were cryoprotected by brief transfer

into a solution of 1.5 M sodium citrate buffer, pH 5.5, containing glycerol (10% v/v). Diffraction

data were collected at Sector 21 of the Life Sciences Collaborative Access Team (LS-CAT) at the

Advanced Photon Source of Argonne National Laboratory (Argonne, IL). Data were reduced using

HKL2000 (Tables A.S2-A.S 1 0).465 Boronate ester restraints were obtained by measuring the bond

lengths and angles from ten CSD small-molecule structures (Table A.S1). Initial phases were

obtained by molecular replacement using the protein atoms of Protein Data Bank (PDB) entry

2qgb as a model. 450 Refinement and model building were conducted with the programs Phenix and

Coot (Tables A.S2-A.S 10 and Figures A. S4-A.S 12).228-229 Ligand models in idealized geometries

308

we-re prepanredr withi thei prngram WebAM ,nd res:1i"t weipipdin Phenix w i he rrgraMI - ' I-~ - - - ' P ..-' %4A9 TV L IVAJ', (LLI.L L%'.JLAL Z1iIL VV% AL F' ,JJJ. ,%. AAAL '. A I 1 '..L ,~ VVALL LAII %.'LJI L I

eLBOW. Restraints generated by eLBOW were modified to impose planarity on the four carbon

atoms in the olefin of the stilbene and on the carbon, boron, and two oxygen atoms in a boronic

acid group. Short interatomic distances in TTR ligand complexes are listed in Table A.S11.

Atomic coordinates and structure factors for all nine TTRdligand complexes have been deposited

in the PDB.

309

A.5 Conclusion

A series of paired stilbenes was designed, synthesized, and tested as ligands for TTR. Each ligand

contained either a phenolic hydroxyl group or a phenylboronic acid group, which was intended to

bind within the inner pocket of the T4-binding site in the dimer-dimer interface. We found that the

functional groups on the stilbene can alter the binding mode, precluding a rigorous thermodynamic

analysis. Nevertheless, our boronic acids are the first ligands observed to form a reversible

covalent bond with TTR-one with a serine residue deep in the inner pocket. Crystal structures

also revealed the first sp2-hybridized boronic esters observed with a protein. These stilbene boronic

acids inhibit the TTR fibril-formation that leads to amyloidosis. Their efficacy extends to V30M

TTR, which is a common disease-related variant. We envision that the unique attributes of boronic

acid groups could find utility in pharmacological stabilizers of other proteins.

310

A . A k nowedg*ets

We are grateful to Drs. R. M. Murphy and C. L. Jenkins for contributive discussions. I.W.W. was

supported by Biotechnology Training Grant T32 GM008349 (NIH) and a Genentech Predoctoral

Fellowship. This work was supported by Grants RO 1 GM044783 (NIH) and MCB 1518160 (NSF),

and made use of NMRFAM (University of Wisconsin-Madison), which is supported by Grant P41

GM103399 (NIH).

311

Table A.1

Table A.1. Interaction of Diphenols 1-4 with Wild-type TTR and its V30M Variant

wild-type TTR V30M TTR

compound Kd,2 (pM) %FF 1:la %FF 2:la %FF 1:la %FF 2:la binding modeb

1 (resveratrol) 0.47 0.03 28 8 9 2 53 12 26 4 forward

2 0.47 0.02 21 4 9 2 48 11 23 6 reverse

3 0.73 0.02 22 4 7 3 44 10 14 4 forward

4 0.44 0.01 19 4 4 2 36 8 12 3 reverse

a%FF, percent fibril formation. b"forward": upper phenyl ring depicted in Chart A.2 lies in the

outer pocket of the T4-binding site, according to X-ray diffraction analysis (Figures A.2 and A.3);

"reverse": upper ring lies in the inner pocket.413

312

Tnhh- A.7

Table A.2. Interactions of Carboxylic Acids 5-8 with Wild-type TTR and its V30M Variant

wild-type TTR V30M TTR

compound Kd,2 (pM) %FF 1:la %FF 2:la %FF 1:la %FF 2:la binding modeb

5 1.8 0.1 75 24 77 14 112 25 120 32 forward

6 0.99 0.02 21 4 11 2 48 12 28 8 reverse

7 0.47 0.01 21 4 3 1 41 10 9 2 forward

8 0.45 0.01 16 4 4 1 32 9 10 2 reverse

a%FF, percent fibril formation. b,"forward": upper phenyl ring depicted in Chart A.2 lies in the

outer pocket of the T4-binding site, according to X-ray diffraction analysis (Figures A.2 and

A.3); "reverse": upper ring lies in the inner pocket.413

313

Table A.3

Table A.3. Interactions of Compounds 9-12 with Wild-type TTR and its V30M Variant

wild-type TTR V30M TTR

compound Kd,2(PM) %FF 1:la %FF2:1a %FF 1:la %FF2:la bindingmodeh

9 0.82 0.01 30 6 12 2 62 16 27 10 NDC

10 0.47 0.03 20 3 3 3 32 9 8 3 covalent

11 0.79 0.03 26 4 10 3 54 16 16 5 forward/covalent

12 (tafamidis) 0.37 0.01 22 5 0 6 5 13 8 7 forwardd

a%FF, percent fibril formation. b,"forward": upper phenyl ring depicted in Chart A.3 lies in the

outer pocket of the T4-binding site, according to X-ray diffraction analysis (Figure A.2); 413

"covalent": formation of a boronate ester with Ser 17. CND, not determined. dRef. 417.

314

Table A.S1

Table A.S1. Bond Angles and Bond Lengths of Planar Boronic Esters in Small-molecule CrystalStructures in the Cambridge Structural Database (CSD) and in Protein Co-crystal Structures Reportedin this Work. The parameter n refers to the number of B-OR' bonds in the structure. The means valuesof the bond angles a and f, and the bond length rB-OR' from these small-molecule structures were used inthe refinement of X-ray diffraction data from the TTR-10 and TTR-11 complexes with the programphenix refine.

SR'

CSD entry

DOFLIUDOFLOADOFLUGHOXPOALUKWUKNEYVIXQEHMATREZYEATOMKAJWUMCAK

a (0)124125122120118119119119118118

/f (0)123124123120120121120122112109

rB-OR' (A)1.371.371.381.371.361.361.351.361.391.37

n2242I11421

Mean SD 120 3 119 5 1.37 0.02

Ligand Chain a (0) 6 (0) rB-OR' (A)10 A 116 109 1.44

10 B 116 108 1.3711 A 121 116 1.4211 B 124 109 1.43

315

Table A.S2

Table A.S2. Crystallographic Data Collection and Refinement Statistics

for the TTR 2 Complex.

Complex 2PDB Code 5u48

Data CollectionX-Ray SourceDetectorWavelength (A)Resolution, last shell (A)Space group

Unit cell, a, b, c (A)Unit cell, a, p6, y (0)No. of ReflectionsNo. of Unique ReflectionsRedundancy (last shell)Mean I/a (last shell)Completeness (last shell)R-meas (last shell)R-pim (last shell)Wilson B-factorAverage Mosaicity (0)

RefinementWorking Set (last shell)Test Set (last shell)Rwork (last shell)Rftee (last shell)RMSD of Bond Lengths (A)RMSD of Bond Angles (0)

Total Number of AtomsProtein ResiduesProteinLigandWater

Average B-factorProteinLigandWater

Ramachandran Favored, Allowed, Outliers(%) from MolProbity

LS-CAT 21-ID-GMAR 300 CCD0.9785735.5-1.50 (1.55-1.50)P21 21 21a = 43.228, b = 84.947c = 64.606a =,8= y = 90284,04039,026 (1904)7.3 (7.3)28.6 (2.6)99.9 (100.0)0.056 (0.738)0.021 (0.272)18.160.2

35,141 (3422)3843 (376)0.178 (0.233)0.205 (0.260)0.0071.05198523118263812123.923.422.431.9

98.7, 1.3, 0

316

Tahle A S3

Table A.S3. Data Collection and Refinement Statistics for the TTR 3

Complex.

ComplexPDB Code

35u49

Data CollectionX-Ray SourceDetectorWavelength (A)Resolution, last shell (A)Space group

Unit cell, a, b, c (A)Unit cell, a, f, y (0 )No. of ReflectionsNo. of Unique ReflectionsRedundancy (last shell)Mean I/ (last shell)Completeness (last shell)R-meas (last shell)R-pim (last shell)Wilson B-factorAverage Mosaicity (0)

RefinementWorking Set (last shell)Test Set (last shell)Rwork (last shell)Rfree (last shell)RMSD of Bond Lengths (A)RMSD of Bond Angles (0)

Total Number of AtomsProtein ResiduesProteinLigandWater

Average B-factorProteinLigandWater

Ramachandran Favored, Allowed, Outliers(%) from MolProbity

LS-CAT 21-ID-DDetris Eiger 9M1.23980133.0-2.22 (2.30-2.22)12 2 2a = 44.517, b 65.895c = 84.594a ='8 = y = 9039,1095956 (300)6.6 (4.4)19.2 (2.3)98.7 (98.0)0.395 (1.722)0.151 (0.757)38.31.3

5598 (1311)621 (146)0.202 (0.249)0.275 (0.318)0.0081.04925116896181141.241.339.538.5

96.5, 3.5, 0

317

Table A.S4

Table A.S4. Crystallographic Data Collection and Refinement Statisticsfor the TTR 4 Complex.Complex 4PDB Code 5u4a

Data CollectionX-Ray SourceDetectorWavelength (A)Resolution, last shell (A)Space group

Unit cell, a, b, c (A)Unit cell, a, ,8, y (0)No. of ReflectionsNo. of Unique ReflectionsRedundancy (last shell)Mean I/- (last shell)Completeness (last shell)R-meas (last shell)R-pim (last shell)Wilson B-factorAverage Mosaicity (0)

RefinementWorking Set (last shell)Test Set (last shell)Rwork (last shell)Rfree (last shell)RMSD of Bond Lengths (A)RMSD of Bond Angles (0)

Total Number of AtomsProtein ResiduesProteinLigandWater

Average B-factorProteinLigandWater

Ramachandran Favored, Allowed, Outliers(%) from MolProbity

LS-CAT 21-ID-GMAR 300 CCD0.9785735.5-1.90 (1.96-1.90)P21 2j 21a = 42.822, b 84.928c = 64.19a =,8 = y = 90137,45619,212 (936)7.2 (7.2)19.4 (2.3)99.8 (100.0)0.097 (0.787)0.037 (0.289)33.61.4

17,310 (1291)1859 (143)0.223 (0.247)0.276 (0.279)0.0071.0319072321792407539.038.932.444.6

97.4, 2.2, 0.4

318

Table A.ql

Table A.S5. Crystallographic Data Collection and Refinement Statistics

for the TTR-5 Complex.

ComplexPDB Code

55u4b

Data CollectionX-Ray SourceDetectorWavelength (A)Resolution, last shell (A)Space group

Unit cell, a, b, c (A)Unit cell, a,,8, y (0)

No. of ReflectionsNo. of Unique ReflectionsRedundancy (last shell)Mean I/- (last shell)Completeness (last shell)R-meas (last shell)R-pim (last shell)Wilson B-factorAverage Mosaicity (0)

RefinementWorking Set (last shell)Test Set (last shell)Rwork (last shell)Rfee (last shellRMSD of Bond Lengths (A)RMSD of Bond Angles (0)

Total Number of AtomsProtein ResiduesProteinLigandWater

Average B-factorProteinLigandWater

Ramachandran Favored, Allowed, Outliers(%) from MolProbity

LS-CAT 21-ID-GMAR 300 CCD0.9785738.5-1.45 (1.5-1.45)P 2 1 2, 2,a = 43.052, b = 85.446c = 64.107a='p=y=90311,25242,801 (2112)7.3 (7.2)25.4 (2.7)99.9 (100.0)0.065 (0.582)0.024 (0.214)15.40.2

38,552 (3756)4230 (405)0.186 (0.241)0.207 (0.269)0.0071.03205123118583615719.618.827.327.2

99.2, 0.8, 0

319

Table A.S6

Table A.S6. Crystallographic Data Collection and Refinement Statistics

for the TTR-6 Complex.Complex 6PDB Code 5u4c

Data CollectionX-Ray SourceDetectorWavelength (A)Resolution, last shell (A)Space group

Unit cell, a, b, c (A)Unit cell, a, p, y (0)No. of ReflectionsNo. of Unique ReflectionsRedundancy (last shell)Mean I/- (last shell)Completeness (last shell)R-meas (last shell)R-pim (last shell)Wilson B-factorAverage Mosaicity (0)

RefinementWorking Set (last shell)Test Set (last shell)Rwork (last shell)Rfree (last shell)RMSD of Bond Lengths (A)RMSD of Bond Angles (0)

Total Number of AtomsProtein ResiduesProteinLigandWater

Average B-factorProteinLigandWater

Ramachandran Favored, Allowed, Outliers(%) from MolProbity

LS-CAT 21-ID-GMAR 300 CCD0.9785736.0-1.7 (1.76-1.70)P 21 2, 2,a = 42.924, b = 85.580c = 63.762a =8= y = 90191,62326,541 (1303)7.2 (7.1)23.9 (2.0)99.6 (99.1)0.065 (0.818)0.024 (0.301)19.40.6

23,900 (2334)2593 (224)0.176 (0.225)0.204 (0.271)0.0071.061204323118694013423.823.730.830.1

97.9. 1.7. 0.4

320

Thh A.S7

Table A.S7. Crystallographic Data Collection and Refinement Statistics

for the TTR-7 Complex.

Complex 7PDB Code 5u4d

Data CollectionX-Ray SourceDetectorWavelength (A)Resolution, last shell (A)Space groupUnit cell, a, b, c (A)

Unit cell, a,f8, y (')No. of ReflectionsNo. of Unique ReflectionsRedundancy (last shell)Mean I/- (last shell)Completeness (last shell)R-meas (last shell)R-pim (last shell)Wilson B-factorAverage Mosaicity (0)

RefinementWorking Set (last shell)Test Set (last shell)Rwork (last shell)Rfree (last shell)RMSD of Bond Lengths (A)RMSD of Bond Angles (0)

Total Number of AtomsProtein ResiduesProteinLigandWater

Average B-factorProteinLigandWater

Ramachandran Favored, Allowed, Outliers(%) from MolProbity

LS-CAT 21-ID-FMAR 225 CCD0.9787238.5-1.55 (1.60-1.55)P2, 21 2a = 43.207, b = 84.814c = 64.618a =#= y = 9021,607435,258 (1735)6.1 (5.2)23.7 (2.1)99.8 (99.5)0.072 (0.675)0.028 (0.291)19.20.2

31740 (3067)3474 (364)0.206 (0.303)0.243 (0.356)0.0071.038198923118213813026.425.826.634.1

97.5, 2.5, 0

321

Table A.S8

Table A.S8. Crystallographic Data Collection and Refinement Statistics

for the TTR 8 Complex.

Complex 8PDB Code 5u4e

Data CollectionX-Ray SourceDetectorWavelength (A)Resolution, last shell (A)Space group

Unit cell, a, b, c (A)Unit cell, a, 8, ' (0)No. of ReflectionsNo. of Unique ReflectionsRedundancy (last shell)Mean I/a (last shell)Completeness (last shell)R-meas (last shell)R-pim (last shell)Wilson B-factorAverage Mosaicity (0)

RefinementWorking Set (last shell)Test Set (last shell)Rwork (last shell)Rfree (last shell)RMSD of Bond Lengths (A)RMSD of Bond Angles (0)

Total Number of AtomsProtein ResiduesProteinLigandWater

Average B-factorProteinLigandWater

Ramachandran Favored, Allowed, Outliers(%) from MolProbity

LS-CAT 21-ID-GMAR 300 CCD0.9785726.0-1.45 (1.50-1.45)P21 21 21a = 43.029, b = 85.861c = 63.831a=/=y=90309,84942,788 (2094)7.2 (7.1)30.2 (2.5)100.0 (99.9)0.055 (0.659)0.20 (0.243)15.40.4

38,504 (3747)4229 (405)0.18 (0.259)0.198 (0.290)0.0071.163215423119018416919.819.220.128.6

98.4, 1.2, 0.4

322

Tabhh A.Q9

Table A.S9. Crystallographic Data Collection and Refinement Statisticsfor the TTR -10 Complex.

ComplexPDB Code

105u4f

Data CollectionX-Ray SourceDetectorWavelength (A)Resolution, last shell (A)Space group

Unit cell, a, b, c (A)Unit cell, a, ,3, y (0)No. of ReflectionsNo. of Unique ReflectionsRedundancy (last shell)Mean I/- (last shell)Completeness (last shell)R-meas (last shell)R-pim (last shell)Wilson B-factorAverage Mosaicity (0)

RefinementWorking Set (last shell)Test Set (last shell)Rwork (last shell)Rfree (last shell)RMSD of Bond Lengths (A)RMSD of Bond Angles (0)

Total Number of AtomsProtein ResiduesProteinLigandWater

Average B-factorProteinLigandWater

Ramachandran Favored, Allowed, Outliers(%) from MolProbity

LS-CAT 21 -ID-DDetris Eiger 9M1.23980138.5-1.50 (1.55-1.50)P21 21 21a = 42.920, b = 85.223c = 63.942a =8= y = 90228,24338,097 (1877)6.0 (6.1)26.3 (3.9)99.5 (99.6)0.089 (0.466)0.035 (0.184)22.220.6

36,057 (3506)2000 (195)0.193 (0.247)0.220 (0.300)0.0071.12197723118124212326.926.429.335.0

98.7, 1.3, 0

323

Table A.S1O

Table A.S10. Crystallographic Data Collection and Refinement Statistics

for the TTR-11 Complex.ComplexPDB Code

115u4g

Data CollectionX-Ray SourceDetectorWavelength (A)Resolution, last shell (A)Space group

Unit cell, a, b, c (A)Unit cell, a, ,, y (0)No. of ReflectionsNo. of Unique ReflectionsRedundancy (last shell)Mean I/- (last shell)Completeness (last shell)R-meas (last shell)R-pim (last shell)Wilson B-factorAverage Mosaicity (0)

RefinementWorking Set (last shell)Test Set (last shell)Rwork (last shell)Rfree (last shellRMSD of Bond Lengths (A)RMSD of Bond Angles (0)

Total Number of AtomsProtein ResiduesProteinLigandWater

Average B-factorProteinLigandWater

Ramachandran Favored, Allowed, Outliers(%) from MolProbity

LS-CAT 2 1-ID-FMAR 225 CCD0.9787238.5-1.8 (1.86-1.80)P21 21 21a = 43.019, b = 85.118c = 64.023a =,8 = y = 90163,19022,450 (1087)7.3 (7.3)29.4 (3.1)99.7 (99.4)0.057 (0.513)0.021 (0.187)22.80.6

20,232 (1898)2189 (199)0.188 (0.246)0.223 (0.281)0.0091.207194723117994010827.627.330.532.6

97.9, 1.7, 0.4

324

Table A.1I

Table A.S11. Non-covalent Interactions and Distances in TTR Ligand Complexes.Ligand Interaction type Ligand atom Protein atom Distance (A)2 Dative BO1/A NZ, A/15 Lys/A 2.82 Dative BO1/B NZ, A/15 Lys/B 3.22 Dative Average 3.02 Hydrogen bond 004/A NZ, A/15 Lys'/A 2.62 Hydrogen bond 003/B NZ, A/15 Lys'/B 2.22 Hydrogen bond Average - 2.4

Hydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bond

DativeDativeDative

Hydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHdr b d A

002/A002/A001/A001/A002/A002/A001/A001/A

Average002/ABO1/ABO1/B

Average003/A003/B

Average001/A021/A002/B001/B

Average001,A/A001,A/A001,B/A001,B/A001,A/B001,A/B00IB/B001,B/B

OGA/117 Ser/AOG,B/117 Ser/AOGA/117 Ser'/AOG,B/117 Ser'/AOGA/117 Ser/BOGB/I 17 Ser/BOG,A/117 Ser'/BOG,B/117 Ser'/B

OGA/1 17 Ser/ANZ, A/15 Lys/ANZ, A/15 Lys/B

222222222344444444444555555555

OGA/1 17OGB/1 17OGA/1 17OGB/I 17OG,A/1 17OGA/1 17OGA/1 17OG,A/1 17

2.73.02.72.73.02.72.92.62.82.43.44.43.93.43.03.22.23.02.12.82.52.72.92.42.53.02.63.32.8

Ser'/BSer'/BSer'/BSer'/BSer'/ASer'/ASer'/ASer'/A

I y urgen Unu Ave rmage - .85 Hydrogen bond 003/B NZ, A/15 Lys/A 2.55 Hydrogen bond 002/B NZ, A/15 Lys'/A 3.65 Hydrogen bond Average - 3.16 Dative BOI/A NZ, A/15 Lys/A 2.96 Dative BO1/B NZ, A/15 Lys'/B 3.06 Hydrogen bond Average - 3.0

Hydrogen bond 002/A NZ, A/15 Lys'/A 3.0

325

NZ, A/15 Lys'/ANZ, A/15 Lys'/B

OGA/117 Ser/AOGA/1 17 Ser'/AOG,A/1 17 Ser/BOGA/117 Ser'/B

6

11 Hydrogen bond Average

NZ, A/15 Lys/BHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bond

DativeDativeCative

Hydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bondHydrogen bond

DativeHydrogen bondHydrogen bondHydrogen bondHydrogen bond

DativeHydrogen bondHydrogen bond

001/BAverage003/A003/A004/A004/A003/B003/B004/B004/B

Average003/A003/B

Average001/A002/A

AverageBO1,A/ABOIA/BAverage002,A/A

001/BAverage003/A003/A004/A004/A003/B003/B004/B004/B

AverageB02/A003/A002/A003/B

AverageBOi/A002/A0/04/B

OG, A/i 17 Ser'/AOG, A/i 17 Ser/B

NZ, A/1- Lys/ANZ, A/15 Lys'/A

NZ, A/15 Lys/ANZ, A/- Lys'/B

NZ, A/15 Lys'/ANZ, A/15 Lys/B

OGA/117 Ser'/AOG,B/117 Ser'/AOGA/117 Ser'/AOG,B/117 Ser'/AOGA/117 Ser/BOG,B/117 Ser/BOG,A/117 Ser/BOG,B/117 Ser/B

NZ-, A/i Lys/ANZ, A/15 Lys'/ANZ, A/15 Lys'/ANZ, A/15 Lys'/B

NZ, A/15 Lys/A 3.2OG,B/117 Ser'/A 2.8OGB/1i7 Ser'/B 3.0

2.9

326

111111

OGA/1 17OG,B/1 17OG,A/1 17OG,B/1 17OG,A/1 17OG,B/1 17OGA/1 17OG,B/1 17

Ser'/ASer'/ASer'/ASer'/ASer/BSer/BSer/BSer/B

4.03.53.12.92.72.33.03.12.42.52.82.22.32.32.72.32.52.92.92.93.03.03.02.92.62.73.02.83.02.52.72.83.53.62.72.62.7

Tahle A.'1)

Table A.S12. Observed o-hole Bond Lengths and Bond Angles in TTR-Ligand Complexes

Ligand Ligand atom Protein residue rci... o (A) Oc-ci o ( ) *.o=c ( )

10 C102/A 108 Ala'/A 3.6 175.4 83.410 C102/B 108 Ala'/B 3.6 174.9 87.811 CIO1/A 108 Ala'/A 3.7 173.4 85.111 C1OI/B 108 Ala'/B 3.7 175.3 84.3Average 3.7 174.8 85.2

327

Figure A.1

rN

C

328

Figure A.1. Three-dimensional Structure of the TTR-Resveratrol Complex. TTR monomers (tan,

red, green, and purple ribbons) have a P-sandwich fold and assemble into a tetramer, which binds

to two molecules of resveratrol (ball-and-stick). The rings of resveratrol (1) occupy inner and outer

pockets of the two T4-binding sites at the dimer-dimer interfaces. The image was created with the

program PyMOL and PDB entry 1 dvs. 4 00

329

Figure A.2

A TTR-2

C TTR-6

E TTR-10

B TTR-4

LU I -oo

F TTR-11

330

Figure A.2. Three-dimensional Structures of TTR Ligand Complexes that Contain a Boronic Acid

Group. One monomer (chain B) of the TTR tetramer is shown, and is in the same orientation in

each panel. The main chain of TTR is rendered as a ribbon, and the side chains of Lys 15 and

Serl 17 are shown explicitly. Ligands are depicted in a ball-and-stick rendition with CPK coloring

and boron atoms labeled explicitly. Alternative conformations of Ser 17 or the ligand are shown

in some panels. Arrows indicate the O117 -B bond in the TTR- 10 and TTR-11 complexes. Images

were created with the program PyMOL. (A) TTR 2 (PDB entry 5u48). (B) TTR-4 (5u4a). (C)

TTR-6 (5u4c). (D) TTR-8 (5u4e). (E) TTR-10 (5u4f). (F) TTR-11 (5u4g).

331

Figure A.3

A TTR-3

B TTR-5

C TTR-7

332

Figure A.3. Three-dimensional Structures of TTR-Ligand Complexes that do not Contain a

Boronic Acid Group. One monomer of the TTR tetramer is shown, and is in the same orientation

in each panel. The main chain of TTR is rendered as a ribbon, and the side chains of Lys15 and

Ser 17 are shown explicitly. Ligands are depicted in a ball-and-stick rendition with CPK coloring.

Alternative conformations of Ser 17 or the ligand are shown in panels B and C. Images were

created with the program PyMOL. (A) TTR-3 (PDB entry 5u49, chain A). (B) TTR 5 (5u4b,

chain B). (C) TTR -7 (5u4d, chain B).

333

Figure A.4

B

TTR-11

334

A

TTR-10

Figure A.4. Halogen-bonding Interactions in the TTR 10 and TTR 11 Complexes. One monomer

(chain B) of the TTR tetramer is shown. Chloro groups in the two ester-forming boronates exhibit

C-Cl -0108' angles that are nearly linear and Cl - 0108' distances (dashed yellow lines) that are

3.6-3.8 A (Table S 12). Images were created with the program PyMOL. (A) TTR -10 (PDB entry

5u4f). (B) TTR 11 (5u4g).

335

Figure A.S1

100- Diphenols 1-4

80

cj 60-

40-D2

20 ~--- 3-64

0--9 -8 -7 -6 -5

log[ligand]

100- Carboxylic Acids 5-8

80-

D60-

- 540-

-+- 6

c 20---8

0 --9 -8 -7 -6 -5

log[ligand]

100- Compounds 9-12

80-

60-

40 --10

S0)

20 -#- 12

0--9 -8 -7 -6 -5

log[ligand]

336

Figure A.S1. Graphs Showing the Results of ANS Competition Assays. (A) Compounds 1-4.

(B) Compounds 5-8. (C) Compounds 9-12. Data were fitted to eq 1 to derive values of K,2 (Table

1); R2 > 0.99 for each dataset.

337

Figure A.S2

100- Diphenols 1-4 M 1

80m4

60-

40 d

20-

0-

wild-type TTR V30M HTR

160 Carboxylic Acids 5-8 m140

S120CUmE 1000 S80-

60-

40-

20

0-1:1wild-

100-

80-

60-

40-

20d

0-

wild-ty~

1:2 1:1 1:2type HTR V30M TTR

Compounds 9-12

m

1p:2 1 31 1:2)e TTR V30M TR

9I10

1112

338

C0

1-

0U-

U-

C:0

0L

-o

Figure A.S2. Graphs Showing the Results of 96-h Fibril-formation Assays at Two Different

TTR:Compound Ratios. (A) Compounds 1-4. (B) Compounds 5-8. (C) Compounds 9-12. Error

bars represent the standard deviation of 16 measurements propagated through eq 2.

339

Figure A.S3

13952.400

13942.163

a ~ m12500 13000 13500 14600 14500 15000 15500 16600 16500

28237.175

42359.733

56344.407

42236.33256152.383

340

A

5-

C',CcDC

2000-

1500-

1000-

500-

B

300

200.

100

0-

20000 25000 30000 35000 40000 45000 50000 55000 60000

m/z

. . - -.. I i

"I , 1.

I IIIIII'm I

Figure A.S3. MALDI-TOF Mass Spectra to Probe the Reversibility of TTR Inhibition. TTR (3.6

tM) was treated with compound 10 (360 ptM) (red) or with buffer alone (black), both containing

DMF (5% v/v). No significant shift in mass was observed for monomeric TTR (A) or its higher

molecular mass oligomers (B), indicating that the TTR -10 complex dissociates.

341

Figure A.S4

A

Er,

k

B

Er

alAW

kI

342

S

Figure A.S4. Electron Density in the TTR-2 Complex. (A) Final structure. Blue: 2Fo - Fc

contoured at 1.0; red and green: Fo - F, contoured at -3.0O and 3.O, respectively. (B) Final

structure after ligand removal and refinement by simulated annealing. Green: F - F. contoured at

3.0a.

343

Figure A.S5

A B

344

Figure A.S5. Electron Density in the TTR-3 Complex. (A) Final structure. Blue: 2F - Fe

contoured at 1.0a; red and green: Fo - F, contoured at -3.Ou and 3.0a, respectively. (B) Final

structure after ligand removal and refinement by simulated annealing. Green: Fo - Fc contoured at

3.0-.

345

Figure A.S6

A B

'(

I

I

346

Ni

N

[a AM]

Figure A.S6. Electron Density in the TTR-4 Complex. (A) Final structure. Blue: 2F - Fc

contoured at 1.0a; red and green: Fo - Fc contoured at -3.0a and 3.0u, respectively. (B) Final

structure after ligand removal and refinement by simulated annealing. Green: Fo - Fe contoured at

3.0u.

347

Figure A.S7

A

V

/

N4

I

B

IIA

I

I

348

Am

6

Figure A.S7. Electron Density in the TTR-5 Complex. (A) Final structure. Blue: 2Fo - F,

contoured at 1.0a; red and green: F, - Fc contoured at -3.0a and 3.0u, respectively. (B) Final

structure after ligand removal and refinement by simulated annealing. Green: Fo - Fc contoured at

3.0u.

349

Figure A.S8

A

w

N

'F

350

7v tB

/

Y

/-7 //

44

I

Figure A.S8. Electron Density in the TTR-6 Complex. (A) Final structure. Blue: 2Fo - Fc

contoured at 1.0a; red and green: Fo - Fc contoured at -3.0a and 3.0a, respectively. (B) Final

structure after ligand removal and refinement by simulated annealing. Green: Fo - Fc contoured at

3.0a.

351

Figure A.S9

A

04

/

~1

6

B

352

J a,

'All4

.1;4:V

ii

480

Figure A.S9. Electron Density in the TTR-7 Complex. (A) Final structure. Blue: 2Fo - Fc

contoured at 1.0u; red and green: Fo - Fc contoured at -3.0O and 3.0u, respectively. (B) Final

structure after ligand removal and refinement by simulated annealing. Green: Fo - Fc contoured at

3.0u.

353

Figure A.S1O

A

4~

B

Ai

4~

Ai

Ioa

AA

354

Q jb

0

Figure A.S1O. Electron Density in the TTR-8 Complex. (A) Final structure. Blue: 2Fo - Fc

contoured at 1.0u; red and green: Fo - F, contoured at -3.0a and 3.0u, respectively. (B) Final

structure after ligand removal and refinement by simulated annealing. Green: Fo - Fc contoured at

3.0u.

355

Figure A.S11

A B

356

Figure A.S11. Electron Density in the TTR 10 Complex. (A) Final structure. Blue: 2F - Fc

contoured at 1.0a; red and green: F - F, contoured at -3.0c and 3.0u, respectively. (B) Final

structure after ligand removal and refinement by simulated annealing. Green: Fo - Fc contoured at

3.0u.

357

Figure A.S12

A B

4

[

4

y

358

Figure A.S12. Electron Density in the TTR-11 Complex. (A) Final structure. Blue: 2Fo - Fc

contoured at 1.0a; red and green: Fo - Fc contoured at -3.0a and 3.0u, respectively. (B) Final

structure after ligand removal and refinement by simulated annealing. Green: Fo - Fe contoured at

3.0u.

359

Chart A.l. Diphenol Ligands

HO OH

NI

OH

I (resveratrol)

HO OH

NI

HOB'H

2

HO OH

IN

NI

OH

3

HO OH

I

BH0'B'OH

4

360

Chart A.2. Carboxylic Acid Ligands

0

OHI-

OH

5

0

OH

I

HO' BOH

6

0

OHI-

C

OH

0OH

'N OH

HO' ~OH

7 8

361

Chart A.3. Diboronic Acid and Related Ligands

OH

CI

CI

OH

OH

9

HO, B'OH

CI

CI

HO

H O'BOH

10

OHBB OH

CI

HO 'BOH

11

0OH

N O

CI CI

12 (tafamidis)

362

Scheme A.1. Routes for the Synthesis of Stilbenes 2-6

I I1. triethylphosphlite (neat) 0 N HO OH

150C B8r3 N2. NaH, DMF - CM

0 *C to rt O*C to rt

89% (2 steps) 70%

Br Br2a

1. trtethylphosphlta (neat) 0 0 H150 *C BBr 32. NaH, DMF DCM0 *C to rt 0 C tort

91% (2 steps) 72%CI

Br3a

2b

0 OH

C

Br3b

bis(pinacolato)diboron HO OHKOAc, Pd(dppf)C1

2dioxane80 IC

85%

2c

bis(pinacolato)dlboron HO OHKOAc, Pd(dppf)C1

2dioxane80 *C

89%1 C

c

HO OHNaIb 4I M HCIH2O/THF

67%

Ho' BOH2

0-N+ HO OH

DCM

70%|CI

OH3

HO OHNaO

4 N1MHCIH2OrTHF

88%

HO' B'OH

1. triethylphosphite (neat)0 150*C

2. NaH, DMF0 0 C to rt

Br 83% (2 steps)

Br Br

0N ~

Br5a

0

OH2 M NaOHTHF/EtOH

96%

Br

5b

bls(pinacolato)diboronKOAc, Pd(dppf)C6dioxane80 *C

73%

0

OH

Sc

0-yN+

DCM

88%

0

OH

OH5

0

NaNO4 OH

1 M HCIH2OTTHF

90%

HO'B,OH

6

363

B

lr Br

Br

Cl

Br

Scheme A.2. Routes for the Synthesis of Stilbenes 7-11

r C

Br1. KMnO4tBuOH/H20 96%2. HCtIMeOH (2 steps)

reflux

0 LIBH4 HO PDC 0THF/Ma0H CM

C1 0 .C to rt r95% 88%

Br Br Br7a 7b 70

1. 0

-~ 0'

Br

triehylphosphite (neat) 0150 1C

2. NsH, DMF 2 M NaOH0 *C to rt THF/EtOH

83% (2 steps) 95%

Br7d

1. Br 1. Br

Cl 89% BrBr 72% (2 steps)

(2 steps) triethylphosphite (neat)iethylphasphite (neat) 150 C150 1C 2. NaH, DMF

2. NaH, DMF 0 *C to rtOCtort

Br

Cl %

C1

Br9a

bls(pinacotato)dlboronKOAc, Pd(dppf)C

2 92%dioxaneBa .C .1

0'B

Cl

- CI

B'

S1Bi1

Br bIs(pInacotuto)dlboronKOAc, Pd(dpPf)C 2

- diaxane80 1C

87%- Ct

Br11a

N.

Cl

0,B,0

11b

OH

C

Br

7.

NatO41 M HCI

H 2O/THF83%

bis(pinacolato)diboronKOAc, Pd(dppf)C 2dioxane80 *C

82%

0- 0

DCM72%

0 aOH

OHN. 7

C1 0

N. NatO4 N. OH

0, , H20THF N

75% a

7f

HO'B OH

OHB'OH

Cl

HO' ,OH

11

0- OHN.

C

DCM80% C1

H

HO, BOH

NatO41 MHCI CIH2OiTHF N

74% C1

HO' 'OH10

364

A.5 HPLC Traces

Compound 2 2.12.01.91.81.71.6.1.51.41.31.01.11.00.90.80.70.6

N 0.50.40.30.20.1

L - 0.0

5 10 15 20 25 30 35 40

time (min)

Compounc 3 2.1.2.01.9.1.81.7-1.8.1.51.41.31.21.11.00.90.80.70.50.50.40.30.20.1

.asi Za 0.0

5 10 15 20 25 30 35 40

time (min)

Compound 5

5 10 15 20 25 30 35 40

time (min)

365

2.12.01.91.81.71.61.5

. 1.4

1.31.2

-1.0Q 0.9

0.80.70.60.50.40.30.20.10.0

A.6 NMR Spectra

'H NMR (CDC13) and 13 C NMR (CDC13) of Compound 2a

II 1 1I

0

1i 11 I v I8 .5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5s 2.0 1.5 O ~ 0. 0.0 -0.5 -1.0

(n 40 -0 arc I" Li(p~

0%t . -40 5

165 160 155 150 145 140 135 130 125 120 115 110 105 100 915 9'0 85 00 75 70 65 600 5'5fl (PPMt)

366

'H NMR (CD30D) and 13 C NMR (CD30D) of Compound 2bI0 "It US R

U 3

LM14 1-

9.5 9.0 8.5 8.0 7.S 7.0 6.5 6.0 5.5fl (ppm)

44 a "N N LAN -L4.. fi 7! ! 'o 0 -

a* r4 0v M g

5.0 4.5 aC D I0004

-A M~ -

11 I

210 200 190 180 170 160150 140 130 120 110 10 90 80 70 60 50 40 30 20 10 0 -10f6 (ppm)

367

7

SD O 3.0S000

aq ID V100 00

I

2.5 2.0 1.5

11

'H NMR (CD 30D) and 13C NMR (CD30D) of Compound 2c

rlNO 8O NP% N N40 41-4 u~ v 0 1-1.9- N

I I r I

III ~ itYY A l 407

9.0 8.5 g8.0 75 ! G 6.0

170 160 150 140 130 120 110 100 90 80fl (pp.)7

3.5 3.0 2.5 2.0 1.5 1.0 0.5

70 60 50

0.0 -0.5

40 30 20 10 0

368

T

n p l s i g gp msg n r-1 , ,ni sy , , , my ,r4

fl (ppmV

'H NMR (CD30D) and 13 C NMR (CD30D) of Compound 2

C4 0*M - | o a*6*0

I I

it I~ I

TNv -4 -4 4 0

InInInW!l

, I I"

4 4 mm N4 N4-

11-1 1 1-1\

5.5 4.5 , 0fl (Im)

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.

in

1......LLI i__________________________ _______________

170 160 150 140 130 120 110 100 90 80Ri (PPM*)

70 60 50 40 30

369

8.5 8.00; 7.5

20 10 0

-4 04

II

'H NMR (CDC13) and 13 C NMR (CDCL3) of Compound 3aU, " 0acMtow o- N M vr!rn! nt!nI": ft! In asC!'R V l

n t w 8 SW 0c-4e -4-4 O

y'a6

0000 0000000o 000

.YS -9 ) -2 2 T . - !

9.0 8.5 8.0 7.5 7.0 1 6.5 560 0 I M W 5.0 fl4M 4.0 3.5 3."- rtvZID

AM em C) 0\ \ O0

J.,wN W INSl mtwPM*ruPI t. lTIt.

(n 1.5 1.0 0.5 0.0

210 200 190 180 170 160 150 140 130 120 110 100f, (ppst)

90 80 70 60 50 40 30 20 10 0 -10

370

A&I

'H NMR (CD 30D) and 13C NMR (CD30D) of Compound 3b

47k 3 N, . A - NP M

/fI//

KIsLs's- r vfm rqc - 0q~~~ c.RR

7. P a 5.5004 ++4+ -40 r 4

M.L4 \4 r4 r4 V

.0 a 4.5 4.0 9.5

II

3.0 2.5 2.0 1.5 1.0 0.5

-l fl -b s a- ,w , i . ..a -n .. s - - - .mn

570 160 150 140 130 120 110 100 90 80fi (ppm)

70 60 50 40 30 20 50

371

8.5 8.0 0.0

0

AFAW" W*Wdolv#~ Aft

LI! I

7.5

I

'H NMR (CD 30D) and 1 3 C NMR (CD 30D) of Compound 3cM5 N0

CoI

I

T,

5.0 4.S 4 0 3.5f1 (pp.4)

NO/

I I

8.5 8.0 7.S 7.0 6.5 6.0 5.5

v0 .-4 \ -4 / 0 M "

Lq \l /ic

0 0(I!

2.5 .

000

190 110 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0fl (ppm)

372

V 05. " " O $A M M &AV4: t q Ui wi N %!N 4040404wto 0 t

/ /

4,4ll , lI l

At 10 1 . . . I I I I I . I .

3.0 1.5 1.0 0 .5 0.0

'H NMR (CD 3OD) and 13 C NMR (CD3OD) of Compound 3- " 11 M"9N 91 a, 0SS0

S WWCON..-l.iN %D w w ws

y %ell ' -~

/

- ;XF-U

I

LryO LAi WA N"

CSP~ a00Nr IL

7n r4ss ry P

5.0 4.5fl (pp.)4.0 3.S 3.0

000'

oh o ow77

2.5 2.0 1.5 1.0 0.5 0.0

0 0 a000

00 00v0V, -

T= = -- .- -- , -- 1 "

i8o 170 160 150 140 130 120 110 000 90 80fl (pp.)

70 60 50 40 30

373

9.5 9.0 8.S 0.0 7.5 7.0 6.5 6.0 5.5

'34at a

LA U

01 0

i

I

20 10 0

U.-

'H NMR (CD 30D) and "C NMR (CD 30D) of Compound 4

0e - C @.

8.0 7.5 7.0 6.5 6.0 55 5.0 4.51 (p )40 3.

00

*MMM"V6N ccr4 -1 /1 4r 4- 4

a0

210 200 190 180 170 160150 140 130 120 110 100 90 00 70 60 50 40 30 20 10 0 -10f1 (PPM)

374

;*. d- M 0V

111/ P.i i .P r ,

Ch

LA

-7 71 :-! i -:= L 1 ,

I

I . I L

3.0 2.5 2.0 1.5 1.0 O.5

'H NMR (CD 30D) and 1 3 C NMR (CD 30D) of Compound 5a

as Limos A A s0 "

If i fHllI 1

I" itj7. 70 6.N5A

@4 NLA 404M5IM4000 0 Lfl0 V V4 CN N. N N4 NCN

I I 'iii

5.5 5.0 4.5 0f1 (pow)

00 eN,

I0 N

L

U0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

4- q

7777 ... - ...assa nna aI s m M aa A-I

70 160 150 140 130 120 110 100 90 80fl (ppm)

70 60 50 40 30 20 10 0

375

7

..0 8.5 8.0 6.0

.1

'H NMR (DMSO) and 3C NMR (DMSO) of Compound 5b

It'13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.S 7.0 6S 6.0 5.5 S.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

81 (Pp )

IR Pl iq 7 R0

..P . . II. wi M .- 00 00

376

7

~~~2~~

6

180 170 100 150 040 130 120 010 100 90 80 70 60 50 40 30 20 10 0fl (ppm)

'H NMR (CDC13) and ' 3 C NMR (CDCL 3) of Compound 5c

/ 1 / // I

I I I .!R *

.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0Qi (pp.)

3.0 2.5 2.0 1.5 1.0 0 .5 0.0

0 1(U)4

d;3 rmL Am ad a go N&N

III

70 160 150 140 130 120 IN 110 ;0 o) 70 60 50 40 30 20 00 0

377

C

11 L"T - - -- 7'=7=Z--7-1'1 L.

8

c;I

I3.5

P.

'H NMR (CD 30D) and 3 C NMR (CD30D) of Compound 5

l i i v i I le_ _ __ _ _ Ir I-

p 8d4,b

6.5 6.0 5.5 5.0 4.5 40 3.5 3.0 2.5 2.0 15 1.0 0.5 0.0f5 lPp5)

a I05

0; 0f%4 0 0 $A U

0

0

10 170 100 150 140 130 120 110 100 90 ' 0 70 60 50 40 30 20 10

fl(0058)

378

'H NMR (CD 30D) and "C NMR (CD30D) of Compound 6

$I 7

8 8 I_

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 .0

K3 N .4

mm M MN "'I

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0

~wawm-aa-

170 160 150 140 130 120 110 100 90 80 70 60 00 40 30 00 10 0f1 (ppM6)

379

NSN*m.elrlrlSNNNNNNNNNN N N N N N

Jill'

--.- J-" P, -Ml 0 0 " ON A j I I -04000APOP

'H NMR (CD 30D) and 13 C NMR (CD 30D) of Compound 7a

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.1

00000000000000w w vN 0 4

w413 to rDOMaca co 40 0

r" en ~ Mv

Li~/S--

70 160 150 140 130 120 110 100 90 80 70 60fl (ppm)

380

t, - v M M

I'

4.5 1pp4.8.0 7.5 7.0

50 40 30 20 00 C

6.5 6.0 5.5 5 .0

20 10C

'HNMR (CDC13) and 13 CNMR (CDC1 3) of Compound 7b

MZ a. N N

II I

c ct~

7.0 6.5 6. 5.5 5.0 4.5 40

U

8:0 7'5

P'. VMS fm M4 wIN- r

I I

3.S 3.0 2.5 2.0 1.5 1.0 0.5 0.0

L)

140 130 120 110 100 90 0 70fl (PPMo)

60 S0 40 30 20 10

381

S1.4 F%

i

LM

0

'H NMR (CDC1 3) and '3C NMR (CDC13) of Compound 7c

%D LAr4N

8.0 7.5 7.0 6.S 6.0 S.S 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.(I (Pppw

LA

382

-I.0

190 180 170 160 150 140 130 120 110 0 90 00 70 60 50 40 30 20 ;0 0fl (p.)

. 10.5 10.O 9,5 9,0 8.5

Z

ch LA LA foo

'H NMR (CDC13) and 13C NMR (CDCL3) of Compound 7d

I I I L I

I I1k

1~

epil.-JI-,M 4d 3i-.' ;

9.0 8. 8.0 7.5 7.06.5 6.0 5.5 5.0 4.5 4.0f1 (pV)

3.5 3.0 2.5 2.0 0.5 1.0 0.5 0.0

0IA fa 61%N a M @OANf M0 NOsMNM A -4 4 Mm 0-440

170 160 150 140 130 120 00 1;0 0 0f1 (ppoo)

70 60 50 40 00 20 10 0

383

S

08uuNUUU0

I

i

'H NMR (DMSO) and "C NMR (DMSO) of Compound 7e

fl ~ ~ ~ ~ & C VOOOfd~tfO * -N

7C

illi _______

13.5 13.0 12.0 12.0 10.0 0.0 10.5 10.0 9.0 9.0 8.0 8.0 7.5 7.0 0.0 6.0 5.5 5.0 4.5 4.0 3.5f1 (pp;;)

3.0 2.5 2.0 1.5 1.0 0.5 0.0

N RCm.-8-4*CCLAN MM-dN

-wdl--OMAN-

110 100 90 80 70 60 50 40 30 20 10 0fl (pp.)

C

g

I1111i170 160 I0 140 130 120

384

1 - I .- [ 117 77= _7 -_

I

I

- L_ ... 91.J

'H NMR (CD 30D) and 1 3 C NMR (CD 30D) of Compound 7f

q;; 4 4 ;

T

/ Ill//I /

________________________

00000

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 45f1 (Pp )

gg w104N scorn0

.0- !I - t

A-'- i6-@5

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10fl (ppm)

385

MD

14

4-0 3.S 3.0 i.s 2.0 1.5 1.0 05 0.0

'H NMR (CD 30D) and 13C NMR (CD30D) of Compound 7

rr0 N P N N P% P.18

9.0 8.5 8.0 7.5 7.0 6.S 6.0 5.5 5.0 4.54 40fl (pp.,)

2 15 . 0

0o0

Ol W l

I I, -,- I [ ll 1 1,

.0

'0

200 1;0 180 170 160 150 140 130 120 110 10 80fl(0743)8070605040302010

0

386

3.S 3 0 2.5 2.0 1.5 1.0 D.5 0.0

j, 7 O 6 'O 5 4 3'0 20 10 0

'H NMR (CD 30D) and 13 C NMR (CD30D) of Compound 8

Oi

0.~

0 15 1. s 00

0

03

---- U--

100 170 160 150' - ' - - - ' I ' ' ' " '

140 130 120 110 100 90 80 70 60 S0 40 30 20 0 0f, (pp.s)

387

1I11

"I i - -i

S. 1 4 ( 40 . 30 .

.4

-. 0 Mviomwevvin

I

9:0 8's 8,0 7'5 7'0 6'5 6'0 5's

N M &A N 14 M N M M 1-0Nli IR R . O V:

N &A V M N fM 0 0 0 0 0

4 4 -4 14 14 4 -4 -4 4 4 -6

'H NMR (CDC13) and 13 C NMR (CDC13) of Compound 9a

tM vvNNNN,Ln ' t In r

1-4 f-,

od~r F% r4q

.0 9.5 8.0 7:S 7:0 6'S 6.0 5.s 5 0 4.5 4,0

22

U

MWOM -I -- .j -I P. -ift

IDID

150 140 130 120 10 100 90 80 70fl (ppM) 60 s0 40 30 20 10 0

388

6- - - -- I " - -- 6, !-2- -

3.s 3.0 2.S 2 0 1.5 1.0 O.S 0.0 -O.5

I"

F" f" M r" r4 C4

1H NMR (CDC1 3) and 13C NMR (CDCb3) of Compound 9b

Mm @ \

*.J~l~ 1,

5.5 0.0 7.5 7.0

l l-' 1

6.5 6.0 5.5 s.0 4.s 4*0)

3.5 3.0 2.5 2.0 Is

MMMUUE

150 140 130 120 110 100 90 80 70

389

80

6A

1:0 O's 00

6 ;0 s ;0 40 30 20 10 0

'H NMR (CD30D) and 13C NMR (CD30D) of Compound 9

Fn W F

-z-

8.0 7.5 7.0 6.5 6.0 55 5.0 4.5 4 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

coo000

M toN 0 f F i

M r41 r,.,4

180 170 160 150 140 130 120 110 100 90 8 70 6'0 50 40 3'0 20 1 0

390

'H NMR (CD30D) and 13C NMR (CD30D) of Compound 10

SO

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 50 4.5 4.0 3.5 3.0

00

WN1 M L

2.5 2.0 1.5 1.0 0.5 0.0 -

cl

180 170 160 150 140 030 120 110 100 00 80 70 00 00 40 30 20 10 0

180 170 160 150 140 1;0 120 110 100 90 80 70 60 50 4'0 3'0 2'0 1031 (PPM)

391

'H NMR (CD 30D) and 13C NMR (CD30D) of Compound 11a

I I lI i

6.0 55 50 45 40 3.5 3'0 2'5 2.0 1.5 1.0 0.5 0.0

®R MMO W 0 *S 1 --- ---

160 ISO 140 130 120 110 100 90 s0 70 60 50R0 (pp.s)

392

A 4

v y N

c:i M r4

id-0 A R-m-- - ---- 0 41PV444"MM IN ww

| ' | ' | ' | '

L- -.0 85 8.0 7.5 7.0 6.5

40 30 20 10 0

'H NMR (CD 30D) and 13C NMR (CD30D) of Compound lib

7

liii, 1/

8.0 7. 5 7.0 f.5 6.0 5.5

'll " 'A , I"

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

inoIYA

0 0i0

toI

II II

130 120 110 00 90 00 A)f0 (ppos) 00 50 40 30 20 10 0

393

I

140

W! Wi

I

5.0

-JJ A

'H NMR (CD30D) and 13C NMR (CD 30D) of Compound 11

Ch Mr AM"$6 N Pl. *

I P L 1 I""CYY

cJ a R4.1

4.0

4.51(pp)4.08. .. 0 7.5 7.0 6.5 6.0

10 t L !L ol:

11,1

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

a

.11II

0

394

170 160 150 140 130 120 110 100 90 80 70 10 50 40 30 20 10 0f1 (p.0

" C4 q

S.5 5.0

1. UNAIDS, Fact Sheet - World AIDS Day 2018. 2018.2. Centers for Disease, C., Pneumocystis pneumonia--Los Angeles. MMWR Morb. Mortal.Wkly. Rep. 1981, 30 (21), 250-2.3. Jaffe, H. W., The early days of the HIV-AIDS epidemic in the USA. Nat. Immunol. 2008,9 (11), 1201-3.4. Barre-Sinoussi, F.; Chermann, J. C.; Rey, F.; Nugeyre, M. T.; Chamaret, S.; Gruest, J.;Dauguet, C.; Axler-Blin, C.; Vezinet-Brun, F.; Rouzioux, C.; Rozenbaum, W.; Montagnier, L.,Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiencysyndrome (AIDS). Science 1983, 220 (4599), 868-71.5. Levy, J. A.; Hoffman, A. D.; Kramer, S. M.; Landis, J. A.; Shimabukuro, J. M.; Oshiro, L.S., Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science1984, 225 (4664), 840-2.6. Popovic, M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C., Detection, isolation, andcontinuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 1984, 224 (4648), 497-500.7. Faria, N. R.; Rambaut, A.; Suchard, M. A.; Baele, G.; Bedford, T.; Ward, M. J.; Tatem, A.J.; Sousa, J. D.; Arinaminpathy, N.; Pepin, J.; Posada, D.; Peeters, M.; Pybus, 0. G.; Lemey, P.,HIV epidemiology. The early spread and epidemic ignition of HIV-1 in human populations.Science 2014, 346 (6205), 56-61.8. Derache, A.; Wallis, C. L.; Vardhanabhuti, S.; Bartlett, J.; Kumarasamy, N.; Katzenstein,D., Phenotype, genotype, and drug resistance in subtype C HIV-1 infection. J Infect. Dis. 2016,213 (2), 250-6.9. Mu, Y.; Kodidela, S.; Wang, Y.; Kumar, S.; Cory, T. J., The dawn of precision medicinein HIV: state of the art of pharmacotherapy. Expert Opin. Pharmacother. 2018, 19 (14), 1581-1595.10. Hall, H. I.; Holtgrave, D. R.; Maulsby, C., HIV transmission rates from persons living withHIV who are aware and unaware of their infection. AIDS 2012, 26 (7), 893-6.11. Pilcher, C. D.; Eron, J. J., Jr.; Galvin, S.; Gay, C.; Cohen, M. S., Acute HIV revisited: newopportunities for treatment and prevention. J Clin. Invest. 2004, 113 (7), 937-45.12. Dalgleish, A. G.; Beverley, P. C.; Clapham, P. R.; Crawford, D. H.; Greaves, M. F.; Weiss,R. A., The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus.Nature 1984, 312 (5996), 763-7.13. Berger, E. A.; Doms, R. W.; Fenyo, E. M.; Korber, B. T.; Littman, D. R.; Moore, J. P.;Sattentau, Q. J.; Schuitemaker, H.; Sodroski, J.; Weiss, R. A., A new classification for HIV-1.Nature 1998, 391 (6664), 240.14. Mao, Y.; Wang, L.; Gu, C.; Herschhorn, A.; Xiang, S. H.; Haim, H.; Yang, X.; Sodroski,J., Subunit organization of the membrane-bound HIV-1 envelope glycoprotein trimer. Nat. Struct.Mol. Biol. 2012, 19 (9), 893-9.15. Gallo, S. A.; Reeves, J. D.; Garg, H.; Foley, B.; Doms, R. W.; Blumenthal, R., Kineticstudies of HIV-1 and HIV-2 envelope glycoprotein-mediated fusion. Retrovirology 2006, 3, 90.

395

16. di Marzo Veronese, F.; Copeland, T. D.; DeVico, A. L.; Rahman, R.; Oroszlan, S.; Gallo,R. C.; Sarngadharan, M. G., Characterization of highly immunogenic p66/p51 as the reversetranscriptase of HTLV-III/LAV. Science 1986, 231 (4743), 1289-9 1.17. Bowerman, B.; Brown, P. 0.; Bishop, J. M.; Varmus, H. E., A nucleoprotein complexmediates the integration of retroviral DNA. Genes Dev. 1989, 3 (4), 469-78.18. Schroder, A. R.; Shinn, P.; Chen, H.; Berry, C.; Ecker, J. R.; Bushman, F., HIV-1integration in the human genome favors active genes and local hotspots. Cell 2002, 110 (4), 521-9.19. Louis, J. M.; Clore, G. M.; Gronenborn, A. M., Autoprocessing of HIV-1 protease is tightlycoupled to protein folding. Nat. Struct. Biol. 1999, 6 (9), 868-75.20. Mervis, R. J.; Ahmad, N.; Lillehoj, E. P.; Raum, M. G.; Salazar, F. H.; Chan, H. W.;Venkatesan, S., The gag gene products of human immunodeficiency virus type 1: alignment withinthe gag open reading frame, identification of posttranslational modifications, and evidence foralternative gag precursors. Journal of virology 1988, 62 (11), 3993-4002.21. Jacks, T.; Power, M. D.; Masiarz, F. R.; Luciw, P. A.; Barr, P. J.; Varmus, H. E.,Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature 1988, 331(6153), 280-3.22. Checkley, M. A.; Luttge, B. G.; Freed, E. 0., HIV-1 envelope glycoprotein biosynthesis,trafficking, and incorporation. Journal of molecular biology 2011, 410 (4), 582-608.23. Morita, E.; Sandrin, V.; McCullough, J.; Katsuyama, A.; Baci Hamilton, I.; Sundquist, W.I., ESCRT-III protein requirements for HIV-1 budding. Cell Host Microbe 2011, 9 (3), 23 5-242.24. Schimer, J.; Pavova, M.; Anders, M.; Pachl, P.; Sacha, P.; Cigler, P.; Weber, J.; Majer, P.;Rezacova, P.; Krausslich, H. G.; Muller, B.; Konvalinka, J., Triggering HIV polyproteinprocessing by light using rapid photodegradation of a tight-binding protease inhibitor. Nat.Commun. 2015, 6, 6461.25. Yang, H.; Nkeze, J.; Zhao, R. Y., Effects of HIV-1 protease on cellular functions and theirpotential applications in antiretroviral therapy. Cell Biosci 2012, 2 (1), 32.26. Quan, Y.; Rong, L.; Liang, C.; Wainberg, M. A., Reverse transcriptase inhibitors canselectively block the synthesis of differently sized viral DNA transcripts in cells acutely infectedwith human immunodeficiency virus type 1. Journal of virology 1999, 73 (8), 6700-7.27. De Clercq, E., The role of non-nucleoside reverse transcriptase inhibitors (NNRTIs) in thetherapy of HIV-1 infection. Antiviral Res. 1998, 38 (3), 153-79.28. Espeseth, A. S.; Felock, P.; Wolfe, A.; Witmer, M.; Grobler, J.; Anthony, N.; Egbertson,M.; Melamed, J. Y.; Young, S.; Hamill, T.; Cole, J. L.; Hazuda, D. J., HIV-1 integrase inhibitorsthat compete with the target DNA substrate define a unique strand transfer conformation forintegrase. Proc. Natl. Acad. Sci. USA 2000, 97 (21), 11244-9.29. Tsiang, M.; Jones, G. S.; Goldsmith, J.; Mulato, A.; Hansen, D.; Kan, E.; Tsai, L.; Bam,R. A.; Stepan, G.; Stray, K. M.; Niedziela-Majka, A.; Yant, S. R.; Yu, H.; Kukolj, G.; Cihlar, T.;Lazerwith, S. E.; White, K. L.; Jin, H., Antiviral activity of bictegravir (GS-9883), a novel potentHIV-1 integrase strand transfer inhibitor with an improved resistance profile. Antimicrob. AgentsChemother. 2016, 60 (12), 7086-7097.30. Jacobson, J. M.; Kuritzkes, D. R.; Godofsky, E.; DeJesus, E.; Larson, J. A.; Weinheimer,S. P.; Lewis, S. T., Safety, pharmacokinetics, and antiretroviral activity of multiple doses of

396

ibalizumab (formerly TNX-355), an anti-CD4 monoclonal antibody, in huiman immunodeficiencyvirus type 1-infected adults. Antimicrob. Agents Chemother. 2009, 53 (2), 450-7.31. Van Der Ryst, E., Maraviroc - a CCR5 antagonist for the treatment of HIV-1 infection.Front Immunol. 2015, 6, 277.32. Furuta, R. A.; Wild, C. T.; Weng, Y.; Weiss, C. D., Capture of an early fusion-activeconformation of HIV-1 gp4l. Nat. Struct. Biol. 1998, 5 (4), 276-9.33. Wild, C. T.; Shugars, D. C.; Greenwell, T. K.; McDanal, C. B.; Matthews, T. J., Peptidescorresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp4lare potent inhibitors of virus infection. Proc. Natl. Acad Sci. USA 1994, 91 (21), 9770-4.34. Saag, M. S.; Benson, C. A.; Gandhi, R. T.; Hoy, J. F.; Landovitz, R. J.; Mugavero, M. J.;Sax, P. E.; Smith, D. M.; Thompson, M. A.; Buchbinder, S. P.; Del Rio, C.; Eron, J. J., Jr.;Fatkenheuer, G.; Gunthard, H. F.; Molina, J. M.; Jacobsen, D. M.; Volberding, P. A., Antiretroviraldrugs for treatment and prevention of HIV infection in adults: 2018 recommendations of theInternational Antiviral Society-USA Panel. JAMA 2018, 320 (4), 379-396.35. Mills, E. J.; Nachega, J. B.; Bangsberg, D. R.; Singh, S.; Rachlis, B.; Wu, P.; Wilson, K.;Buchan, I.; Gill, C. J.; Cooper, C., Adherence to HAART: a systematic review of developed anddeveloping nation patient-reported barriers and facilitators. PLoS Med 2006, 3 (11), e438.36. Abram, M. E.; Ferris, A. L.; Shao, W.; Alvord, W. G.; Hughes, S. H., Nature, position, andfrequency of mutations made in a single cycle of HIV- 1 replication. Journal of virology 2010, 84(19), 9864-78.37. Chen, L.; Kwon, Y. D.; Zhou, T.; Wu, X.; O'Dell, S.; Cavacini, L.; Hessell, A. J.; Pancera,M.; Tang, M.; Xu, L.; Yang, Z. Y.; Zhang, M. Y.; Arthos, J.; Burton, D. R.; Dimitrov, D. S.; Nabel,G. J.; Posner, M. R.; Sodroski, J.; Wyatt, R.; Mascola, J. R.; Kwong, P. D., Structural basis ofimmune evasion at the site of CD4 attachment on HIV-1 gp120. Science 2009, 326 (5956), 1123-7.38. Zhou, T.; Doria-Rose, N. A.; Cheng, C.; Stewart-Jones, G. B. E.; Chuang, G. Y.;Chambers, M.; Druz, A.; Geng, H.; McKee, K.; Kwon, Y. D.; O'Dell, S.; Sastry, M.; Schmidt, S.D.; Xu, K.; Chen, L.; Chen, R. E.; Louder, M. K.; Pancera, M.; Wanninger, T. G.; Zhang, B.;Zheng, A.; Farney, S. K.; Foulds, K. E.; Georgiev, I. S.; Joyce, M. G.; Lemmin, T.; Narpala, S.;Rawi, R.; Soto, C.; Todd, J. P.; Shen, C. H.; Tsybovsky, Y.; Yang, Y.; Zhao, P.; Haynes, B. F.;Stamatatos, L.; Tiemeyer, M.; Wells, L.; Scorpio, D. G.; Shapiro, L.; McDermott, A. B.; Mascola,J. R.; Kwong, P. D., Quantification of the impact of the HIV-1-glycan shield on antibodyelicitation. Cell Rep. 2017, 19 (4), 719-732.39. Barouch, D. H.; Whitney, J. B.; Moldt, B.; Klein, F.; Oliveira, T. Y.; Liu, J.; Stephenson,K. E.; Chang, H. W.; Shekhar, K.; Gupta, S.; Nkolola, J. P.; Seaman, M. S.; Smith, K. M.;Borducchi, E. N.; Cabral, C.; Smith, J. Y.; Blackmore, S.; Sanisetty, S.; Perry, J. R.; Beck, M.;Lewis, M. G.; Rinaldi, W.; Chakraborty, A. K.; Poignard, P.; Nussenzweig, M. C.; Burton, D. R.,Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys. Nature 2013, 503 (7475), 224-8.40. Murray, A. J.; Kwon, K. J.; Farber, D. L.; Siliciano, R. F., The latent reservoir for HIV-1:how immunologic memory and clonal expansion contribute to HIV-1 persistence. J Immunol.2016, 197 (2), 407-17.

397

41. Ryscavage, P.; Kelly, S.; Li, J. Z.; Harrigan, P. R.; Taiwo, B., Significance and clinicalmanagement of persistent low-level viremia and very-low-level viremia in HIV-1-infectedpatients. Antimicrob. Agents Chemother. 2014, 58 (7), 3585-98.42. Margolis, D. M.; Garcia, J. V.; Hazuda, D. J.; Haynes, B. F., Latency reversal and viralclearance to cure HIV-1. Science 2016, 353 (6297), aaf6517.43. Shirakawa, K.; Chavez, L.; Hakre, S.; Calvanese, V.; Verdin, E., Reactivation of latentHIV by histone deacetylase inhibitors. Trends Microbiol. 2013, 21 (6), 277-85.44. Costin, J. M., Cytopathic mechanisms of HIV-1. Virol. J 2007, 4, 100.45. Darcis, G.; Van Driessche, B.; Van Lint, C., HIV latency: should we shock or lock? TrendsImmunol. 2017, 38 (3), 217-228.46. Mitchell, A. R., Bruce Merrifield and solid-phase peptide synthesis: a historicalassessment. Biopolymers 2008, 90 (3), 175-84.47. Inouye, K.; Voynick, I. M.; Delpierre, G. R.; Fruton, J. S., New synthetic substrates forpepsin. Biochemistry 1966, 5 (7), 2473-83.48. Matayoshi, E. D.; Wang, G. T.; Krafft, G. A.; Erickson, J., Novel fluorogenic substratesfor assaying retroviral proteases by resonance energy transfer. Science 1990, 247 (4945), 954-8.49. Toth, M. V.; Marshall, G. R., A simple, continuous fluorometric assay for HIV protease.International journal ofpeptide and protein research 1990, 36 (6), 544-50.50. Miller, M., The early years of retroviral protease crystal structures. Biopolymers 2010, 94(4), 521-9.51. Wlodawer, A.; Miller, M.; Jaskolski, M.; Sathyanarayana, B. K.; Baldwin, E.; Weber, I.T.; Selk, L. M.; Clawson, L.; Schneider, J.; Kent, S. B., Conserved folding in retroviral proteases:crystal structure of a synthetic HIV-1 protease. Science 1989, 245 (4918), 616-21.52. Dunn, B. M.; Goodenow, M. M.; Gustchina, A.; Wlodawer, A., Retroviral proteases.Genome Biol. 2002, 3 (4), REVIEWS3006.53. Siva, M.; Svoboda, M.; Veverka, V.; Trempe, J. F.; Hofmann, K.; Kozisek, M.; Hexnerova,R.; Sedlak, F.; Belza, J.; Brynda, J.; Sacha, P.; Hubalek, M.; Starkova, J.; Flaisigova, I.;Konvalinka, J.; Saskova, K. G., Human DNA-damage-inducible 2 protein is structurally andfunctionally distinct from Its yeast ortholog. Sci. Rep. 2016, 6, 30443.54. Revuelta, M. V.; van Kan, J. A.; Kay, J.; Ten Have, A., Extensive expansion of Al familyaspartic proteinases in fungi revealed by evolutionary analyses of 107 complete eukaryoticproteomes. Genome Biol. Evol. 2014, 6 (6), 1480-94.55. Sparks, M. A.; Crowley, S. D.; Gurley, S. B.; Mirotsou, M.; Coffman, T. M., Classicalrenin-angiotensin system in kidney physiology. Compr. Physiol. 2014, 4 (3), 1201-28.56. Fruton, J. S., A history of pepsin and related enzymes. Q. Rev. Biol. 2002, 77 (2), 127-47.57. Strisovsky, K.; Tessmer, U.; Langner, J.; Konvalinka, J.; Krausslich, H. G., Systematicmutational analysis of the active-site threonine of HIV-I proteinase: rethinking the "fireman's grip"hypothesis. Protein Sci 2000, 9 (9), 1631-41.58. Galiano, L.; Bonora, M.; Fanucci, G. E., Interflap distances in HIV-1 protease determinedby pulsed EPR measurements. J Am. Chem. Soc. 2007, 129 (36), 11004-5.59. Hornak, V.; Okur, A.; Rizzo, R. C.; Simmerling, C., HIV-1 protease flaps spontaneouslyopen and reclose in molecular dynamics simulations. Proc. Natl. Acad Sci. USA 2006, 103 (4),915-20.

398

60. Radzicka, A.; Wolfenden, R., Rates of uncatalyzed peptide bond hydrolysis in neutralsolution and the transition state affinities of proteases. J Am. Chem. Soc. 1996, 118 (26), 6105-6109.61. Szecsi, P. B., The aspartic proteases. Scand J Clin. Lab. Invest. Suppl. 1992, 210, 5-22.62. Rodriguez, E. J.; Angeles, T. S.; Meek, T. D., Use of nitrogen-15 kinetic isotope effects toelucidate details of the chemical mechanism of human immunodeficiency virus 1 protease.Biochemistry 1993, 32 (46), 12380-5.63. Liu, F.; Kovalevsky, A. Y.; Louis, J. M.; Boross, P. I.; Wang, Y. F.; Harrison, R. W.;Weber, I. T., Mechanism of drug resistance revealed by the crystal structure of the unligandedHIV-1 protease with F53L mutation. Journal of molecular biology 2006, 358 (5), 1191-9.64. Sayer, J. M.; Liu, F.; Ishima, R.; Weber, I. T.; Louis, J. M., Effect of the active site D25Nmutation on the structure, stability, and ligand binding of the mature HIV-1 protease. J Biol.Chem. 2008, 283 (19), 13459-13470.65. Prabu-Jeyabalan, M.; Nalivaika, E.; Schiffer, C. A., Substrate shape determines specificityof recognition for HIV-1 protease: analysis of crystal structures of six substrate complexes.Structure 2002, 10 (3), 369-381.66. Kovalevsky, A. Y.; Chumanevich, A. A.; Liu, F.; Louis, J. M.; Weber, I. T., Caught in theact: the 1.5 A resolution crystal structures of the HIV-1 protease and the 154V mutant reveal atetrahedral reaction intermediate. Biochemistry 2007, 46 (51), 14854-64.67. Shen, C. H.; Tie, Y.; Yu, X.; Wang, Y. F.; Kovalevsky, A. Y.; Harrison, R. W.; Weber, I.T., Capturing the reaction pathway in near-atomic-resolution crystal structures of HIV- 1 protease.Biochemistry 2012, 51 (39), 7726-32.68. Das, A.; Prashar, V.; Mahale, S.; Serre, L.; Ferrer, J. L.; Hosur, M. V., Crystal structure ofHIV- 1 protease in situ product complex and observation of a low-barrier hydrogen bond betweencatalytic aspartates. Proc. Natl. Acad Sci. USA 2006, 103 (49), 18464-9.69. Tozser, J.; Weber, I. T.; Gustchina, A.; Blaha, I.; Copeland, T. D.; Louis, J. M.; Oroszlan,S., Kinetic and modeling studies of S3-S3' subsites of HIV proteinases. Biochemistry 1992, 31(20), 4793-800.70. Szeltner, Z.; Polgar, L., Rate-determining steps in HIV-1 protease catalysis. The hydrolysisof the most specific substrate. J Biol. Chem. 1996, 271 (50), 32180-32184.71. Hyland, L. J.; Tomaszek, T. A., Jr.; Roberts, G. D.; Carr, S. A.; Magaard, V. W.; Bryan,H. L.; Fakhoury, S. A.; Moore, M. L.; Minnich, M. D.; Culp, J. S.; et al., Human immunodeficiencyvirus-I protease. 1. Initial velocity studies and kinetic characterization of reaction intermediatesby 180 isotope exchange. Biochemistry 1991, 30 (34), 8441-53.72. Kipp, D. R.; Hirschi, J. S.; Wakata, A.; Goldstein, H.; Schramm, V. L., Transition states ofnative and drug-resistant HIV-1 protease are the same. Proc. Natl. Acad. Sci. USA 2012, 109 (17),6543-8.73. Wang, W.; Liang, T. C., Substrate specificity of porcine renin: P1', P1, and P3 residues ofrenin substrates are crucial for activity. Biochemistry 1994, 33 (48), 14636-41.74. Dhanaraj, V.; Dealwis, C. G.; Frazao, C.; Badasso, M.; Sibanda, B. L.; Tickle, I. J.; Cooper,J. B.; Driessen, H. P.; Newman, M.; Aguilar, C.; et al., X-ray analyses of peptide-inhibitorcomplexes define the structural basis of specificity for human and mouse renins. Nature 1992, 357(6378), 466-72.

399

75. Shintani, T.; Nomura, K.; Ichishima, E., Engineering of porcine pepsin. Alteration of SIsubstrate specificity of pepsin to those of fungal aspartic proteinases by site-directed mutagenesis.J Biol. Chem. 1997, 272 (30), 18855-61.76. Ali, A.; Bandaranayake, R. M.; Cai, Y.; King, N. M.; Kolli, M.; Mittal, S.; Murzycki, J. F.;Nalam, M. N.; Nalivaika, E. A.; Ozen, A.; Prabu-Jeyabalan, M. M.; Thayer, K.; Schiffer, C. A.,Molecular basis for drug resistance in HIV-1 protease. Viruses 2010, 2 (11), 2509-35.77. Weber, I. T.; Wu, J.; Adomat, J.; Harrison, R. W.; Kimmel, A. R.; Wondrak, E. M.; Louis,J. M., Crystallographic analysis of human immunodeficiency virus 1 protease with an analog ofthe conserved CA-p2 substrate -- interactions with frequently occurring glutamic acid residue atP2' position of substrates. Eur. J Biochem. 1997, 249 (2), 523-530.78. Tozser, J.; Bagossi, P.; Weber, I. T.; Louis, J. M.; Copeland, T. D.; Oroszlan, S., Studieson the symmetry and sequence context dependence of the HIV-1 proteinase specificity. J Biol.Chem. 1997, 272 (27), 16807-14.79. Potempa, M.; Lee, S. K.; Kurt Yilmaz, N.; Nalivaika, E. A.; Rogers, A.; Spielvogel, E.;Carter, C. W., Jr.; Schiffer, C. A.; Swanstrom, R., HIV-1 protease uses bi-specific S2/S2' subsitesto optimize cleavage of two classes of target sites. Journal ofmolecular biology 2018.80. Tozser, J., Comparative studies on retroviral proteases: substrate specificity. Viruses 2010,2 (1), 147-65.81. Miller, M.; Schneider, J.; Sathyanarayana, B. K.; Toth, M. V.; Marshall, G. R.; Clawson,L.; Selk, L.; Kent, S. B.; Wlodawer, A., Structure of complex of synthetic HIV-1 protease with asubstrate-based inhibitor at 2.3 A resolution. Science 1989, 246 (4934), 1149-52.82. Prabu-Jeyabalan, M.; Nalivaika, E.; Schiffer, C. A., How does a symmetric dimerrecognize an asymmetric substrate? A substrate complex of HIV-1 protease. Journal of molecularbiology 2000, 301 (5), 1207-20.83. Palella, F. J., Jr.; Delaney, K. M.; Moorman, A. C.; Loveless, M. 0.; Fuhrer, J.; Satten, G.A.; Aschman, D. J.; Holmberg, S. D., Declining morbidity and mortality among patients withadvanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl.J Med 1998, 338 (13), 853-60.84. Paton, N. I.; Stohr, W.; Oddershede, L.; Arenas-Pinto, A.; Walker, S.; Sculpher, M.; Dunn,D. T., The protease inhibitor monotherapy versus ongoing triple therapy (PIVOT) trial: arandomised controlled trial of a protease inhibitor monotherapy strategy for long-termmanagement of human immunodeficiency virus infection. Health Technol. Assess. 2016, 20 (21),1-158.85. Nachega, J. B.; Marconi, V. C.; van Zyl, G. U.; Gardner, E. M.; Preiser, W.; Hong, S. Y.;Mills, E. J.; Gross, R., HIV treatment adherence, drug resistance, virologic failure: evolvingconcepts. Infect. Disord. Drug Targets 2011, 11 (2), 167-74.86. Umezawa, H.; Aoyagi, T.; Morishima, H.; Matsuzaki, M.; Hamada, M., Pepstatin, a newpepsin inhibitor produced by Actinomycetes. J Antibiot. 1970, 23 (5), 259-62.87. Marciniszyn, J., Jr.; Hartsuck, J. A.; Tang, J., Mode of inhibition of acid proteases bypepstatin. J Biol. Chem. 1976, 251 (22), 7088-94.88. Dreyer, G. B.; Metcalf, B. W.; Tomaszek, T. A., Jr.; Carr, T. J.; Chandler, A. C., 3rd;Hyland, L.; Fakhoury, S. A.; Magaard, V. W.; Moore, M. L.; Strickler, J. E.; et al., Inhibition ofhuman immunodeficiency virus 1 protease in vitro: rational design of substrate analogue inhibitors.Proc. Natl. Acad Sci. USA 1989, 86 (24), 9752-6.

400

89. Rich, D. H.;- Green, J.; Toth, M. V.; Marshall, G. R.; Kent, S. B., Hydroxyethylamineanalogues of the p1 7/p2 4 substrate cleavage site are tight-binding inhibitors of HIV protease. JMed. Chem. 1990, 33 (5), 1285-8.90. Boger, J.; Lohr, N. S.; Ulm, E. H.; Poe, M.; Blaine, E. H.; Fanelli, G. M.; Lin, T. Y.; Payne,L. S.; Schorn, T. W.; LaMont, B. I.; Vassil, T. C.; Stabilito, II; Veber, D. F.; Rich, D. H.; Bopari,A. S., Novel renin inhibitors containing the amino acid statine. Nature 1983, 303 (5912), 81-4.91. Roberts, N. A.; Martin, J. A.; Kinchington, D.; Broadhurst, A. V.; Craig, J. C.; Duncan, I.B.; Galpin, S. A.; Handa, B. K.; Kay, J.; Krohn, A.; et al., Rational design of peptide-based HIVproteinase inhibitors. Science 1990, 248 (4953), 358-61.92. Merry, C.; Barry, M. G.; Mulcahy, F.; Ryan, M.; Heavey, J.; Tjia, J. F.; Gibbons, S. E.;Breckenridge, A. M.; Back, D. J., Saquinavir pharmacokinetics alone and in combination withritonavir in HIV-infected patients. AIDS 1997, 11 (4), F29-33.93. Noor, M. A.; Lo, J. C.; Mulligan, K.; Schwarz, J. M.; Halvorsen, R. A.; Schambelan, M.;Grunfeld, C., Metabolic effects of indinavir in healthy HIV-seronegative men. AIDS 2001, 15 (7),F11-8.94. Romines, K. R.; Watenpaugh, K. D.; Howe, W. J.; Tomich, P. K.; Lovasz, K. D.; Morris,J. K.; Janakiraman, M. N.; Lynn, J. C.; Horng, M. M.; Chong, K. T.; et al., Structure-based designof nonpeptidic HIV protease inhibitors from a cyclooctylpyranone lead structure. J Med. Chem.1995, 38 (22), 4463-73.95. Ghosh, A. K.; Thompson, W. J.; McKee, S. P.; Duong, T. T.; Lyle, T. A.; Chen, J. C.;Darke, P. L.; Zugay, J. A.; Emini, E. A.; Schleif, W. A.; et al., 3-Tetrahydrofuran and pyranurethanes as high-affinity P2-ligands for HIV-1 protease inhibitors. J Med. Chem. 1993, 36 (2),292-4.96. Kim, E. E.; Baker, C. T.; Dwyer, M. D.; Murcko, M. A.; Rao, B. G.; Tung, R. D.; Navia,M. A., Crystal-structure of HIV-1 protease in complex with VX-478, a potent and orallybioavailable inhibitor of the enzyme. J Am. Chem. Soc. 1995, 117 (3), 1181-1182.97. Ghosh, A. K., Aspartic Acid Proteases as Therapeutic Targets. Wiley-VCH: Weinheim,Germany, 2010; p xxiii, 615 p.98. Condra, J. H.; Schleif, W. A.; Blahy, 0. M.; Gabryelski, L. J.; Graham, D. J.; Quintero, J.C.; Rhodes, A.; Robbins, H. L.; Roth, E.; Shivaprakash, M.; et al., In vivo emergence of HIV-1variants resistant to multiple protease inhibitors. Nature 1995, 374 (6522), 569-71.99. Falcoz, C.; Jenkins, J. M.; Bye, C.; Hardman, T. C.; Kenney, K. B.; Studenberg, S.; Fuder,H.; Prince, W. T., Pharmacokinetics of GW433908, a prodrug of amprenavir, in healthy malevolunteers. J Clin. Pharmacol. 2002, 42 (8), 887-98.100. Hester, E. K.; Chandler, H. V.; Sims, K. M., Fosamprenavir: drug development foradherence. Ann. Pharmacother. 2006, 40 (7-8), 1301-10.101. Sham, H. L.; Kempf, D. J.; Molla, A.; Marsh, K. C.; Kumar, G. N.; Chen, C. M.; Kati, W.;Stewart, K.; Lal, R.; Hsu, A.; Betebenner, D.; Korneyeva, M.; Vasavanonda, S.; McDonald, E.;Saldivar, A.; Wideburg, N.; Chen, X.; Niu, P.; Park, C.; Jayanti, V.; Grabowski, B.; Granneman,G. R.; Sun, E.; Japour, A. J.; Leonard, J. M.; Plattner, J. J.; Norbeck, D. W., ABT-378, a highlypotent inhibitor of the human immunodeficiency virus protease. Antimicrob. Agents Chemother.1998, 42 (12), 3218-24.102. Bold, G.; Fassler, A.; Capraro, H. G.; Cozens, R.; Klimkait, T.; Lazdins, J.; Mestan, J.;Poncioni, B.; Rosel, J.; Stover, D.; Tintelnot-Blomley, M.; Acemoglu, F.; Beck, W.; Boss, E.;

401

Eschbach, M.; Hurlimann, T.; Masso, E.; Roussel, S.; Ucci-Stoll, K.; Wyss, D.; Lang, M., Newaza-dipeptide analogues as potent and orally absorbed HIV-1 protease inhibitors: candidates forclinical development. J Med Chem. 1998, 41 (18), 3387-401.103. Turner, S. R.; Strohbach, J. W.; Tommasi, R. A.; Aristoff, P. A.; Johnson, P. D.; Skulnick,H. I.; Dolak, L. A.; Seest, E. P.; Tomich, P. K.; Bohanon, M. J.; Horng, M. M.; Lynn, J. C.; Chong,K. T.; Hinshaw, R. R.; Watenpaugh, K. D.; Janakiraman, M. N.; Thaisrivongs, S., Tipranavir(PNU-140690): a potent, orally bioavailable nonpeptidic HIV protease inhibitor of the 5,6-dihydro-4-hydroxy-2-pyrone sulfonamide class. J Med Chem. 1998, 41 (18), 3467-76.104. Poppe, S. M.; Slade, D. E.; Chong, K. T.; Hinshaw, R. R.; Pagano, P. J.; Markowitz, M.;Ho, D. D.; Mo, H.; Gorman, R. R., 3rd; Dueweke, T. J.; Thaisrivongs, S.; Tarpley, W. G., Antiviralactivity of the dihydropyrone PNU-140690, a new nonpeptidic human immunodeficiency virusprotease inhibitor. Antimicrob. Agents Chemother. 1997, 41 (5), 1058-63.105. Ghosh, A. K.; Kincaid, J. F.; Cho, W.; Walters, D. E.; Krishnan, K.; Hussain, K. A.; Koo,Y.; Cho, H.; Rudall, C.; Holland, L.; Buthod, J., Potent HIV protease inhibitors incorporating high-affinity P2-ligands and (R)-(hydroxyethylamino)sulfonamide isostere. Bioorg. Med. Chem. Lett.1998, 8 (6), 687-90.106. Ghosh, A. K.; Kincaid, J. F.; Walters, D. E.; Chen, Y.; Chaudhuri, N. C.; Thompson, W.J.; Culberson, C.; Fitzgerald, P. M.; Lee, H. Y.; McKee, S. P.; Munson, P. M.; Duong, T. T.; Darke,P. L.; Zugay, J. A.; Schleif, W. A.; Axel, M. G.; Lin, J.; Huff, J. R., Nonpeptidal P2 ligands forHIV protease inhibitors: structure-based design, synthesis, and biological evaluation. J MedChem. 1996, 39 (17), 3278-90.107. De Meyer, S.; Azijn, H.; Surleraux, D.; Jochmans, D.; Tahri, A.; Pauwels, R.; Wigerinck,P.; de Bethune, M. P., TMC 114, a novel human immunodeficiency virus type 1 protease inhibitoractive against protease inhibitor-resistant viruses, including a broad range of clinical isolates.Antimicrob. Agents Chemother. 2005, 49 (6), 2314-21.108. Hayashi, H.; Takamune, N.; Nirasawa, T.; Aoki, M.; Morishita, Y.; Das, D.; Koh, Y.;Ghosh, A. K.; Misumi, S.; Mitsuya, H., Dimerization of HIV-1 protease occurs through two stepsrelating to the mechanism of protease dimerization inhibition by darunavir. Proc. Natl. Acad Sci.USA 2014, 111 (33), 12234-9.109. Weber, I. T.; Agniswamy, J., HIV-1 protease: structural perspectives on drug resistance.Viruses 2009, 1 (3), 1110-36.110. Rick, S. W.; Topol, I. A.; Erickson, J. W.; Burt, S. K., Molecular mechanisms of resistance:free energy calculations of mutation effects on inhibitor binding to HIV-1 protease. Protein Sci1998, 7 (8), 1750-6.111. Ragland, D. A.; Whitfield, T. W.; Lee, S. K.; Swanstrom, R.; Zeldovich, K. B.; Kurt-Yilmaz, N.; Schiffer, C. A., Elucidating the interdependence of drug resistance from combinationsof mutations. J. Chem. Theory Comput. 2017, 13 (11), 5671-5682.112. Van Marck, H.; Dierynck, I.; Kraus, G.; Hallenberger, S.; Pattery, T.; Muyldermans, G.;Geeraert, L.; Borozdina, L.; Bonesteel, R.; Aston, C.; Shaw, E.; Chen, Q.; Martinez, C.; Koka, V.;Lee, J.; Chi, E.; de Bethune, M. P.; Hertogs, K., The impact of individual humanimmunodeficiency virus type 1 protease mutations on drug susceptibility is highly influenced bycomplex interactions with the background protease sequence. Journal of virology 2009, 83 (18),9512-20.

402

113. Piana, S.; Carloni, P.; Rothlisberger, U., Drug resistance in HIV-lprotease: fexibility-assisted mechanism of compensatory mutations. Protein Sci 2002, 11 (10), 2393-402.114. Kolli, M.; Stawiski, E.; Chappey, C.; Schiffer, C. A., Human immunodeficiency virus type1 protease-correlated cleavage site mutations enhance inhibitor resistance. Journal of virology2009, 83 (21), 11027-42.115. Kurt Yilmaz, N.; Swanstrom, R.; Schiffer, C. A., Improving viral protease inhibitors tocounter drug resistance. Trends Microbiol. 2016, 24 (7), 547-557.116. Liu, Z.; Huang, X.; Hu, L.; Pham, L.; Poole, K. M.; Tang, Y.; Mahon, B. P.; Tang, W.; Li,K.; Goldfarb, N. E.; Dunn, B. M.; McKenna, R.; Fanucci, G. E., Effects of hinge-region naturalpolymorphisms on human immunodeficiency virus-type 1 protease structure, dynamics, and drugpressure evolution. J Biol. Chem. 2016, 291 (43), 22741-22756.117. Martin, P.; Vickrey, J. F.; Proteasa, G.; Jimenez, Y. L.; Wawrzak, Z.; Winters, M. A.;Merigan, T. C.; Kovari, L. C., "Wide-open" 1.3 A structure of a multidrug-resistant HIV-1 proteaseas a drug target. Structure 2005, 13 (12), 1887-95.118. Agniswamy, J.; Shen, C. H.; Aniana, A.; Sayer, J. M.; Louis, J. M.; Weber, I. T., HIV-1protease with 20 mutations exhibits extreme resistance to clinical inhibitors through coordinatedstructural rearrangements. Biochemistry 2012, 51 (13), 2819-28.119. de Vera, I. M.; Smith, A. N.; Dancel, M. C.; Huang, X.; Dunn, B. M.; Fanucci, G. E.,Elucidating a relationship between conformational sampling and drug resistance in HIV-1protease. Biochemistry 2013, 52 (19), 3278-88.120. Chetty, S.; Bhakat, S.; Martin, A. J.; Soliman, M. E., Multi-drug resistance profile of PR20HIV-1 protease is attributed to distorted conformational and drug binding landscape: moleculardynamics insights. J Biomol. Struct. Dyn. 2016, 34 (1), 135-51.121. Louis, J. M.; Roche, J., Evolution under drug pressure remodels the folding free-energylandscape of mature HIV-1 protease. Journal of molecular biology 2016, 428 (13), 2780-92.122. Zhang, Y.; Chang, Y. C.; Louis, J. M.; Wang, Y. F.; Harrison, R. W.; Weber, I. T.,Structures of darunavir-resistant HIV-1 protease mutant reveal atypical binding of darunavir towide open flaps. A CS Chem. Biol. 2014, 9 (6), 1351-8.123. Aoki, M.; Hayashi, H.; Yedidi, R. S.; Martyr, C. D.; Takamatsu, Y.; Aoki-Ogata, H.;Nakamura, T.; Nakata, H.; Das, D.; Yamagata, Y.; Ghosh, A. K.; Mitsuya, H., C-5-modifiedtetrahydropyrano-tetrahydofuran-derived protease inhibitors (PIs) exert potent inhibition of thereplication of HIV-1 variants highly resistant to various PIs, including darunavir. Journal ofvirology 2015, 90 (5), 2180-94.124. DeFrancesco, H.; Dudley, J.; Coca, A., Boron chemistry: an overview. In Boron Reagentsin Synthesis, American Chemical Society: 2016; Vol. 1236, pp 1-25.125. Lennox, A. J.; Lloyd-Jones, G. C., Selection of boron reagents for Suzuki-Miyauracoupling. Chem. Soc. Rev. 2014, 43 (1), 412-43.126. Smoum, R.; Rubinstein, A.; Dembitsky, V. M.; Srebnik, M., Boron containing compoundsas protease inhibitors. Chem. Rev. 2012, 112 (7), 4156-220.127. Tomsho, J. W.; Pal, A.; Hall, D. G.; Benkovic, S. J., Ring structure and aromatic substituenteffects on the pKa of the benzoxaborole pharmacophore. ACS Med Chem. Lett. 2012, 3 (1), 48-52.128. Lorand, J. P.; Edwards, J. 0., Polyol complexes and structure of the benzeneboronate ion.J Org. Chem. 1958, 24 (6), 769-774.

403

129. Babcock, L.; Pizer, R., Dynamics of boron acid complexation reactions. Formation of 1:1boron acid-ligand complexes. Inorg. Chem. 1980, 19 (1), 56-61.130. Yan, J.; Springsteen, G.; Deeter, S.; Wang, B., The relationship among pKa, pH, andbinding constants in the interactions between boronic acids and diols-it is not as simple as itappears. Tetrahedron 2004, 60 (49), 11205-11209.131. Bromba, C.; Carrie, P.; Chui, J. K. W.; Fyles, T. M., Phenyl boronic acid complexes ofdiols and hydroxyacids. Supramol. Chem. 2009, 21 (1-2), 81-88.132. Dowlut, M.; Hall, D. G., An improved class of sugar-binding boronic acids, soluble andcapable of complexing glycosides in neutral water. J Am. Chem. Soc. 2006, 128 (13), 4226-4227.133. Bosch, L. I.; Fyles, T. M.; James, T. D., Binary and ternary phenylboronic acid complexeswith saccharides and Lewis bases. Tetrahedron 2004, 60 (49), 11175-11190.134. Diaz, D. B.; Yudin, A. K., The versatility of boron in biological target engagement. Nat.Chem. 2017, 9 (8), 731-742.135. Lagoutte, R.; Patouret, R.; Winssinger, N., Covalent inhibitors: an opportunity for rationaltarget selectivity. Curr. Opin. Chem. Biol. 2017, 39, 54-63.136. Simon, G. M.; Cravatt, B. F., Activity-based proteomics of enzyme superfamilies: serinehydrolases as a case study. J Biol. Chem. 2010, 285 (15), 11051-5.137. Fu, H.; Fang, H.; Sun, J.; Wang, H.; Liu, A.; Sun, J.; Wu, Z., Boronic acid-based enzymeinhibitors: a review of recent progress. Curr. Med. Chem. 2014, 21 (28), 3271-80.138. Zervosen, A.; Herman, R.; Kerff, F.; Herman, A.; Bouillez, A.; Prati, F.; Pratt, R. F.; Frere,J. M.; Joris, B.; Luxen, A.; Charlier, P.; Sauvage, E., Unexpected tricovalent binding mode ofboronic acids within the active site of a penicillin-binding protein. J Am. Chem. Soc. 2011, 133(28), 10839-48.139. Lienhard, G. E.; Koehler, K. A., 2-Phenylethaneboronic acid, a possible transition-stateanalog for chymotrypsin. Biochemistry 1971, 10 (13), 2477-2483.140. Matthews, D. A.; Alden, R. A.; Birktoft, J. J.; Freer, S. T.; Kraut, J., X-ray crystallographicstudy of boronic acid adducts with subtilisin BPN' (Novo). A model for the catalytic transitionstate. J. Biol. Chem. 1975, 250 (18), 7120-6.141. Pekol, T.; Daniels, J. S.; Labutti, J.; Parsons, I.; Nix, D.; Baronas, E.; Hsieh, F.; Gan, L. S.;Miwa, G., Human metabolism of the proteasome inhibitor bortezomib: identification of circulatingmetabolites. Drug Metab. Dispos. 2005, 33 (6), 771-777.142. Raedler, L. A., Ninlaro (Ixazomib): first oral proteasome inhibitor approved for thetreatment of patients with relapsed or refractory multiple myeloma. Am. Health Drug Benefits2016, 9 (Spec Feature), 102-5.143. Vogl, D. T.; Martin, T. G.; Vij, R.; Hari, P.; Mikhael, J. R.; Siegel, D.; Wu, K. L.; Delforge,M.; Gasparetto, C., Phase I/I study of the novel proteasome inhibitor delanzomib (CEP-18770)for relapsed and refractory multiple myeloma. Leuk. Lymphoma 2017, 58 (8), 1872-1879.144. Schrader, J.; Henneberg, F.; Mata, R. A.; Tittmann, K.; Schneider, T. R.; Stark, H.;Bourenkov, G.; Chari, A., The inhibition mechanism of human 20S proteasomes enables next-generation inhibitor design. Science 2016, 353 (6299), 594.145. Vanderheyden, P. M.; Fierens, F. L.; De Backer, J.; Vauquelin, G., Reversible and syntopicinteraction between angiotensin receptor antagonists on Chinese hamster ovary cells expressinghuman angiotensin II type 1 receptors. Biochem. Pharmacol. 2000, 59 (8), 927-35.

404

146. Rock, F L.; Mao, W.; Yaremhk, A.; Tunalo, M.; Crepin, T.; Zh i H.; 7hng, Y. K.;Hernandez, V.; Akama, T.; Baker, S. J.; Plattner, J. J.; Shapiro, L.; Martinis, S. A.; Benkovic, S.J.; Cusack, S.; Alley, M. R., An antifungal agent inhibits an aminoacyl-tRNA synthetase bytrapping tRNA in the editing site. Science 2007, 316 (5832), 1759-61.147. Elewski, B. E.; Aly, R.; Baldwin, S. L.; Gonzalez Soto, R. F.; Rich, P.; Weisfeld, M.; Wiltz,H.; Zane, L. T.; Pollak, R., Efficacy and safety of tavaborole topical solution, 5%, a novel boron-based antifungal agent, for the treatment of toenail onychomycosis: results from 2 randomizedphase-III studies. J Am. Acad Dermatol. 2015, 73 (1), 62-69.148. Freund, Y. R.; Akama, T.; Alley, M. R.; Antunes, J.; Dong, C.; Jarnagin, K.; Kimura, R.;Nieman, J. A.; Maples, K. R.; Plattner, J. J.; Rock, F.; Sharma, R.; Singh, R.; Sanders, V.; Zhou,Y., Boron-based phosphodiesterase inhibitors show novel binding of boron to PDE4 bimetalcenter. FEBS Lett. 2012, 586 (19), 3410-4.149. Nazarian, R.; Weinberg, J. M., AN-2728, a PDE4 inhibitor for the potential topicaltreatment of psoriasis and atopic dermatitis. Curr. Opin. Investig. Drugs 2009, 10 (11), 1236-42.150. Ajami, D.; Dube, H.; Rebek, J., Jr., Boronic acid hydrogen bonding in encapsulationcomplexes. J Am. Chem. Soc. 2011, 133 (25), 9689-91.151. Akama, T.; Dong, C.; Virtucio, C.; Sullivan, D.; Zhou, Y.; Zhang, Y. K.; Rock, F.; Freund,Y.; Liu, L.; Bu, W.; Wu, A.; Fan, X. Q.; Jarnagin, K., Linking phenotype to kinase: identificationof a novel benzoxaborole hinge-binding motif for kinase inhibition and development of high-potency rho kinase inhibitors. J. Pharmacol. Exp. Ther. 2013, 347 (3), 615-25.152. Lomax, J. E.; Eller, C. H.; Raines, R. T., Rational design and evaluation of mammalianribonuclease cytotoxins. Methods Enzymol. 2012, 502, 273-90.153. Rutkoski, T. J.; Raines, R. T., Evasion of ribonuclease inhibitor as a determinant ofribonuclease cytotoxicity. Curr. Pharm. Biotechnol. 2008, 9 (3), 185-9.154. Raines, R. T., Ribonuclease A. Chem. Rev. 1998, 98 (3), 1045-1066.155. Klink, T. A.; Woycechowsky, K. J.; Taylor, K. M.; Raines, R. T., Contribution of disulfidebonds to the conformational stability and catalytic activity of ribonuclease A. Eur. J. Biochem.2000, 267 (2), 566-72.156. Ruoppolo, M.; Vinci, F.; Klink, T. A.; Raines, R. T.; Marino, G., Contribution of individualdisulfide bonds to the oxidative folding of ribonuclease A. Biochemistry 2000, 39 (39), 12033-42.157. Kartha, G.; Bello, J.; Harker, D., Tertiary structure of ribonuclease. Nature 1967, 213(5079), 862-5.158. Zito, E.; Melo, E. P.; Yang, Y.; Wahlander, A.; Neubert, T. A.; Ron, D., Oxidative proteinfolding by an endoplasmic reticulum-localized peroxiredoxin. Mol. Cell 2010, 40 (5), 787-97.159. delCardayre, S. B.; Ribo, M.; Yokel, E. M.; Quirk, D. J.; Rutter, W. J.; Raines, R. T.,Engineering ribonuclease A: production, purification and characterization of wild-type enzymeand mutants at Gln1 1. Protein Eng. 1995, 8 (3), 261-73.160. Findlay, D.; Herries, D. G.; Mathias, A. P.; Rabin, B. R.; Ross, C. A., The active site andmechanism of action of bovine pancreatic ribonuclease. Nature 1961, 190, 781-84.161. delCardayre, S. B.; Raines, R. T., The extent to which ribonucleases cleave ribonucleicacid. Analytical biochemistry 1995, 225 (1), 176-8.162. Kelemen, B. R.; Schultz, L. W.; Sweeney, R. Y.; Raines, R. T., Excavating an active site:the nucleobase specificity of ribonuclease A. Biochemistry 2000, 39 (47), 14487-94.

405

163. Thompson, J. E.; Kutateladze, T. G.; Schuster, M. C.; Venegas, F. D.; Messmore, J. M.;Raines, R. T., Limits to catalysis by ribonuclease A. Bioorg. Chem. 1995, 23 (4), 471-481.164. Leland, P. A.; Staniszewski, K. E.; Park, C.; Kelemen, B. R.; Raines, R. T., Theribonucleolytic activity of angiogenin. Biochemistry 2002, 41 (4), 1343-50.165. Hoang, T. T.; Raines, R. T., Molecular basis for the autonomous promotion of cellproliferation by angiogenin. Nucleic Acids Res. 2017, 45 (2), 818-831.166. Notomista, E.; Catanzano, F.; Graziano, G.; Piaz, F. D.; Barone, G.; D'Alessio, G.; DiDonato, A., Onconase: an unusually stable protein. Biochemistry 2000, 39 (30), 8711-8718.167. Reitsma, S.; Slaaf, D. W.; Vink, H.; van Zandvoort, M. A.; oude Egbrink, M. G., Theendothelial glycocalyx: composition, functions, and visualization. Pflugers Arch. 2007, 454 (3),345-59.168. Sundlass, N. K.; Eller, C. H.; Cui, Q.; Raines, R. T., Contribution of electrostatics to thebinding of pancreatic-type ribonucleases to membranes. Biochemistry 2013, 52 (37), 6304-12.169. Chao, T. Y.; Lavis, L. D.; Raines, R. T., Cellular uptake of ribonuclease A relies on anionicglycans. Biochemistry 2010, 49 (50), 10666-73.170. Fuchs, S. M.; Raines, R. T., Pathway for polyarginine entry into mammalian cells.Biochemistry 2004, 43 (9), 2438-44.171. Richard, J. P.; Melikov, K.; Brooks, H.; Prevot, P.; Lebleu, B.; Chernomordik, L. V.,Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis andheparan sulfate receptors. J Biol. Chem. 2005, 280 (15), 15300-6.172. Chao, T. Y.; Raines, R. T., Mechanism of ribonuclease A endocytosis: analogies to cell-penetrating peptides. Biochemistry 2011, 50 (39), 8374-82.173. Varkouhi, A. K.; Scholte, M.; Storm, G.; Haisma, H. J., Endosomal escape pathways fordelivery of biologicals. J Control Release 2011, 151 (3), 220-8.174. L6nn, P.; Kacsinta, A. D.; Cui, X.-S.; Hamil, A. S.; Kaulich, M.; Gogoi, K.; Dowdy, S. F.,Enhancing endosomal escape for intracellular delivery of macromolecular biologic therapeutics.Sci. Rep. 2016, 6, 32301.175. Lee, J. E.; Raines, R. T., Ribonucleases as novel chemotherapeutics: the ranpirnaseexample. BioDrugs 2008, 22 (1), 53-8.176. Haigis, M. C.; Kurten, E. L.; Raines, R. T., Ribonuclease inhibitor as an intracellular sentry.Nucleic Acids Res. 2003, 31 (3), 1024-32.177. Dickson, K. A.; Haigis, M. C.; Raines, R. T., Ribonuclease inhibitor: structure andfunction. Prog. Nucleic Acid Res. Mol. Biol. 2005, 80, 349-74.178. Johnson, R. J.; Lavis, L. D.; Raines, R. T., Intraspecies regulation of ribonucleolyticactivity. Biochemistry 2007, 46 (45), 13131-40.179. Lomax, J. E.; Bianchetti, C. M.; Chang, A.; Phillips, G. N., Jr.; Fox, B. G.; Raines, R. T.,Functional evolution of ribonuclease inhibitor: insights from birds and reptiles. Journal ofmolecular biology 2014, 426 (17), 3041-56.180. Kobe, B.; Deisenhofer, J., A structural basis of the interactions between leucine-richrepeats and protein ligands. Nature 1995, 374 (6518), 183-6.181. Turcotte, R. F.; Lavis, L. D.; Raines, R. T., Onconase cytotoxicity relies on the distributionof its positive charge. FEBS] 2009, 276 (14), 3846-57.

406

182. Johnson, R. J.; McCoy, J. G.; Bingman, C. A ; Phillips, G. N., Jr; Raines, R. T., Inhibitionof human pancreatic ribonuclease by the human ribonuclease inhibitor protein. Journal ofmolecular biology 2007, 368 (2), 434-49.183. Johnson, R. J.; Chao, T. Y.; Lavis, L. D.; Raines, R. T., Cytotoxic ribonucleases: thedichotomy of Coulombic forces. Biochemistry 2007, 46 (36), 10308-16.184. Lomax, J. E.; Eller, C. H.; Raines, R. T., Comparative functional analysis of ribonuclease1 homologs: molecular insights into evolving vertebrate physiology. Biochem. J 2017, 474 (13),2219-2233.185. Koczera, P.; Martin, L.; Marx, G.; Schuerholz, T., The ribonuclease A superfamily inhumans: canonical RNases as the buttress of innate immunity. Int. J Mol. Sci. 2016, 17 (8).186. Pulido, D.; Torrent, M.; Andreu, D.; Nogues, M. V.; Boix, E., Two human host defenseribonucleases against mycobacteria, the eosinophil cationic protein (RNase 3) and RNase 7.Antimicrob. Agents Chemother. 2013, 57 (8), 3797.187. Rosenberg, H. F., Eosinophil-derived neurotoxin (EDN/RNase 2) and the mouseeosinophil-associated RNases (mEars): expanding roles in promoting host defense. Int. J Mol. Sci.2015, 16 (7), 15442-55.188. Cocchi, F.; DeVico, A. L.; Lu, W.; Popovic, M.; Latinovic, 0.; Sajadi, M. M.; Redfield,R. R.; Lafferty, M. K.; Galli, M.; Garzino-Demo, A.; Gallo, R. C., Soluble factors from T cellsinhibiting X4 strains of HIV are a mixture of beta chemokines and RNases. Proc. Natl. Acad Sci.USA 2012, 109 (14), 5411-6.189. Youle, R. J.; Wu, Y. N.; Mikulski, S. M.; Shogen, K.; Hamilton, R. S.; Newton, D.;D'Alessio, G.; Gravell, M., RNase inhibition of human immunodeficiency virus infection of H9cells. Proc. Natl. Acad. Sci. USA 1994, 91 (13), 6012-6.190. Bedoya, V. I.; Boasso, A.; Hardy, A. W.; Rybak, S.; Shearer, G. M.; Rugeles, M. T.,Ribonucleases in HIV type 1 inhibition: effect of recombinant RNases on infection of primary Tcells and immune activation-induced RNase gene and protein expression. AIDS Res. Hum.Retroviruses 2006, 22 (9), 897-907.191. Swain, A. L.; Miller, M. M.; Green, J.; Rich, D. H.; Schneider, J.; Kent, S. B.; Wlodawer,A., X-ray crystallographic structure of a complex between a synthetic protease of humanimmunodeficiency virus 1 and a substrate-based hydroxyethylamine inhibitor. Proc. Natl. AcadSci. USA 1990, 87 (22), 8805-9.192. Rahuel, J.; Rasetti, V.; Maibaum, J.; Rueger, H.; Goschke, R.; Cohen, N. C.; Stutz, S.;Cumin, F.; Fuhrer, W.; Wood, J. M.; Grutter, M. G., Structure-based drug design: the discoveryof novel nonpeptide orally active inhibitors of human renin. Chem. Biol. 2000, 7 (7), 493-504.193. Fujinaga, M.; Chernaia, M. M.; Tarasova, N. I.; Mosimann, S. C.; James, M. N., Crystalstructure of human pepsin and its complex with pepstatin. Protein Sci 1995, 4 (5), 960-72.194. Fontecilla-Camps, J. C.; de Llorens, R.; le Du, M. H.; Cuchillo, C. M., Crystal structure ofribonuclease A.d(ApTpApApG) complex. Direct evidence for extended substrate recognition. JBiol. Chem. 1994, 269 (34), 21526-31.195. Brik, A.; Wong, C.-H., HIV-1 protease: mechanism and drug discovery. Org. Biomol.Chem. 2003, 1, 5-14.196. Ghosh, A.; Chapsal, B.; Weber, I.; Mitsuya, H., Design of HIV protease inhibitors targetingprotein backbone: an effective strategy for combating drug resistance. Acc. Chem. Res. 2008, 41,78-86.

407

197. Wensing, A. M. J.; van Maarseveen, N. M.; Nijhuis, M., Fifteen years of HIV proteaseinhibitors: raising the barrier to resistance. Antiviral Res. 2010, 85, 59-74.198. Altman, M. D.; Ali, A.; Reddy, G. S.; Nalam, M. N.; Anjum, S. G.; Cao, H.; Chellappan,S.; Kairys, V.; Fernandes, M. X.; Gilson, M. K.; Schiffer, C. A.; Rana, T. M.; Tidor, B., HIV-1protease inhibitors from inverse design in the substrate envelope exhibit subnanomolar binding todrug-resistant variants. J Am. Chem. Soc. 2008, 130 (19), 6099-113.199. Ghosh, A. K.; Dawson, Z. L.; Mitsuya, H., Darunavir, a conceptually new HIV-1 proteaseinhibitor for the treatment of drug-resistant HIV. Bioorg. Med Chem. 2007, 15 (24), 7576-80.200. King, N. M.; Prabu-Jeyabalan, M.; Nalivaika, E. A.; Wigerinck, P.; de Bethune, M. P.;Schiffer, C. A., Structural and thermodynamic basis for the binding of TMC 114, a next-generationhuman immunodeficiency virus type 1 protease inhibitor. Journal of virology 2004, 78 (21),12012-21.201. Hazen, R.; Harvey, R.; Ferris, R.; Craig, C.; Yates, P.; Griffin, P.; Miller, J.; Kaldor, I.;Ray, J.; Samano, V.; Furfine, E.; Spaltenstein, A.; Hale, M.; Tung, R.; St Clair, M.; Hanlon, M.;Boone, L., In vitro antiviral activity of the novel, tyrosyl-based human immunodeficiency virus(HIV) type 1 protease inhibitor brecanavir (GW640385) in combination with other antiretroviralsand against a panel of protease inhibitor-resistant HIV. Antimicrob. Agents Chemother. 2007, 51(9), 3147-54.202. Burlingham, B. T.; Widlanski, T. S., An intuitive look at the relationship of Ki and IC5 0 : amore general use for the Dixon plot. J Chem Educ 2003, 80 (2), 214-218.203. Williams, J. W.; Morrison, J. F., The kinetics of reversible tight-binding inhibition.Methods Enzymol. 1979, 63, 437-467.204. Murphy, D. J., Determination of accurate K values for tight-binding enzyme inhibitors:An in silico study of experimental error and assay design. Analytical biochemistry 2004, 327 (1),61-7.205. Velazquez-Campoy, A.; Freire, E., Isothermal titration calorimetry to determineassociation constants for high-affinity ligands. Nature protocols 2006, 1 (1), 186-91.206. Hanlon, M. H.; Porter, D. J.; Furfine, E. S.; Spaltenstein, A.; Carter, H. L.; Danger, D.;Shu, A. Y.; Kaldor, I. W.; Miller, J. F.; Samano, V. A., Inhibition of wild-type and mutant humanimmunodeficiency virus type 1 proteases by GW0385 and other arylsulfonamides. Biochemistry2004, 43 (45), 14500-7.207. Beck, Z. Q.; Hervio, L.; Dawson, P. E.; Elder, J. H.; Madison, E. L., Identification ofefficiently cleaved substrates for HIV- 1 protease using a phage display library and use in inhibitordevelopment. Virology 2000, 274 (2), 391-401.208. Wu, G., Assay Development: Fundamentals and Practices. John Wiley & Sons: Hoboken,NJ, 2010.209. Lavis, L. D.; Raines, R. T., Bright ideas for chemical biology. ACS Chem. Biol. 2008, 3,142-155.210. Lavis, L. D.; Rutkoski, T. J.; Raines, R. T., Tuning the pKa of fluorescein to optimizebinding assays. Anal. Chem. 2007, 79, 6775-6782.211. Sun, W.-C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P., Synthesis of fluorinatedfluoresceins. J Org. Chem. 1997, 62, 6469-6475.

408

212. Ingr, M.; Uhlikova, T.; Strisovsky, K.; Majerova, E.; Konvalinka, J., Kinetics of thedimerization of retroviral proteases: the "fireman's grip" and dimerization. Protein Sci. 2003, 12(10), 2173-82.213. Todd, M. J.; Semo, N.; Freire, E., The structural stability of the HIV-1 protease. Journalof molecular biology 1998, 283 (2), 475-88.214. Tie, Y.; Boross, P. I.; Wang, Y. F.; Gaddis, L.; Hussain, A. K.; Leshchenko, S.; Ghosh, A.K.; Louis, J. M.; Harrison, R. W.; Weber, I. T., High resolution crystal structures of HIV-1 proteasewith a potent non-peptide inhibitor (UIC-94017) active against multi-drug-resistant clinicalstrains. Journal of molecular biology 2004, 338 (2), 341-52.215. Gibson, D. G.; Young, L.; Chuang, R. Y.; Venter, J. C.; Hutchison, C. A., 3rd; Smith, H.0., Enzymatic assembly of DNA molecules up to serval hundred kilobases. Nat. Methods 2009, 6,343-345.216. Hui, J. 0.; Tomasselli, A. G.; Reardon, I. M.; Lull, J. M.; Brunner, D. P.; Tomich, C. S.;Heinrikson, R. L., Large scale purification and refolding of HIV- 1 protease from Escherichia coliinclusion bodies. Journal ofprotein chemistry 1993, 12 (3), 323-7.217. Artimo, P.; Jonnalagedda, M.; Arnold, K.; Baratin, D.; Csardi, G.; de Castro, E.; Duvaud,S.; Flegel, V.; Fortier, A.; Gasteiger, E.; Grosdidler, A.; Hernandez, C.; loannidis, V.; Kuznetsov,D.; Liechti, R.; Moretti, S.; Mostaguir, K.; Redaschi, N.; Rossier, G.; Xenarios, I.; Stockinger, H.,ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 2012, 40, W597-W603.218. Prabu-Jeyabalan, M.; Nalivaika, E. A.; King, N. M.; Schiffer, C. A., Viability of a drug-resistant human immunodeficiency virus type 1 protease variant: structural insights for betterantiviral therapy. Journal of virology 2003, 77 (2), 1306-1315.219. Liu, Z.; Wang, Y.; Brunzelle, J.; Kovari, I. A.; Kovari, L. C., Nine crystal structuresdetermine the substrate envelope of the MDR HIV-1 protease. Protein J. 2011, 30 (3), 173-183.220. Tie, Y.; Boross, P. I.; Wang, Y. F.; Gaddis, L.; Liu, F.; Chen, X.; Tozser, J.; Harrison, R.W.; Weber, I. T., Molecular basis for substrate recognition and drug resistance from 1.1 to 1.6angstroms resolution crystal structures of HIV- 1 protease mutants with substrate analogs. FEBS J.2005, 272 (20), 5265-77.221. Lee, S. K.; Potempa, M.; Swanstrom, R., The choreography of HIV-1 proteolyticprocessing and virion assembly. J. Biol. Chem. 2012, 287 (49), 40867-40874.222. Altman, M. D.; Nalivaika, E. A.; Prabu-Jeyabalan, M.; Schiffer, C. A.; Tidor, B.,Computational design and experimental study of tighter binding peptides to an inactivated mutantof HIV-1 protease. Proteins 2008, 70 (3), 678-694.223. Windsor, I. W.; Raines, R. T., Fluorogenic assay for inhibitors of HIV-1 protease with sub-picomolar affinity. Sci. Rep. 2015, 5, 11286.224. Feldblum, E. S.; Arkin, I. T., Strength of a bifurcated H bond. Proc. Natl. Acad. Sci. USA2014,1]] (11), 4085-4090.225. Ridky, T. W.; Cameron, C. E.; Cameron, J.; Leis, J.; Copeland, T.; Wlodawer, A.; Weber,I. T.; Harrison, R. W., Human immunodeficiency virus, type 1 protease substrate specificity islimited by interactions between substrate amino acids bound in adjacent enzyme subsites. J Biol.Chem. 1996, 271 (9), 4709-4717.226. Ozen, A.; Haliloglu, T.; Schiffer, C. A., Dynamics of preferential substrate recognition inHIV-1 protease: redefining the substrate envelope. Journal of molecular biology 2011, 410 (4),726-744.

409

227. Velazquez-Campoy, A.; Todd, M. J.; Vega, S.; Freire, E., Catalytic efficiency and vitalityof HIV-1 proteases from African viral subtypes. Proc. Natl. Acad Sci. USA 2001, 98 (11), 6062-6067.228. Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd,J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner,R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H., PHENIX: Acomprehensive Python-based system for macromolecular structure solution. Acta Crystallogr.2010, D66 (Pt 2), 213-221.229. Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K., Features and development of Coot.A cta Crystallogr. 2010, D66 (Pt 4), 486-50 1.230. de Oliveira, T.; Engelbrecht, S.; van Rensburg, E. J.; Gordon, M.; Bishop, K.; zur Megede,J.; Barnett, S. W.; Cassol, S., Variability at human immunodeficiency virus type 1 subtype Cprotease cleavage sites: an indication of viral fitness? Journal of virology 2003, 77, 9422-9430.231. Knowles, J. R., On the mechanism of action of pepsin. Phil. Trans. Roy. Soc. Lond. 1970,B257, 135-146.232. Dunn, B. M., Structure and mechanism of the pepsin-like family of aspartic peptidases.Chem. Rev. 2002, 102 (12), 4431-4458.233. Wlodawer, A.; Gustchina, A., Structural and biochemical studies of retroviral proteases.Biochimica et biophysica acta 2000, 1477 (1-2), 16-34.234. Li, M.; Dimaio, F.; Zhou, D.; Gustchina, A.; Lubkowski, J.; Dauter, Z.; Baker, D.;Wlodawer, A., Crystal structure of XMRV protease differs from the structures of otherretropepsins. Nat. Struct. Mol. Biol. 2011, 18 (2), 227-229.235. Zhou, A.; Carrell, R. W.; Murphy, M. P.; Wei, Z.; Yan, Y.; Stanley, P. L. D.; Stein, P. E.;Broughton Pipkin, F.; Read, R. J., A redox switch in angiotensinogen modulates angiotensinrelease. Nature 2010, 468 (7320), 108-111.236. Green, D. W.; Aykent, S.; Gierse, J. K.; Zupec, M. E., Substrate specificity of recombinanthuman renal renin: effect of histidine in the P2 subsite on pH dependence. Biochemistry 1990, 29(12), 3126-3133.237. Tbzser, J., Comparative studies on retroviral proteases: Substrate specificity. Viruses 2010,2 (1), 147-165.238. Laco, G. S.; Schalk-Hihi, C.; Lubkowski, J.; Morris, G.; Zdanov, A.; Olson, A.; Elder, J.H.; Wlodawer, A.; Gustchina, A., Crystal structures of the inactive D30N mutant of felineimmunodeficiency virus protease complexed with a substrate and an inhibitor. Biochemistry 1997,36 (35), 10696-10708.239. Tyndall, J. D.; Nall, T.; Fairlie, D. P., Proteases universally recognize beta strands in theiractive sites. Chem. Rev. 2005, 105, 973-999.240. Dall'Acqua, W.; Carter, P., Substrate-assisted catalysis: molecular basis and biologicalsignificance. Protein Sci. 2000, 9, 1-9.241. Note3, 2018.242. Kolli, M.; Ozen, A.; Kurt-Yilmaz, N.; Schiffer, C. A., HIV-1 protease-substratecoevolution in nelfinavir resistance. Journal of virology 2014, 88 (13), 7145-7154.243. Tyndall, J. D.; Pattenden, L. K.; Reid, R. C.; Hu, S. H.; Alewood, D.; Alewood, P. F.;Walsh, T.; Fairlie, D. P.; Martin, J. L., Crystal structures of highly constrained substrate and

410

hydrolysis products bound to HIV-1 protease. Implicatinc fror the -t1Ayti-7 dxT caism.Biochemistry 2008, 47 (12), 3736-3744.244. Prabu-Jeyabalan, M.; Nalivaika, E. A.; King, N. M.; Schiffer, C. A., Structural basis forcoevolution of a human immunodeficiency virus type 1 nucleocapsid-p I cleavage site with a V82Adrug-resistant mutation in viral protease. Journal of virology 2004, 78, 12446-12454.245. Prabu-Jeyabaian, M.; Nalivaika, E. A.; Romano, K.; Schiffer, C. A., Mechanism ofsubstrate recognition by drug-resistant human immunodeficiency virus type 1 protease variantsrevealed by a novel structural intermediate. Journal of virology 2006, 80, 3607-3616.246. Bandaranayake, R. M.; Prabu-Jeyabalan, M.; Kakizawa, J.; Sugiura, W.; Schiffer, C. A.,Structural analysis of human immunodeficiency virus type 1 CRF01 _AE protease in complex withthe substrate p I-p6 . Journal of virology 2008, 82, 6762-6766.247. Ozen, A.; Lin, K. H.; Kurt Yilmaz, N.; Schiffer, C. A., Structural basis and distal effectsof Gag substrate coevolution in drug resistance to HIV-1 protease. Proc. Natl. Acad Sci. USA2014, 111 (45), 15993-15998.248. Perskie, L. L.; Street, T. 0.; Rose, G. D., Structures, basins, and energies: a deconstructionof the Protein Coil Library. Protein Sci. 2008, 17 (7), 1151-1161.249. Nemethy, G.; Printz, M. P., The y turn, a possible folded conformation of the polypeptidechain. comparison with the p turn. Macromolecules 1972, 5 (6), 755-758.250. Note2, 2018.251. Pettit, S. C.; Simsic, J.; Loeb, D. D.; Everitt, L.; Hutchison, C. A., 3rd; Swanstrom, R.,Analysis of retroviral protease cleavage sites reveals two types of cleavage sites and the structuralrequirements of the P1 amino acid. J Biol. Chem. 1991, 266 (22), 14539-14547.252. Newlander, K. A.; Callahan, J. F.; Moore, M. L.; Tomaszek, T. A.; Huffman, W. F., Anovel constrained reduced-amide inhibitor of HIV-1 protease derived from the sequentialincorporation of 7-tum mimetics into a model substrate. J Med. Chem. 1993, 36 (16), 2321-233 1.253. Moore, M. L.; Dreyer, G. B., Substrate-based inhibitors of HIV-1 protease. Perspect. DrugDiscov. Des. 1993, 1 (1), 85-108.254. Hoog, S. S.; Zhao, B.; Winborne, E.; Fisher, S.; Green, D. W.; DesJarlais, R. L.;Newlander, K. A.; Callahan, J. F.; Abdel-Meguid, S. S.; Moore, M. L.; Huffman, W. F., A checkon rational drug design: crystal structure of a complex of human immunodeficiency virus type 1protease with a novel y-turn mimetic inhibitor. J Med Chem. 1995, 38 (17), 3246-3252.255. Wilhelm, P.; Lewandowski, B.; Trapp, N.; Wennemers, H., A crystal structure of anoligoproline PPII-helix, at last. J Am. Chem. Soc. 2014, 136 (45), 15829-15832.256. Adzhubei, A. A.; Sternberg, M. J.; Makarov, A. A., Polyproline-I helix in proteins:structure and function. Journal of molecular biology 2013, 425 (12), 2100-2132.257. Horng, J. C.; Raines, R. T., Stereoelectronic effects on polyproline conformation. ProteinSci. 2006, 15 (1), 74-83.258. Singh, S. K.; Das, A., The n--*w* interaction: a rapidly emerging non-covalent interaction.Phys. Chem. Chem. Phys. 2015, 17, 9596-9612.259. Newberry, R. W.; Raines, R. T., The n-sc* interaction. Acc. Chem. Res. 2017, 50 (8),1838-1846.260. Bartlett, G. J.; Choudhary, A.; Raines, R. T.; Woolfson, D. N., n-oc* interactions inproteins. Nat. Chem. Biol. 2010, 6 (8), 615-620.

411

261. Shi, Z.; Chen, K.; Liu, Z.; Kallenbach, N. R., Conformation of the backbone in unfoldedproteins. Chem. Rev. 2006, 106 (5), 1877-1897.262. Cubellis, M. V.; Caillez, F.; Blundell, T. L.; Lovell, S. C., Properties of polyproline II, asecondary structure element implicated in protein-protein interactions. Proteins 2005, 58 (4), 880-892.263. Brown, A. M.; Zondlo, N. J., A propensity scale for type II polyproline helices (PPII):aromatic amino acids in proline-rich sequences strongly disfavor PPII due to proline-aromaticinteractions. Biochemistry 2012, 51 (25), 5041-5051.264. Windsor, I. W.; Raines, R. T., A substrate selected by phage display exhibits enhancedside-chain hydrogen bonding to HIV-1 protease. Acta Crystallogr. 2018, D74, 690-694.265. Coates, L.; Tuan, H. F.; Tomanicek, S.; Kovalevsky, A.; Mustyakimov, M.; Erskine, P.;Cooper, J., The catalytic mechanism of an aspartic proteinase explored with neutron and X-raydiffraction. J Am. Chem. Soc. 2008, 130 (23), 7235-7237.266. Weinhold, F., Natural bond orbital analysis: a critical overview of relationships toalternative bonding perspectives. J Comput. Chem. 2012, 33 (30), 2363-2379.267. Weinhold, F.; Landis, C. R., Discovering Chemistry with Natural Bond Orbitals. JohnWiley & Sons: Hoboken, NJ, 2012.268. Choudhary, A.; Fry, C. G.; Kamer, K. J.; Raines, R. T., An n--->* interaction reduces theelectrophilicity of the acceptor carbonyl group. Chem. Commun. 2013, 49, 8166-8168.269. Robertus, J. D.; Kraut, J.; Alden, R. A.; Birktoft, J., Subtilisin; a stereochemical mechanisminvolving transition-state stabilization. Biochemistry 1972, 11, 4293-4303.270. Kraut, J., Serine proteases: structure and mechanism of catalysis. Annu. Rev. Biochem.1977, 46, 331-358.271. Henderson, R.; Wright, C. S.; Hess, G. P.; Blow, D. M., a-Chymotrypsin: what can welearn about catalysis from X-ray diffraction? Cold Spring Harb. Symp. Quant. Biol. 1972, 36, 63-69.272. Bryan, P.; Pantoliano, M. W.; Quill, S. G.; Hsiao, H.-Y.; Poulos, T., Site-directedmutagenesis and the role of the oxyanion hole in subtilisin. Proc. Natl. Acad. Sci. USA 1986, 83,3743-3745.273. Henderson, R., Structure of crystalline a-chymotrypsin. IV. The structure ofindoleacryloyl-a-chymotrypsin and its relevance to the hydrolytic mechanism of the enzyme.Journal of molecular biology 1970, 54, 341-354.274. Simon, L.; Goodman, J. M., Enzyme catalysis by hydrogen bonds: The balance betweentransition state binding and substrate binding in oxyanion holes. J Org. Chem. 2010, 75 (6), 1831-1840.275. Pauling, L., Chemical achievement and hope for the future. Am. Sci. 1948, 36, 51-58.276. Jencks, W. P., Strain and conformation change in enzymatic catalysis. In Current AspectsofBiochemical Energetics, Kaplan, N. 0.; Kennedy, E. P., Eds. Academic Press: New York, NY,1966; pp 273-298.277. Albery, W. J.; Knowles, J. R., Evolution of enzyme function and the development ofcatalytic efficiency. Biochemistry 1976, 15, 5631-5640.278. Lienhard, G. E., Enzymatic catalysis and transition-state theory. Science 1973, 180, 149-154.

412

279. Pauling, L., Nature of forces between large m t4oles nf 1ilngical interest. Nature 1948,161, 707-709.280. Pauling, L., Molecular architecture and biological reactions. Chem. Eng News 1946, 24,1375-1377.281. Kirby, A. J.; Komarov, I. V.; Wothers, P. D.; Feeder, N., The most twisted amide: structureand reactions. Angew. Chem. Int. Ed. 1998, 37, 785-786.282. Mujika, J. I.; Mercero, J. A.; Lopez, Z., Water-promoted hydrolysis of a highly twistedamide: rate acceleration caused by the twist of the amide bond. J. Am. Chem. Soc. 2005, 127, 445-4453.283. Aube, J., A new twist on amide solvolysis. Angew. Chem. Int. Ed. 2012, 51, 3063-3065.284. Pauling, L., In Nature of the Chemical Bond, Cornell University Press: Ithaca, NY, 1939;pp 192-193.285. Pearl, L. H., The catalytic mechanism of aspartic proteases. FEBS Lett. 1987, 214, 8-12.286. Tozser, J.; Blaha, I.; Copeland, T. D.; Wondrak, E. M.; Oroszlan, S., Comparison of theHIV- 1 and HIV-2 proteinases using oligopeptide substrates representing cleavage sites in Gag andGag-Pol polyproteins. FEBS Lett. 1991, 281 (1-2), 77-80.287. Krzemin'ska, A.; Moliner, V.; $widerek, K., Dynamic and electrostatic effects on thereaction catalyzed by HIV-1 protease. J. Am. Chem. Soc. 2016, 138 (50), 16283-16298.288. Ribeiro, A. J. M.; Santos-Martins, D.; Russo, N.; Ramos, M. J.; Fernandes, P. A.,Enzymatic flexibility and reaction rate: A QM/MM study of HIV- 1 protease. ACS Catal. 2015, 5(9), 5617-5626.289. Garrec, J.; Sautet, P.; Fleurat-Lessard, P., Understanding the HIV-1 protease reactivity withDFT: what do we gain from recent functionals? J Phys. Chem. B 2011, 115 (26), 8545-8558.290. Carnevale, V.; Raugei, S.; Piana, S.; Carloni, P., On the nature of the reaction intermediatein the HIV-1 protease: A quantum chemical study. Comput. Phys. Commun. 2008, 179 (1-3), 120-123.291. Calixto, A. R.; Bras, N. F.; Fernandes, P. A.; Ramos, M. J., Reaction mechanism of humanrenin studied by quantum mechanics/molecular mechanics (QM/MM) calculations. ACS Catal.2014, 4 (11), 3869-3876.292. Bras, N. F.; Ramos, M. J.; Fernandes, P. A., The catalytic mechanism of mouse reninstudied with QM/MM calculations. Phys. Chem. Chem. Phys. 2012, 14 (36), 12605-12613.293. Calixto, A. R.; Ramos, M. J.; Fernandes, P. A., Influence of frozen residues on theexploration of the PES of enzyme reaction mechanisms. J. Chem. Theory Comput. 2017, 13 (11),5486-5495.294. Tomasi, J.; Mennucci, B.; Cammi, R., Quantum mechanical continuum solvation models.Chem. Rev. 2005, 105, 2999-3093.295. Gaussian, 2016.296. E. D. Glendening, J., K. Badenhoop, A. E. Reed, J. E. Carpenter, J. A. Bohmann, C. M.Morales, C. R. Landis, and F. Weinhold NBO 6.0, Theoretical Chemistry Institute, University ofWisconsin: Madison, WI, 2013.297. Wendt, M.; Weinhold, F. NBOView 1.1, Theoretical Chemistry Institute, University ofWisconsin-Madison: Madison, WI, 2001.298. Akgun, B.; Hall, D. G., Fast and tight boronate formation for click bioorthogonalconjugation. Angew. Chem. Int. Ed. 2016, 55, 3909-3913.

413

299. Fitzgerald, P. M.; McKeever, B. M.; VanMiddlesworth, J. F.; Springer, J. P.; Helmbach, J.C.; Leu, C.-T.; Herger, W. K.; Dixon, R. A.; Darke, P. L., Crystallographic analysis of a complexbetween human immunodeficiency virus type 1 protease and acetyl-pepstatin at 2.0-A resolution.J Biol. Chem. 1990, 25, 14209-14219.300. Vondrasek, J.; Wlodawer, A., Inhibitors of HIV-1 protease: a major success of structure-assisted drug design. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 249-284.301. Ripka, A. S.; Rich, D. H., Peptidomimetic design. Curr. Opin. Chem. Biol. 1998, 2 (4),441-452.302. Koh, Y.; Nakata, H.; Maeda, K.; Ogata, H.; Bilcer, G.; Devasamudram, T.; Kincaid, J. F.;Boross, P.; Wang, Y. F.; Tie, Y.; Volarath, P.; Gaddis, L.; Harrison, R. W.; Weber, I. T.; Ghosh,A. K.; Mitsuya, H., Novel bis-tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor(PI) UIC-94017 (TMC 114) with potent activity against multi-PI-resistant humanimmunodeficiency virus in vitro. Antimicrob. Agents Chemother. 2003, 47 (10), 3123-3129.303. de Vera, I. M.; Blackburn, M. E.; Fanucci, G. E., Correlating conformational shift inductionwith altered inhibitor potency in a multidrug resistant HIV-1 protease variant. Biochemistry 2012,51 (40), 7813-7815.304. King, N. M.; Prabu-Jeyabalan, M.; Nalivaika, E. A.; Schiffer, C. A., Combatingsusceptibility to drug resistance: Lessons from HIV-1 protease. Chem. Biol. 2004, 11 (10), 1333-1338.305. Ghosh, A. K.; Sridhar, P. R.; Leshchenko, S.; Hussain, A. K.; Li, J.; Kovalevsky, A. Y.;Walters, D. E.; Wedekind, J. E.; Grum-Tokars, V.; Das, D.; Koh, Y.; Maeda, K.; Gatanaga, H.;Weber, I. T.; Mitsuya, H., Structure-based design of novel HIV-1 protease inhibitors to combatdrug resistance. J. Med Chem. 2006, 49 (17), 5252-61.306. Lefebvre, E.; Schiffer, C. A., Resilience to resistance of HIV-1 protease inhibitors: profileof darunavir. AIDS Rev. 2008, 10 (3), 131-142.307. Nalam, M. N.; Ali, A.; Reddy, G. S.; Cao, H.; Anjum, S. G.; Altman, M. D.; Yilmaz, N.K.; Tidor, B.; Rana, T. M.; Schiffer, C. A., Substrate envelope-designed potent HIV-1 proteaseinhibitors to avoid drug resistance. Chem. Biol. 2013, 20 (9), 1116-1124.308. Tzeli, D.; Theodorakopoulos, G.; Petsalakis, I. D.; Ajami, D.; Rebek, J., Theoretical studyof hydrogen bonding in homodimers and heterodimers of amide, boronic acid, and carboxylic acid,free and in encapsulation complexes. J. Am. Chem. Soc. 2011, 133 (42), 16977-16985.309. Hall, D. G., Boronic Acids: Preparation and Applications in Organic Synthesis, Medicineand Materials, 2nd ed Wiley-VCH: Weinheim, Germany, 2011.310. Kovalevsky, A. Y.; Tie, Y.; Liu, F.; Boross, P. I.; Wang, Y.-F.; Leshchenko, S.; Ghosh, A.K.; Harrison, R. W.; Weber, I. T., Effectiveness of nonpeptide clinical inhibitor TMC- 114 on HIV-1 protease with highly drug resistant mutations D30N, I50V, and L90M. J Med Chem. 2006, 49(4), 1379-1387.311. Perrin, C. L., Are short, low-barrier hydrogen bonds unusually strong? Acc. Chem. Res.2010, 43 (12), 1550-1557.312. Cleland, W. W.; Frey, P. A.; Gerlt, J. A., The low barrier hydrogen bond in enzymaticcatalysis. J. Biol. Chem. 1998, 273, 25529-25532.313. Bader, R. F. W., A quantum theory of molecular structure and its applications. Chem. Rev.1991, 91 (5), 893-928.

414

314. Schitt, B.; Iversen Nadrn (- V - arn P V ric T. C., On the electronicnature of low-barrier hydrogen bonds in enzymatic reactions. Proc. Nati. Acad. Sci. USA 1998, 95(22), 12799-12802.315. pKa, 2018.316. Gilli, G.; Bellucci, F.; Ferretti, V.; Bertolasi, V., Evidence for resonance-assisted hydrogenbonding from crystal-structure correlations on the enol form of the f-diketone fragment. J Am.Chem. Soc. 1989, 111, 1023-1028.317. Grosch, A. A.; van der Lubbe, S. C. C.; Fonseca, G. C., Nature of intramolecular resonanceassisted hydrogen bonding in malonaldehyde and its saturated analogue. J Phys. Chem. A 2018,122, 1813-1820.318. G6ra, R. W.; Maj, M.; Grabowski, S. J., Resonance-assisted hydrogen bonds revisited.Resonance stabilization vs. charge delocalization. Phys. Chem. Chem. Phys. 2013, 15, 2514-2522.319. Gentile, J. M.; Gentile, G. J.; Plewa, M. J., Mutagenicity of selected aniline derivatives toSalmonella following plant activation and mammalian hepatic activation. Mutat. Res. 1987, 188,185-196.320. Bomhard, E. M.; Herbold, B. A., Genotoxic activities of aniline and its metabolites andtheir relationship to the carcinogenicity of aniline in the spleen of rat. Crit. Rev. Toxicol. 2005, 35,783-835.321. Makhdoumi, P.; Limoee, M.; Ashraf, G. M.; Hossini, H., Molecular mechanism of anilineinduced spleen toxicity and neuron toxicity in experimental rat exposure: a review. Curr.Neuropharmacol. 2018, DOI: 10.2174/1570159X16666180803164238.322. Bu, W.; Akama, T.; Chanda, S.; Sullivan, D.; Ciaravino, V.; Jarnagin, K.; Freund, Y.;Sanders, V.; Chen, C.-W.; Fan, X.; Heyman, I.; Liu, L., Early rapid identification of in vivo ratmetabolites of AN6414, a novel boron-containing PDE4 inhibitor by QTRAP LC/MS/MS tosupport drug discovery. J Pharm. Biomed. Anal. 2012, 70, 344-353.323. drug, 2018.324. Surleraux, D. L.; Tahri, A.; Verschueren, W. G.; Pille, G. M.; de Kock, H. A.; Jonckers, T.H.; Peeters, A.; De Meyer, S.; Azijn, H.; Pauwels, R.; de Bethune, M. P.; King, N. M.; Prabu-Jeyabalan, M.; Schiffer, C. A.; Wigerinck, P. B., Discovery and selection of TMC 114, a nextgeneration HIV-1 protease inhibitor. J Med. Chem. 2005, 48 (6), 1813-1822.325. Greco, W. R.; Hakala, M. T., Evaluation of methods for estimating the dissociationconstant of tight binding enzyme inhibitors. J Biol. Chem. 1979, 254, 12104-12109.326. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; G. E. Scuseria; Robb, M. A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A.V.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.;Izmaylov, A. F.; Sonnenberg, J. L.; D. Williams-Young; Ding, F.; Lipparini, F.; Egidi, F.; Goings,J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.;Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;Nakajima, T.; Honda, Y.; Kitao, 0.; Nakai, H.; Vreven, T.; Throssell, K.; J. A. Montgomery, J.;Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J. J.; Brothers, E. N.; Kudin, K. N.; Staroverov,V. N.; Keith, T. A.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.;Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski,J. W.; Martin, R. L.; Morokuma, K.; Farkas, 0.; Foresman, J. B.; Fox, D. J., Gaussian 16, RevisionA.03. Gaussian, Inc.: Wallingford CT, 2016.

415

327. Molkanov, K.; Jelsch, C.; Wenger, E.; Stare, J.; Madsen, A. 0.; Kojid-Prodid, B.,Experimental evidence of a 3-centre, 2-electron covalent bond character of the central 0-H-Ofragment on the Zundel cation in crystals of Zundel nitranilate tetrahydrate. CrystEngComm 2017,19 (28), 3898-3901.328. Yedidi, R. S.; Maeda, K.; Fyvie, W. S.; Steffey, M.; Davis, D. A.; Palmer, I.; Aoki, M.;Kaufman, J. D.; Stahl, S. J.; Garimella, H.; Das, D.; Wingfield, P. T.; Ghosh, A. K.; Mitsuya, H.,P2' benzene carboxylic acid moiety is associated with decrease in cellular uptake: evaluation ofnovel nonpeptidic HIV-1 protease inhibitors containing P2 bis-tetrahydrofuran moiety.Antimicrob. Agents Chemother. 2013, 57 (10), 4920-4927.329. Ghosh, A. K.; Rao, K. V.; Nyalapatla, P. R.; Osswald, H. L.; Martyr, C. D.; Aoki, M.;Hayashi, H.; Agniswamy, J.; Wang, Y. F.; Bulut, H.; Das, D.; Weber, I. T.; Mitsuya, H., Designand development of highly potent HIV-1 protease inhibitors with a crown-like oxotricyclic coreas the P2-ligand to combat multidrug-resistant HIV variants. J Med Chem. 2017, 60 (10), 4267-4278.330. Suzuki, A., Cross-coupling reactions of organoboranes: an easy way to construct C-Cbonds (Nobel lecture). Angew. Chem. Int. Ed. 2011, 50, 6723-6737.331. Das, B. C.; Thapa, P.; Karki, R.; Schinke, C.; Das, S.; Kambhampati, S.; Banerjee, S. K.;Van Veldhuizen, P.; Verma, A.; Weiss, L. M.; Evans, T., Boron chemicals in diagnosis andtherapeutics. Future Med Chem. 2013, 5 (6), 653-676.332. Ni, N.; Wang, B., Applications of boronic acids in chemical biology and medicinalchemistry. In Boronic Acids: Preparation and Applications in Organic Synthesis, Medicine andMaterials, Wiley-VCH: Weinheim, Germany, 2011; pp 591-620.333. Hall, D. G.; Akgun, B., Boronic acids as bioorthogonal probes in site-selective labeling ofproteins. Angew. Chem. Int. Ed. 2018, 57, In Press.334. Liu, J.; Lavigne, J. J., Boronic acids in materials chemistry. In Boronic Acids: Preparationand Applications in Organic Synthesis, Medicine and Materials, 2nd ed, Hall, D. G., Ed. Wiley-VCH: Weinheim, Germany, 2011; pp 621-676.335. Brooks, W. L. A.; Sumerlin, B. S., Synthesis and applications of boronic acid-containingpolymers: from materials to medicine. Chem. Rev. 2015, 116 (3), 13 75-1397.336. Li, D.; Chen, Y.; Liu, Z., Boronate affinity materials for separation and molecularrecognition: structure, properties and applications. Chem. Soc. Rev. 2015, 44, 8097-8123.337. Severin, K., Boronic acids as building blocks for molecular nanostructures and polymericmaterials. Dalton Trans. 2009, 5254-5264.338. Trippier, P. C.; McGuigan, C., Boronic acids in medicinal chemistry: anticancer,antibacterial and antiviral applications. MedChemComm 2010, 1 (3), 183-198.339. Adams, J.; Behnke, M.; Chen, S.; Cruickshank, A. A.; Dick, L. R.; Grenier, L.; Klunder, J.M.; Ma, Y.-T.; Plamondon, L.; Stein, R. L., Potent and selective inhibitors of the proteasome:dipeptidyl boronic acids. Bioorg. Med Chem. Lett. 1998, 8, 333-338.340. Adamczyk-Wozniak, A.; Cyranfski, M. K.; Zubrowska, A.; Sporzyfnski, A.,Benzoxaboroles-old compounds with new applications. J Organomet. Chem. 2009, 694 (22),3533-3541.341. Adamczyk-Wozniak, A.; Borys, K. M.; Sporzyn'ski, A., Recent developments in thechemistry and biological applications of benzoxaboroles. Chem. Rev. 2015, 115 (11), 5224-5247.

416

342. Liu, C. T.; Tomsho, J. WV.; Bernkovic, 5. J., The unique chemistry of benzoxaboroles:current and emerging applications in biotechnology and therapeutic treatments. Bioorg. MedChem. 2014, 22 (16), 4462-4473.343. Cox, P. A.; Reid, M.; Leach, A. G.; Campbell, A. D.; King, E. J.; Lloyd-Jones, G. C., Base-catalyzed aryl-B(OH)2 protodeboronation revisited: from concerted proton-transfer to liberationof a transient arylanion. J. Am. Chem. Soc. 2017, 139 (37), 13156-13165.344. Jacobs, R. T.; Nare, B.; Wring, S. A.; Orr, M. D.; Chen, D.; Sligar, J. M.; Jenks, M. X.;Noe, R. A.; Bowling, T. S.; Mercer, L. T.; Rewerts, C.; Gaukel, E.; Owens, J.; Parham, R.;Randolph, R.; Beaudet, B.; Bacchi, C. J.; Yarlett, N.; Plattner, J. J.; Freund, Y.; Ding, C.; Akama,T.; Zhang, Y.-K.; Brun, R.; Kaiser, M.; Scandale, I.; Don, R., SCYX-7158, an orally-activebenzoxaborole for the treatment of stage 2 human African trypanosomiasis. PLoS Negl. Trop. Dis.2011, 5 (6), e1151.345. Labutti, J.; Parsons, I.; Huang, R.; Miwa, G.; Gan, L. S.; Daniels, J. S., Oxidativedeboronation of the peptide boronic acid proteasome inhibitor bortezomib: contributions fromreactive oxygen species in this novel cytochrome P450 reaction. Chem. Res. Toxicol. 2006, 19 (4),539-546.346. Giorgio, M.; Trinei, M.; Migliaccio, E.; Pelicci, P. G., Hydrogen peroxide: a metabolic by-product or a common mediator of ageing signals? Nat. Rev. Mol. Cell Biol. 2007, 8 (9), 722-728.347. Zhong, Q.; Zhang, C.; Zhang, Q.; Miele, L.; Zheng, S.; Wang, G., Boronic prodrug of 4-hydroxytamoxifen is more efficacious than tamoxifen with enhanced bioavailability independentof CYP2D6 status. BMC Cancer 2015, 15 (1), 625.348. Lippert, A. R.; Van de Bittner, G. C.; Chang, C. J., Boronate oxidation as a bioorthogonalreaction approach for studying the chemistry of hydrogen peroxide in living systems. Acc. Chem.Res. 2011, 44 (9), 793-804.349. Hoang, T. T.; Smith, T. P.; Raines, R. T., A boronic acid conjugate of angiogenin thatshows ROS-responsive neuroprotective activity. Angew. Chem. Int. Ed. 2017, 56 (10), 2619-2622.350. Springsteen, G.; Wang, B., A detailed examination of boronic acid-diol complexation.Tetrahedron 2002, 58 (26), 5291-5300.351. Hashimoto, T.; Gailvez, A. 0.; Maruoka, K., In situ assembled boronate ester assisted chiralcarboxylic acid catalyzed asymmetric trans-aziridinations. J. Am. Chem. Soc. 2013, 135, 17667-17670.352. Simmons, S.; Fratini, A.; Benin, V., A sodium salt of the dimer of boronoterephthalic acidanhydride. Acta Crystallogr. 2011, C67 (5), m123-m125.353. Zhang, L.; Cheng, J.; Carry, B.; Hou, Z., Catalytic boracarboxylation of alkynes withdiborane and carbon dioxide by an N-heterocyclic carbene copper catalyst. J Am. Chem. Soc.2012, 134 (35), 14314-14317.354. Thenard, L., Observations sur l'influence de l'eau dans la formation des acides oxigends.Ann. Chim. Phys. 1818, 9, 314-317.355. Sies, H.; Berndt, C.; Jones, D. P., Oxidative stress. Annu. Rev. Biochem. 2017, 86, 715-748.356. Ainley, A. D.; Challenger, F., CCLXXX.-Studies of the boron-carbon linkage. part I. Theoxidation and nitration of phenylboric acid. J Chem. Soc. 1930, (0), 2171-2180.

417

357. Kuivila, H. G.; Armour, A. G., Electrophilic displacement reactions. IX. Effects ofsubstituents on rates of reactions between hydrogen peroxide and benzeneboronic acid. J Am.Chem. Soc. 1957, 79 (21), 5659-5662.358. Larkin, J. D.; Markham, G. D.; Milkevitch, M.; Brooks, B. R.; Bock, C. W., Computationalinvestigation of the oxidative deboronation of boroglycine, H2N-CH2-B(OH)2, using H20 andH202. J Phys. Chem. A 2009, 113 (41), 11028-11034.359. Zou, Y. Q.; Chen, J. R.; Liu, X. P.; Lu, L. Q.; Davis, R. L.; Jorgensen, K.; Xiao, W. J.,Highly efficient aerobic oxidative hydroxylation of arylboronic acids: photoredox catalysis usingvisible light. Angew. Chem. Int. Ed. 2012, 51 (3), 784-788.360. Sikora, A.; Zielonka, J.; Lopez, M.; Joseph, J.; Kalyanaraman, B., Direct oxidation ofboronates by peroxynitrite: mechanism and implications in fluorescence imaging of peroxynitrite.Free Radic. Biol. Med. 2009, 47 (10), 1401-1407.361. Buncel, E.; Urn, I.-H., The a-effect and its modulation by solvent. Tetrahedron 2004, 60,7801-7825.362. Yamamoto, Y.; Matsumura, T.; Takao, N.; Yamagishi, H.; Takahashi, M.; Iwatsuki, S.;Ishihara, K., Fast trigonal/tetragonal interconversion followed by slow chelate-ring closure in thecomplexation of boronic acids. Inorg. Chim. A cta 2005, 358 (12), 3355-3361.363. Matsumura, T.; Iwatsuki, S.; Ishihara, K., Direct kinetic measurements for the fastinterconversion process between trigonal boronic acid and tetragonal boronate ion at lowtemperatures. Inorg. Chem. Commun. 2005, 8 (8), 713-716.364. Ni, N.; Laughlin, S.; Wang, Y.; Feng, Y.; Zheng, Y.; Wang, B., Probing the general timescale question of boronic acid binding with sugars in aqueous solution at physiological pH. Bioorg.Med Chem. 2012, 20 (9), 2957-2961.365. Kuivila, H. G., Electrophilic displacement reactions. III. Kinetics of the reaction betweenhydrogen peroxide and benzeneboronic acid. J Am. Chem. Soc. 1954, 76 (3), 870-874.366. Toyao, T.; Ueno, N.; Miyahara, K.; Matsui, Y.; Kim, T.-H.; Horiuchi, Y.; Ikeda, H.;Matsuoka, M., Visible-light, photoredox catalyzed, oxidative hydroxylation of arylboronic acidsusing a metal-organic framework containing tetrakis(carboxyphenyl)porphyrin groups. Chem.Commun. 2015, 51 (89), 16103-16106.367. Cox, P. A.; Leach, A. G.; Campbell, A. D.; Lloyd-Jones, G. C., Protodeboronation ofheteroaromatic, vinyl, and cyclopropyl boronic acids: pH-rate profiles, autocatalysis, anddisproportionation. J Am. Chem. Soc. 2016, 138 (29), 9145-9157.368. Ellis, G. A.; Palte, M. J.; Raines, R. T., Boronate-mediated biologic delivery. J Am. Chem.Soc. 2012, 134 (8), 3631-3634.369. Winterbourn, C. C.; Metodiewa, D., Reactivity of biologically important thiol compoundswith superoxide and hydrogen peroxide. Free Radic. Biol. Med 1999, 27 (3-4), 322-328.370. Collaboration, H.-C.; Ray, M.; Logan, R.; Steme, J. A.; Hernandez-Diaz, S.; Robins, J. M.;Sabin, C.; Bansi, L.; van Sighem, A.; de Wolf, F.; Costagliola, D.; Lanoy, E.; Bucher, H. C.; vonWyl, V.; Esteve, A.; Casbona, J.; del Amo, J.; Moreno, S.; Justice, A.; Goulet, J.; Lodi, S.; Phillips,A.; Seng, R.; Meyer, L.; Perez-Hoyos, S.; Garcia de Olalla, P.; Hernan, M. A., The effect ofcombined antiretroviral therapy on the overall mortality of HIV-infected individuals. AIDS 2010,24 (1), 123-37.371. Chun, T. W.; Justement, J. S.; Murray, D.; Hallahan, C. W.; Maenza, J.; Collier, A. C.;Sheth, P. M.; Kaul, R.; Ostrowski, M.; Moir, S.; Kovacs, C.; Fauci, A. S., Rebound of plasma

418

viremia fnhllwing oesstio- n nf ntiretrrvniral ther Aspte prnFrunly low leve1 s f HIV

reservoir: implications for eradication. AIDS 2010, 24 (18), 2803-8.372. Hendrickson, C. J.; Pascoe, S. J. S.; Huber, A. N.; Moolla, A.; Maskew, M.; Long, L. C.;Fox, M. P., "My future is bright...I won't die with the cause of AIDS": ten-year patient ARToutcomes and experiences in South Africa. J Int. AIDS Soc. 2018, 21 (10), e25184.373. Siliciano, R. F.; Greene, W. C., HIV latency. Cold Spring Harb. Perspect. Med 2011, 1(1), a007096.374. Kim, Y.; Anderson, J. L.; Lewin, S. R., Getting the "kill" into "shock and kill": strategiesto eliminate latent HIV. Cell Host Microbe 2018, 23 (1), 14-26.375. Vocero-Akbani, A. M.; Heyden, N. V.; Lissy, N. A.; Ratner, L.; Dowdy, S. F., KillingHIV-infected cells by transduction with an HIV protease-activated caspase-3 protein. Nat. Med.1999, 5 (1), 29-33.376. Plainkum, P.; Fuchs, S. M.; Wiyakrutta, S.; Raines, R. T., Creation of a zymogen. Nat.Struct. Biol. 2003, 10 (2), 115-9.377. Johnson, R. J.; Lin, S. R.; Raines, R. T., A ribonuclease zymogen activated by the NS3protease of the hepatitis C virus. FEBS.J 2006, 273 (23), 5457-65.378. Turcotte, R. F.; Raines, R. T., Design and characterization of an HIV-specific ribonucleasezymogen. AIDS Res. Hum. Retroviruses 2008, 24 (11), 1357-63.379. Callis, M.; Serrano, S.; Benito, A.; Laurents, D. V.; Vilanova, M.; Bruix, M.; Ribo, M.,Towards tricking a pathogen's protease into fighting infection: the 3D structure of a stablecircularly permuted onconase variant cleavedby HIV-1 protease. PLoS ONE 2013, 8 (1), e54568.380. Kanno, A.; Yamanaka, Y.; Hirano, H.; Umezawa, Y.; Ozawa, T., Cyclic luciferase for real-time sensing of caspase-3 activities in living mammals. Angew. Chem. Int. Ed. 2007, 46(40), 7595-9.381. Butler, J. S.; Mitrea, D. M.; Mitrousis, G.; Cingolani, G.; Loh, S. N., Structural andthermodynamic analysis of a conformationally strained circular permutant of barase.Biochemistry 2009, 48 (15), 3497-507.382. Evans, T. C., Jr.; Martin, D.; Kolly, R.; Panne, D.; Sun, L.; Ghosh, I.; Chen, L.; Benner, J.;Liu, X. Q.; Xu, M. Q., Protein trans-splicing and cyclization by a naturally split intein from thednaE gene of Synechocystis species PCC6803. J Biol. Chem. 2000, 275 (13), 9091-4.383. Zettler, J.; Schutz, V.; Mootz, H. D., The naturally split Npu DnaE intein exhibits anextraordinarily high rate in the protein trans-splicing reaction. FEBS Lett. 2009, 583 (5), 909-14.384. Bal, H. P.; Batra, J. K., Human pancreatic ribonuclease--deletion of the carboxyl-terminalEDST extension enhances ribonuclease activity and thermostability. Eur. J. Biochem. 1997, 245(2), 465-9.385. Pous, J.; Mallorqui-Fernandez, G.; Peracaula, R.; Terzyan, S. S.; Futami, J.; Tada, H.;Yamada, H.; Seno, M.; de Llorens, R.; Gomis-Ruth, F. X.; Coll, M., Three-dimensional structureof human RNase 1 delta N7 at 1.9 A resolution. Acta Crystallogr. 2001, D57 (Pt 4), 498-505.386. Fisher, B. M.; Ha, J. H.; Raines, R. T., Coulombic forces in protein-RNA interactions:binding and cleavage by ribonuclease A and variants at Lys7, Arg 10, and Lys66. Biochemistry1998, 37(35), 12121-32.387. Fujii, T.; Ueno, H.; Hayashi, R., Significance of the four carboxyl terminal amino acidresidues of bovine pancreatic ribonuclease A for structural folding. J Biochem. 2002, 131 (2),193-200.

419

388. Leland, P. A.; Staniszewski, K. E.; Kim, B. M.; Raines, R. T., Endowing human pancreaticribonuclease with toxicity for cancer cells. J Biol. Chem. 2001, 276 (46), 43095-102.389. Shah, N. H.; Vila-Perello, M.; Muir, T. W., Kinetic control of one-pot trans-splicingreactions by using a wild-type and designed split intein. Angew. Chem. Int. Ed. 2011, 50 (29),6511-5.390. Kelemen, B. R.; Klink, T. A.; Behlke, M. A.; Eubanks, S. R.; Leland, P. A.; Raines, R. T.,Hypersensitive substrate for ribonucleases. Nucleic A cids Res. 1999, 27 (18), 3696-701.391. Eller, C. H.; Lomax, J. E.; Raines, R. T., Bovine brain ribonuclease is the functionalhomolog of human ribonuclease 1. J Biol.Chem. 2014, 289 (38), 25996-6006.392. O'Meara, M. J.; Leaver-Fay, A.; Tyka, M. D.; Stein, A.; Houlihan, K.; DiMaio, F.; Bradley,P.; Kortemme, T.; Baker, D.; Snoeyink, J.; Kuhlman, B., Combined covalent-electrostatic modelof hydrogen bonding improves structure prediction with Rosetta. J Chem. Theory Comput. 2015,11 (2), 609-22.393. Stein, A.; Kortemme, T., Improvements to robotics-inspired conformational sampling inrosetta. PLoS ONE 2013, 8 (5), e63090.394. Gront, D.; Kulp, D. W.; Vernon, R. M.; Strauss, C. E.; Baker, D., Generalized fragmentpicking in Rosetta: design, protocols and applications. PLoS ONE 2011, 6 (8), e23294.395. Wang, C.; Bradley, P.; Baker, D., Protein-protein docking with backbone flexibility.Journal of molecular biology 2007, 373 (2), 503-19.396. Dobson, C. M., Protein folding and misfolding. Science 2003, 426, 884-890.397. Chiti, F.; Dobson, C. M., Protein misfolding, functional amyloid, and human disease. Annu.Rev. Biochem. 2006, 75, 333-366.398. Knowles, T. P.; Vendruscolo, M.; Dobson, C. M., The amyloid state and its associationwith protein misfolding diseases. Nat. Rev. Mol. Cell Biol. 2014, 15 (6), 3 84-396.399. Richardson, S. J., Cell and molecular biology of transthyretin and thyroid hormones. Int.Rev. Cytol. 2007, 258, 137-193.400. Klabunde, T.; Petrassi, H. M.; Oza, V. B.; Raman, P.; Kelly, J. W.; Sacchettini, J. C.,Rational design of potent human transthyretin amyloid disease inhibitors. Nat. Struct. Biol. 2000,7, 312-321.401. Hamilton, J. A.; Benson, M. D., Transthyretin: a review from a structural perspective. Cell.Mol. Life Sci. 2001, 58, 1491-1521.402. Hund, E., Familial amyloidotic polyneuropathy: current and emerging treatment optionsfor transthyretin-mediated amyloidosis. The application of clinical genetics 2012, 5, 37-41.403. Yang, D. T.; Joshi, G.; Cho, P. Y.; Johnson, J. A.; Murphy, R. M., Transthyretin as both asensor and a scavenger of -amyloid oligomers. Biochemistry 2013, 52, 2849-2861.404. Li, X.; Zhang, X.; Ladiwala, A. R. A.; Du, D.; Yadav, J. K.; Tessier, P. M.; Wright, P. E.;Kelly, J. W.; Buxbaum, J. N., Mechanisms of transthyretin inhibition of P-amyloid aggregation invitro. J. Neurosci. 2013, 33, 19423-19433.405. Stein, T. D.; Anders, N. J.; DeCarli, C.; Chan, S. L.; Mattson, M. P.; Johnson, J. A.,Neutralization of transthyretin reverses the neuroprotective effects of secreted amyloid precursorprotein (APP) in APPSW mice resulting in tau phosphorylation and loss of hippocampal neurons:Support for the amyloid hypothesis. J Neurosci. 2004, 24, 7707-7717.406. Celej, M. S., Protein stability induced by ligand binding correlates with changes in proteinflexibility. Protein Sci. 2003, 12, 1496-1506.

420

407. Hingorani, K. S.; Metcalf, /T. C.; Dmig, n. T.; Garm-an, S. . Powers, E T.L. M., Ligand-promoted protein folding by biased kinetic partitioning. Nat. Chem. Biol. 2017, 13,369-371.408. Martinez Molina, D.; Jafari, R.; Ignatushchenko, M.; Seki, T.; Larsson, E. A.; Dan, C.;Sreekumar, L.; Cao, Y.; Nordlund, P., Monitoring drug target engagement in cells and tissuesusing the cellular thermal shift assay. Science 2013, 341 (6141), 84-7.409. Miller, D. W.; Dill, K. A., Ligand binding to proteins: the binding landscape model. ProteinSci. 1997, 6, 2166-2179.410. O'Sullivan, C.; Tompson, F. W., LX.-Invertase: a contribution to the history of anenzyme or unorganised ferment. J Chem. Soc. 1890, 57, 834-93 1.411. Obici, L.; Merlini, G., An overview of drugs currently under investigation for the treatmentof transthyretin-related hereditary amyloidosis. Expert Opin. Investig. Drugs 2014, 23, 1239-1251.412. Nencetti, S.; Orlandini, E., TTR fibril formation inhibition: is there a SAR? Curr. MedChem. 2012, 19, 2356-2379.413. Johnson, S. M.; Wiseman, R. L.; Sekijima, Y.; Green, N. S.; Adamski-Werner, S. L.; Kelly,J. W., Native state kinetic stabilization as a strategy to ameliorate protein misfolding diseases: afocus on the transthyretin amyloidoses. Acc. Chem. Res. 2005, 38 (12), 911-921.414. Adamski-Werner, S. L.; Palaninathan, S. K.; Sacchettini, J. C.; Kelly, J. W., Diflunisalanalogs stabilize the native state of transthyretin. potent inhibition of amyloidogenesis. J. Med.Chem. 2004, 47, 355-374.415. Castano, A.; Helmke, S.; Alvarez, J.; Delisle, S.; Maurer, M. S., Diflunisal for ATTRcardiac amyloidosis. Congest. Heart Fail. 2012, 18 (6), 315-319.416. Scott, L. J., Tafamidis: a review of its use in familial amyloid polyneuropathy. Drugs 2014,74, 1371-1378.417. Bulawa, C. E.; Connelly, S.; DeVit, M.; Wang, L.; Weigel, C.; Fleming, J. A.; Packman,J.; Powers, E. T.; Wiseman, R. L.; Foss, T. R.; Wilson, I. A.; Kelly, J. W.; Labaudiniere, R.,Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid cascade.Proceedings of the National Academy of Sciences of the United States ofAmerica 2012, 109 (24),9629-9634.418. Sekijima, Y., Recent progress in the understanding and treatment of transthyretinamyloidosis. Journal of clinical pharmacy and therapeutics 2014, 39 (3), 225-23 3.419. Johnson, D. S.; Weerapana, E.; Cravatt, B. F., Strategies for discovering and deriskingcovalent, irreversible enzyme inhibitors. Future Med Chem. 2010, 2 (6), 949-964.420. Baillie, T. A., Targeted covalent inhibitors for drug design. Angew. Chem. Int. Ed. 2016,55, 2-17.421. Miller, R. M.; Taunton, J., Targeting protein kinases with selective and semipromiscuouscovalent inhibitors. Methods Enzymol. 2014, 548, 93-116.422. Johnson, S. M.; Connelly, S.; Fearns, C.; Powers, E. T.; Kelly, J. W., The transthyretinamyloidoses: from delineating the molecular mechanism of aggregation linked to pathology to aregulatory-agency-approved drug. Journal of molecular biology 2012, 421 (2-3), 185-203.423. Choi, S.; Connelly, S.; Reixach, N.; Wilson, I. A.; Kelly, J. W., Chemoselective smallmolecules that covalently modify one lysine in a non-enzyme protein in plasma. Nat. Chem. Biol.2010, 6 (2), 133-139.

421

424. Choi, S.; Ton Ong, D. S.; Kelly, J. W., A stilbene that binds selectively to transthyretin incells and remains dark until it undergoes a chemoselective reaction to create a bright bluefluorescent conjugate. J Am. Chem. Soc. 2010, 132, 16043-16051.425. Suh, E. H.; Liu, Y.; Connelly, S.; Genereux, J. C.; Wilson, I. A.; Kelly, J. W., Stilbenevinyl sulfonamides as fluorogenic sensors of and traceless covalent kinetic stabilizers oftransthyretin that prevent amyloidogenesis. J Am. Chem. Soc. 2013, 135 (47), 17869-17880.426. Grimster, N. P.; Connelly, S.; Baranczak, A.; Dong, J.; Krasnova, L. B.; Sharpless, K. B.;Powers, E. T.; Wilson, I. A.; Kelly, J. W., Aromatic sulfonyl fluorides covalently kineticallystabilize transthyretin to prevent amyloidogenesis while affording a fluorescent conjugate. J Am.Chem. Soc. 2013, 135 (15), 5656-5668.427. Baranczak, A.; Liu, Y.; Connelly, S.; Han Du, W.-G.; Greiner, E. R.; Genereux, J. C.;Wiseman, R. L.; Eisele, Y. S.; Bradbury, N. C.; Dong, J.; Noodleman, L.; Sharpless, K. B.; Wilson,I. A.; Encalada, S. E.; Kelly, J. W., A fluorogenic aryl fluorosulfate for intraorganellar transthyretinimaging in living cells and in Caenorhabditis elegans. J Am. Chem. Soc. 2015, 137 (23), 7404-7414.428. Peters, J. A., Interactions between boric acid derivatives and saccharides in aqueous media:structures and stabilities of resulting esters. Coord. Chem. Rev. 2014, 268, 1-22.429. Adams, J., The development of proteasome inhibitiors as anticancer drugs. Cancer Cell2003, 5, 417-421.430. Antonov, V. K.; Ivanina, T. V.; Berezin, I. V.; Martinek, K., n-Alkylboronic acids asbifunctional reversible inhibitors of c-chymotrypsin. FEBS Lett. 1970, 7, 23-25.431. Kettner, C.; Shenvi, A., Inhibition of the serine proteases leukocyte elastase, pancreaticelastase, cathepsin G, and chymotrypsin by peptide boronic acids. J Biol. Chem. 1984, 259,15106-15114.432. Wang, J.; Wu, W.; Jiang, X., Nanoscaled boron-containing delivery systems andtherapeutic agents for cancer treatment. Nanomedicine 2015, 10 (7), 1149-1163.433. Ma, R.; Shi, L., Phenylboronic acid-based glucose-responsive polymeric nanoparticles:synthesis and applications in drug delivery. Polym. Chem. 2014, 5 (5), 1503.434. Andersen, K. A.; Smith, T. P.; Lomax, J. E.; Raines, R. T., Boronic acid for the tracelessdelivery of proteins into cells. ACS Chem. Biol. 2016, 11, 319-323.435. Wu, X.; Li, Z.; Chen, X. X.; Fossey, J. S.; James, T. D.; Jiang, Y. B., Selective sensing ofsaccharides using simple boronic acids and their aggregates. Chem. Soc. Rev. 2013, 42 (20), 8032-8048.436. Whyte, G. F.; Vilar, R.; Woscholski, R., Molecular recognition with boronic acids-applications in chemical biology. J Chem. Biol. 2013, 6 (4), 161-174.437. Cal, P. M. S. D.; Frade, R. F. M.; Cordeiro, C.; Gois, P. M. P., Reversible lysinemodification on proteins by using functionalized boronic acids. Chem.-Eur. J. 2015, 21, 8182-8187.438. Ak9ay, G.; Belmonte, M. A.; Aquila, B.; Chuaqui, C.; Hird, A. W.; Lamb, M. L.; Rawlins,P. B.; Su, N.; Tentarelli, S.; Grimster, N. P.; Su, Q., Inhibition of Ml-i through covalentmodification of a noncatalytic lysine side chain. Nat. Chem. Biol. 2016, 12, 931-936.439. Bull, S. D.; Davidson, M. G.; van den Elsen, J. M. H.; Fossey, J. S.; Jenkins, A. T. A.;Jiang, Y.-B.; Kubo, Y.; Marken, F.; Sakurai, K.; Zhao, J.; James, T. D., Exploiting the reversible

422

covalent bonding of boronic acids-recognition, sensing, and assembly. Ac. Chem. Res. 2012, 46

(2), 312-326.440. Services, U. S. D. o. H. a. H., Toxicology Profile for Boron. Agency for Toxic Substancesand Disease Registry: Atlanta, GA, 2010.441. Baures, P. W.; Peterson, S. A.; Kelly, J. W., Discovering transthyretin amyloid fibrilinhibitors by limited screening. Bioorg. Med Chem. 1998, 6, 1389-1401.442. Bourgault, S.; Choi, S.; Buxbaum, J. N.; Kelly, J. W.; Price, J. L.; Reixach, N., Mechanismsof transthyretin cardiomyocyte toxicity inhibition by resveratrol analogs. Biochemical andbiophysical research communications 2011, 410 (4), 707-713.443. Johnson, S. M.; Cohen, S. M.; Wilson, I. A.; Kelly, J. W., Toward optimization of thelinker substructure common to transthyretin amyloidogenesis inhibitors using biochemical andstructural studies. J Med. Chem. 2008, 51, 6348-6358.444. Cotrina, E. Y.; Pinto, M.; Bosch, L.; Vila, M.; Blasi, D.; Quintana, J.; Centeno, N. B.;Arsequell, G.; Planas, A.; Valencia, G., Modulation of the fibrillogenesis inhibition properties oftwo transthyretin ligands by halogenation. J Med Chem. 2013, 56 (22), 9110-9121.445. Palaninathan, S. K., Nearly 200 X-ray crystal structures of transthyretin-what do they tellus about this protein and the design of drugs for TTR amyloidoses? Curr. Med Chem. 2012, 19,2324-2342.446. Oza, V. B.; Smith, C.; Raman, P.; Koepf, E. K.; Lashuel, H. A.; Petrassi, H. M.; Chiang,K. P.; Powers, E. T.; Sacchettini, J. C.; Kelly, J. W., Synthesis, structure, and activity of diclofenacanalogues as transthyretin amyloid fibril formation inhibitors. J Med Chem. 2002, 45, 321-332.447. Palaninathan, S. K.; Mohamedmohaideen, N. N.; Orlandini, E.; Ortore, G.; Nencetti, S.;Lapucci, A.; Rossello, A.; Freundlich, J. S.; Sacchettini, J. C., Novel transthyretin amyloid fibrilformation inhibitors: synthesis, biological evaluation, and X-ray structural analysis. PLoS ONE2009, 4 (7), e6290.448. Neumann, P.; Cody, V.; Wojtczak, A., Ligand binding at the transthyretin dimer-dimerinterface: structure of the transthyretin-T4Ac complex at 2.2 A resolution. Acta Crystallogr. 2005,D61, 1313-1319.449. Muzio, T.; Cody, V.; Luft, J. R.; Pangborn, W.; Wojtczak, A., Complex of rat transthyretinwith tetraiodothyroacetic acid refined at 2.1 and 1.8 A resolution. Acta Biochim. Pol. 2001, 48 (4),877-884.450. Johnson, S. M.; Connelly, S.; Wilson, I. A.; Kelly, J. W., Biochemical and structuralevaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors.J Med Chem. 2008, 51, 260-270.451. Note l, 2017.452. Groll, M.; Berkers, C. R.; Ploegh, H. L.; Ovaa, H., Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome. Structure 2006,14 (3), 451-456.453. Bone, R.; Frank, D.; Kettner, C. A.; Agard, D. A., Structural analysis of specificity: a-lyticprotease complexes with analogues of reaction intermediates. Biochemistry 1989, 28 (19), 7600-7609.454. Kawasaki, Y.; Chufan, E. E.; Lafont, V.; Hidaka, K.; Kiso, Y.; Mario Amzel, L.; Freire,E., How much binding affinity can be gained by filling a cavity? Chemical biology & drug design2010, 75 (2), 143-151.

423

455. Eriksson, A. E.; Baase, W. A.; Zhang, X.-J.; Heinz, D. W.; Blaber, M.; Baldwin, E. P.;Matthews, B. W., Response of a protein structure to cavity-creating mutations and its relation tothe hydrophobic effect. Science 1992, 255, 178-183.456. Voth, A. R.; Khuu, P.; Oishi, K.; Ho, P. S., Halogen bonds as orthogonal molecularinteractions to hydrogen bonds. Nat. Chem. 2009, 1, 74-79.457. Riley, K. E.; Merz, K. M., Jr., Insights into the strength and origin of halogen bonding: thehalobenzene-formaldehyde dimer. J Phys. Chem. A 2007, 111 (9), 1688-1694.458. Peters, J. A., Interactions between boric acid derivatives and saccharides in aqueous media:Structures and stabilities of resulting esters. Coord. Chem. Rev. 2015, 268, 1-22.459. Alhamadsheh, M. M.; Connelly, S.; Cho, A.; Reixach, N.; Powers, E. T.; Pan, D. W.;Wilson, I. A.; Kelly, J. W.; Graef, I. A., Potent kinetic stabilizers that prevent transthyretin-mediated cardiomyocyte proteotoxicity. Sci. Transl. Med 2011, 3, 97ra8 1.460. Zhu, C.; Wang, R.; Falck, J. R., Mild and rapid hydroxylation of aryl/heteroaryl boronicacids and boronate esters with N-oxides. Org. Lett. 2012, 14, 3494-3497.461. McCutchen, S. L.; Colon, W.; Kelly, J. W., Transthyretin mutation Leu-55-Prosignificantly alters tetramer stability and increases amyloidogenicity. Biochemistry 1993, 32 (45),12119-12127.462. Grimm, F. A.; Lehmler, H. J.; He, X.; Robertson, L. W.; Duffel, M. W., Sulfatedmetabolites of polychlorinated biphenyls are high-affinity ligands for the thyroid hormonetransport protein transthyretin. Environmental health perspectives 2013, 121 (6), 65 7-662.463. Giraldo, J.; Vivas, N. M.; Vila, E.; Badia, A., Assessing the (a)symmetry of concentration-effect curves: empirical versus mechanistic models. Pharmacology & therapeutics 2002, 95 (1),21-45.464. Miroy, G. J.; Lai, Z.; Lashuel, H. A.; Peterson, S. A.; Strang, C.; Kelly, J. W., Inhibitingtransthyretin amyloid fibril formation via protein stabilization. Proceedings of the NationalAcademy of Sciences of the United States ofAmerica 1996, 93 (26), 15051-15056.465. Otwinowski, Z.; Minor, W., Processing of X-ray diffraction data collected in oscillationmode. Methods Enzymol. 1997, 276, 307-326.

424