Synthesis and Photophysical Studies of Nitrogen Heterocycles Containing Benzothiazines, Benzothiazoles, Indoles and Quinolines

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Ealin N. Patel University of Maine, [email protected]

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Recommended Citation Patel, Ealin N., "Synthesis and Photophysical Studies of Nitrogen Heterocycles Containing Benzothiazines, Benzothiazoles, Indoles and Quinolines" (2021). Electronic Theses and Dissertations. 3385. https://digitalcommons.library.umaine.edu/etd/3385

This Open-Access Thesis is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of DigitalCommons@UMaine. For more information, please contact [email protected]. SYNTHESIS AND PHOTOPHYSICAL STUDIES OF NITROGEN HETEROCYCLES CONTAINING BENZOTHIAZINES, BENZOTHIAZOLES, INDOLES AND QUINOLINES

Ealin Patel

B.A. University at Buffalo, 2013

B.S. University at Buffalo, 2013

A DISSERTATION

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Doctor of Philosophy

(in Chemistry)

The Graduate School

The University of Maine

May 2021

Advisory Committee:

Matthew Brichacek, Assistant Professor of Chemistry, Advisor

Alice E Bruce, Professor of Chemistry

Mitchell R. Bruce, Professor of Chemistry

William M Gramlich, Associate Professor of Chemistry

Michael Kienzler, Assistant Professor of Chemistry

SYNTHESIS AND PHOTOPHYSICAL STUDIES OF NITROGEN HETEROCYCLES CONTAINING BENZOTHIAZINE, BENZOTHIAZOLE, INDOLE AND QUINOLINE

By Ealin Patel

Dissertation Advisor: Dr. Matthew Brichacek

An Abstract of the Dissertation Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy (in Chemistry) May 2021 Nitrogen heterocycles such as quinolines, benzothiazines, benzothiazoles, and indoles are widely found in nature and these compounds have significance in organic and medicinal chemistry, biochemistry as well as materials science. Monomeric units of eumelanin (brominated 5,6- dimethoxyindolecarboxylate derivatives) were synthesized and regioselectively functionalized to form three monobrominated, three dibrominated, and a tribrominated variant using thermally induced nitrene insertions. After deprotection, three monobrominated 5,6-dihydroxyindole carboxylate (DHICA) derivatives were synthesized, and their UV-Vis absorbance was compared to DHICA. Bominated DHICA derivatives showed similar λmax as DHICA (~330 nm) but did not show aerial oxidation as evident by absence of chromophore absorbance at 550 nm. Monomeric synthetic analogues of pheomelanin subunit benzothiazole (six variants) and benzothiazine (four variants) were synthesized with CO2Et and CH2CN sidechain via Sandmeyer reaction, and Cu (II)- catalyzed thiolation-Ullmann coupling, respectively. This monomeric pheomelanoid synthesis was the first successful unified strategy to access this class of molecules. Utilizing these melanin building blocks, synthesis of mixed melanoids was done using Miyaura borylation and Suzuki- coupling. Preliminary photophysical studies of synthesized mixed melanoids showed red shifted UV absorbance (from ~280 nm to 360 nm) due to extended conjugation and these compounds exhibited fluorescence properties as organic chromophores. With a goal of developing material for optical memory, gold-heli viologen was synthesized and characterized successfully via Pd (0)- catalyzed Stille coupling route, or Hiyama-Heck coupling from 3-bromoquionoline as the key step. Temperature variable photoluminescence and quenching experiments were performed to study the photophysical behavior which showed quenching of gold luminescence by heli-viologen involving an electron transfer.

iii

DEDICATION

Dedicated to my parents, Rupal and Nishid Patel, and

my wife Dr. Sutanuka Bhattacharjya

for their love, support, and encouragement

ACKNOWLEDGEMENTS

I would like to express my gratitude to my advisor and mentor Dr. Matthew Brichacek for his unwavering support, guidance, patience, and encouragement through my PhD program.

Special thanks to our collaborators of the Viologen project, Dr. Howard Patterson, Dr. Aaron

Nicholas, and Dr. Robert Arthur for their contributions. I would like to extend my gratitutde to

Dr. Mohammad Omary and Dr. Eric Reinheimer for their contributions to this project and the publication.

I would like to thank my advisory committee Dr. William Gramlich, Dr. Michael Kienzler, Dr.

Mitchell Bruce, and Dr. Alice Bruce for their guidance, advice, and suggestions throughout my

PhD work. I would like to thank Dr. Barbara Cole for allowing me to use the photoreactor and

LC-MS. This work could not have been completed without the help of David LaBrecque with the instruments. I am grateful to all the current and previous members of Brichacek group specially

Billy, Sudeera, Ahmed and Robin.

Thank you to all my friends at UMaine, especially Anwesha and Naveen for all the memories and constant encouragement; Nayeem, Grape, Sadie, Suyog, Melissa, and Kyle for making the grad school experience great. Thank you to all my friends from UB-Aishwarya, Yash, and

Omkar.

I would like to thank my family. My parents, Rupal and Nishid Patel, my sister Aneri, and extended family Neha, Kirit and Vishakha Patel for their support throughout my education and for believing in my success. Finally, thank you to my greatest supporter -my wife Sutanuka who encouraged me to pursue Ph.D.

TABLE OF CONTENTS

ABSTRACT ...... iii

DEDICATION ...... iv

ACKNOWLEDGEMENTS ...... v

LIST OF TABLES ...... xii

LIST OF SCHEMES...... xiii

LIST OF FIGURES ...... xiv

1 INTRODUCTION ...... 1

1.1 Nitrogen heterocycles ...... 1

1.2 Melanin ...... 2

1.2.1 Melanogenesis...... 4

1.2.1.1 Rapor-Mason-Prota pathway of Melanogenesis: ...... 4

1.2.1.2 Kinetics of melanogenesis ...... 6

1.2.1.3 Eumelanogenesis ...... 8

1.2.1.3.1 Eumelanin structure elucidation: ...... 9

1.2.1.4 Pheomelanogenesis ...... 11

1.2.1.4.1 Pheomelanin structre elucidation: ...... 14

1.2.1.5 Mixed melanogenesis ...... 15

1.2.1.5.1 Neuromelanin structure elucidation: ...... 16

1.2.2 Sources and synthesis of Melanins: ...... 18

1.2.2.1 Extraction: ...... 18

1.2.2.2 Chemoenzymatic synthesis: ...... 20

1.2.2.3 Oxidative polymerization: ...... 21

1.2.2.4 Chemical synthesis of melanin subunits: ...... 22

1.2.2.4.1 Synthesis of 5,6-dihydroxyindole carboxylate compounds: ...... 22

1.2.2.4.2 Synthesis of Eumelanoid oligomers ...... 23

1.2.2.4.3 Synthesis of aminobenzothiazole and 1,4-benzothiazine:...... 25

1.2.2.4.4 Synthesis of pheomelanoid oligomers: ...... 26

1.2.3 Properties of Melanins: ...... 27

1.2.3.1 Physiochemical properties ...... 27

1.2.3.2 Spectral and Photophysical properties ...... 28

1.2.4 Significance and applications: ...... 31

1.2.4.1 Biological significance: ...... 31

1.2.4.2 Materials applications: ...... 32

1.3 Viologens ...... 34

1.3.1 Viologen Applications: ...... 34

1.3.2 Ion-Pair Charge Transfer (IPCT) complex: ...... 36

1.3.2.1 Au(CN)2-DNP viologen complex:...... 36

1.3.2.2 Ag(CN)2-DNP viologen complex:...... 38

1.3.3 Helicene viologen ...... 40

vii

1.4 Thesis overview: ...... 41

2 SYNTHESIS, AND PHOTOPHYSICAL STUDIES OF REGIOSELECTIVELY

FUNCTIONALIZED EUMELANOIDS ...... 43

2.1 Introduction ...... 43

2.2 Results and discussion ...... 45

2.3 Conclusions ...... 57

2.4 Synthesis ...... 57

2.4.1 General information ...... 57

2.4.2 Synthesis of DMIC(2-3) and 3’-bromo DMIC (2-4) ...... 58

2.4.3 Synthesis of 4’-bromo DMIC (2-8) ...... 61

2-bromo-3-hydroxy-4-methoxybenzaldehyde (2-5) ...... 61

2.4.4 Synthesis of 7’-bromo DMIC (2-12) ...... 63

2.4.5 Synthesis of di and tri-brominated DMIC (2-14 to 2-17) ...... 66

Synthesis of 3’4’-dibromo DMICA (2-14) ...... 66

Synthesis of 3’-4’-7’-tribromo DMICA (2-16) ...... 67

Synthesis of 4’-7’-dibromo dimethoxyindole carboxylate (2-17) ...... 67

2.4.6 Synthesis of DHICA (2-19) and its mono-brominated derivatives ...... 68

3 SYNTHESIS OF PHEOMELANOID MONOMERS- BENZOTHIAZOLES AND

BENZOTHIAZINES ...... 73

3.1 Introduction ...... 73

viii

3.2 Results and Discussion: ...... 74

3.2.1 ortho-methoxyaniline synthesis ...... 75

3.2.2 Benzothiazole(BTZL) synthesis ...... 78

3.2.3 Synthesis of key intermediate: ortho-aminothiophenol ...... 80

3.2.4 Alkylate condensation:...... 85

3.3 Conclusion and future direction ...... 87

3.4 Synthesis ...... 88

3.4.1 General synthesis procedure ...... 88

3.4.2 Synthesis of ortho-methoxy aniline compound ...... 88

3.4.3 Benzothiazole synthesis ...... 91

3.4.4 Synthesis of ortho-aminothiophenol intermediate ...... 97

4 SYNTHESIS OF REGIOSELECTIVELY FUNCTIONALIZED

EUMELANOIDS MIXED MELANOIDS AND THEIR PHOTOPHYSICAL

STUDIES ...... 111

4.1 Introduction ...... 111

4.2 Results and Discussion: ...... 112

4.2.1 Eumelanoid functionalization ...... 113

4.2.2 Mixed melanoid synthesis via Suzuki Coupling:...... 121

4.2.3 UV-Vis and Fluroscence properties of mixed melanoids ...... 124

4.3 Conclusion and future direction: ...... 126

4.4 Synthesis ...... 127

4.4.1 General Synthetic procedure: ...... 127

4.4.2 Conjugation via oxidation: ...... 128

5 SYNTHESIS, STRUCTURE AND PHOTOPHYSICAL PROPERTIES OF

METAL DICYANIDE DONORS COORDINATED TO AZA[5]HELIZENE

VIOLOGEN ACCEPTORS ...... 136

5.1 Introduction ...... 136

5.2 Results and Discussion ...... 138

5.2.1 Synthesis ...... 138

5.2.2 Single crystal X-ray diffraction ...... 144

5.2.3 Infrared spectroscopy ...... 147

5.2.4 Photophysical studies ...... 148

5.2.5 Electron transfer analysis ...... 151

5.2.6 Ag-Heli Viologen (5-14)...... 153

5.3 Conclusion and Future direction ...... 154

5.4 Experimental ...... 155

5.4.1 General Information ...... 155

5.4.2 Synthesis of (E)-1,2-di(quinoline-3-yl)ethene (5-5)...... 156

5.4.2.1 Step-wise Wittig route ...... 156

5.4.2.2 Stille coupling to synthesize 5-5 ...... 158

5.4.2.3 Hiyama-Heck Coupling ...... 159

5.4.3 Photocyclization to synthesize 5,10-diaza[5]helicene (5-9) ...... 160

5.4.4 Synthesis of dimethylaza[5]helicene ...... 161

5.4.5 Synthesis of Au heli-viologen(5-13) ...... 163

5.4.6 Crystallography ...... 163

5.4.7 Spectroscopy studies ...... 164

5.4.8 Stern–Volmer quenching experiments ...... 165

REFERENCES…………………………………………………………………………………166

APPENDIX A. NMR data for Chapter-2………………………………………………………186

APPENDIX B. NMR data for Chapter-3………………………………………………………210

APPENDIX C. NMR data for Chapter-4………………………………………………………235

APPENDIX D. HPLC chromatograms for Chapter-4………………………………………….246

APPENDIX E. Photophysical data for Chapter-4……………………………………………...248

APPENDIX F. NMR data for Chapter-5……………………………………………………….250

APPENDIX G. IR spectra for Chapter-5……………………………………………………….259

APPENDIX H. Bond lengths and Bond angles for Au-heli viologen………………………….260

APPENDIX I. Computational modeling………………………………………………………..262

APPENDIX J. UV-Vis, DFT and MO for Au-heli viologen…………………………………...266

APPENDIX K. Copyrights and Permissions from Authors and Publishers……………………270

BIOGRAPHY OF THE AUTHOR……………………………………………………………..280

LIST OF TABLES

Table 3-1 Smiles rearrangement-reaction conditions and results ...... 77

Table 3-2 Thiocyanation of protected ortho-methoxyaniline...... 82

Table 4-1 3’-borylation reaction optimization and relevant mole fractions

(from HPLC data)...... 119

Table 5-1 Catalyst optimization for Stille coupling...... 140

Table 5-2 Light source optimization for photocyclization of 5-5 ...... 142

Table H 1 Select bond lengths of Au heli-viologen ...... 260

Table H 2 Select bond angles of Au heli-viologen ...... 261

Table J 1 TD-DFT calculated excited states of of Au heli-viologen with corresponding energy and f-oscillation ...... 267

Table J 2 Calculated MO transitions of Au heli-viologen for excited state at 399 nm with percent contribution ...... 268

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LIST OF FIGURES

Figure 1-1- Pharmaceuticals containing nitrogen Heterocycles4

(Adapted with permission from Njardson et al.)...... 1

Figure 1-2 N-heterocycles of interest...... 2

Figure 1-3 Melanin pigments responsible for hair color...... 3

Figure 1-4 Rapor-Mason-Prota pathway of Mixed melanogenesis.17...... 5

Figure 1-5 Kinetics of melanogenesis.22,29 ...... 7

Figure 1-6 Kinetics of Eumelanogenesis: formation of DHI and DHICA quinones...... 8

Figure 1-7 Oxidative degradation of eumelanin by Alkaline H2O2. (Arrows indicate conjugation/chain proliferation sites of the polymer) ...... 10

Figure 1-8 Nucleophilic addition of cysteine...... 11

Figure 1-9 Pheomelanogenesis: formation of 1,4-benzothiazines.38 ...... 12

Figure 1-10 Oxidation of benzothiazine to benzothiazole.38 ...... 13

Figure 1-11 Pheomelanin degradation by oxidation and reductive hydrolysis...... 14

Figure 1-12 Cysteine dependent mixed melanogenesis.

(Adapted with permission from Ito and Wakamatsu, 2008)20 ...... 15

Figure 1-13 Degradation products of dopamine and cys-dopamine derived melanin...... 17

Figure 1-14 (A-D)SEM images (scale bar-200 nm) of neuromelanin

E) AFM of neuromelanin pigment from premotor cortex F) Wavelength dependent photoelectron emission microscopy data (Adapted from Zecca et al., 2008)53 ...... 19

Figure 1-15 Chemoenzymatic synthesis of melanins...... 20

Figure 1-16 Melanin (polydopamine) synthesis via oxidative polymerization

(Adapted with permission from Ju et al., 2011)88 ...... 21

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Figure 1-17 Methods to synthesize 5,6-dimethoxyindole carboxylate:

(a) Hemetsberger indole synthesis67 (b) Copper catalyzed domino coupling-cyclization69 (c) Horner-Wadsworth-Emmons synthesis-

Cadogan type cyclization.70 ...... 22

Figure 1-18 Strategy to synthesize biindolyls.71,72 ...... 24

Figure 1-19 Suzuki coupling to conjugate indole to a 5,6-Dimethoxyindole carboxylate (DMICA) compound73 ...... 24

Figure 1-20 Methods for aminobenzothiazole and 1,4-benzothiazine synthesis...... 25

Figure 1-21 Synthesis of ∆2,2’-bibenzothiazine via oxidative coupling of 2-phenyl-1,4-benzothiazine. 87 ...... 26

Figure 1-22 DTG and TGA of sepia ink melanin. (Adapted from Praleaet al., 2019)7 ...... 27

Figure 1-23 A) UV-vis absorption spectra of sepia ink melanin and synthetic melanin, B) Linear plot of logarithmic absorbance vs wavelength.

(Adapted from Pralea et al., 2019)7 ...... 29

Figure 1-24 IR absorption spectra of synthetic melanin and sepia ink melanin.

(Adapted from Pralea et al., 2019)7 ...... 30

Figure 1-25 MRI images of substantia nigra and locus coeruleus of healthy individual. (NM present-white) and patient with PD (depleted NM).

(Adapted from Isaias et al., 2016)123 ...... 32

Figure 1-26 Applications of DOPA and dopamine derived melanins.

(Adapted with permission from d’Ischia et al., 2014)62...... 33

xiv

Figure 1-27 Viologen as (a) electron acceptor (b) electron donor

Figure 1-28 Electochromic switching in tri-cat viologen system.

(Adapted with permission from Sun et al., 2015)140 ...... 35

2+ 2- Figure 1-29 Temperature dependent luminescence of DNP -[Au(CN)2]2 .

(Adapted with permission from Abouelwafa et al., 2012)150 ...... 37

Figure 1-30 Energy diagram of the {DNP-[Au(CN)2]2} complex.

(Adapted with permission from Abouelwafa et al., 2012)150 ...... 38

2+ 2- Figure 1-31 Luminescence of DNP -[Ag(CN)2]2 .

(Adapted with permission from Ahern et al., 2014)146 ...... 39

Figure 1-32 Electrostatic potential energy surface and crystal motif of

5,10-diaza[5]helicene. (Adapted with permission from Zhang et al., 2016)141 ...... 40

Figure 1-33 Overview of the thesis...... 42

Figure 2-1 Targeted 5,6-dimethoxyindole carboxylate (DMIC) derivatives...... 44

Figure 2-2 Mechanism of methyl azidoacetate condensation over sidechain aldehyde...... 46

Figure 2-3 Infrared spectrum of azidoacetate condensation product...... 47

Figure 2-4 Mechanism of cyclization via nitrene insertion...... 48

Figure 2-5 7’-bromo DMIC (2-12) synthesis via Hemetsberger-Knittel indole cyclization...... 49

Figure 2-6 Synthesis of 3,4’-dibromo DMIC (2-14) and 3,7’-dibromo DMIC (2-15)...... 50

Figure 2-7 Synthesis of 4,7’-dibromo DMIC (2-17) and 3,4,7’-tribromo DMIC (2-16)...... 50

Figure 2-8 Analytical HPLC chromatogram of regioselective debromination reaction...... 51

Figure 2-9 Mechanism of regioselective debromination of tribromo eumelanoid...... 52

Figure 2-10 Synthesis of mono-brominated 5,6-dihydroxyindole carboxylic acid

(DHICA)- A) NaOH (5 eq.), ethanol:water(1:1), RT, B) BBr3, DCM, -78 °C-RT

C) LiOH, ethanol:water (1:1), RT ...... 53

Figure 2-11 UV-Vis spectra of 4’-bromo DMIC (2-8) and 4’-bromo DHICA (2-21)...... 54

Figure 2-12 UV-Vis absorption spectra of 5,6-dihydroxyindole (DHICA) and its bromo derivatives...... 55

Figure 2-13 UV-Vis spectra of DHICA (2-19) and 4’-bromo DHICA (2-21) at t=5 mins, 1 hr, and 24 hrs...... 56

Figure 3-1 Pheomelanin monomeric units...... 73

Figure 3-2 Unified strategy to access monomeric pheomelanin subunits...... 74

Figure 3-3 Sandmeyer reaction mechanism ...... 79

Figure 3-4 Mechanism of thiolation via Ullman type C-S coupling...... 84

Figure 4-1 Target synthetic mixed melanoid synthesized from synthetic monomeric subunits of pheomelanin and eumelanin...... 112

Figure 4-2 Mechanism of KI catalysed coupling of benzothiazole with aromatic aldehyde.11 ...... 114

Figure 4-3 Mole fractions of 4’-bromo DMIC (2-8) borylation reaction.

(Calculated from HPLC data) ...... 116

Figure 4-4 Borylation and hydrolysis of pinacol ester monitored by HPLC.

(C-18 stationary phase, Mobile phase gradient 50%:50% water:AcN to 100% AcN in 10 minutes) ...... 117

Figure 4-5 Borylation of 7’-bromo DMIC (2-11)...... 118

Figure 4-6 3’-borylation reaction monitored by analytical HPLC...... 119

xvi

Figure 4-7 Borylation of bromobenzothiazole (3-14)...... 120

Figure 4-8 Mechanism of Suzuki coupling...... 121

Figure 4-9 Suzuki coupling to synthesize mixed melanoids...... 122

Figure 4-10 2D HMBC (Indole proton-focused) of mixed melanoid

[ethyl 2-(5,6-dimethoxy-2-(methoxycarbonyl)-1H-indol-4-yl)-4-methoxybenzo

[d]thiazole-6-carboxylate] (4-4)...... 123

Figure 4-11 UV-Vis absorbance of 5,6-DMIC (2-3), BTZL (3-13), and their mixed melanoid (4-10)- 50 µM in ethanol...... 124

Figure 4-12 Fluorescence spectra of 4-10...... 125

Figure 5-1 Previously studied DNP viologen and target Gold-heli viologen (5-13) ...... 137

Figure 5-2 Photocyclization mechanism of 5-5...... 141

Figure 5-3 UV-Vis absorption spectrum of 5-5 ...... 142

Figure 5-4 Single crystal synthesis via vapor-diffusion method...... 144

Figure 5-5 View along the a-axis of Au heli-viologen crystal. The dotted lines

- show dimer interactions among adjacent [Au(CN)2] anions...... 145

Figure 5-6 Infrared spectrum of Au heli viologen (5-13)...... 147

Figure 5-7 Solid state DRS absorption spectrum of solid samples of

Au heli-viologen and MV2+ at 298 K converted via Kubelka-Munk...... 148

Figure 5-8 Luminescence spectra of Au heli-viologen between 10 K and 298 K.

Excitation (dashed) and emission (solid)...... 149

Figure 5-9 Energy diagram of emission pathways for Au heli-viologen...... 150

Figure 5-10 Absorption spectra of potassium dicyanoaurate, heli[5]viologen, and Au heli-viologen in methanol (5 x 10-4 M)...... 151

xvii

Figure 5-11 Stern-Volmer plot for aqueous Au heli-viologen...... 152

Figure 5-12 Infrared spectra of KAg(CN)2 (upper) and potential

Ag-Heli viologen (lower) ...... 154

Figure A 1 1H NMR spectrum of 2-1 ...... 186

Figure A 2 13C NMR spectrum of 2-1...... 186

Figure A 3 1H NMR of 2-2 ...... 187

Figure A 4 13C NMR of 2-2 ...... 187

Figure A 5 1H NMR of 2-3 ...... 188

Figure A 6 13C NMR of 2-3 ...... 188

Figure A 7 1H NMR of 2-18 ...... 189

Figure A 8 13C NMR of 2-18 ...... 189

Figure A 9 1H NMR of 2-19 ...... 190

Figure A 10 13C NMR of 2-19 ...... 190

Figure A 11 1H NMR of 2-4 ...... 191

Figure A 12 13C NMR of 2-4 ...... 191

Figure A 13 1H NMR of 2-22 ...... 192

Figure A 14 13C NMR of 2-22 ...... 192

Figure A 15 1H NMR of 2-23 ...... 193

Figure A 16 13C NMR of 2-23 ...... 193

Figure A 17 1H NMR of 2-5 ...... 194

Figure A 18 13C NMR of 2-5 ...... 194

Figure A 19 1H NMR of 2-6 ...... 195

Figure A 20 13C NMR of 2-6 ...... 195

xviii

Figure A 21 1H NMR of 2-7 ...... 196

Figure A 22 13C NMR of 2-7 ...... 196

Figure A 23 1H NMR of 2-8 ...... 197

Figure A 24 13C NMR of 2-8 ...... 197

Figure A 25 1H NMR of 2-20 ...... 198

Figure A 26 13C NMR of 2-20 ...... 198

Figure A 27 1H NMR of 2-21 ...... 199

Figure A 28 13C NMR of 2-21 ...... 199

Figure A 29 1H NMR of 2-9 ...... 200

Figure A 30 13C NMR of 2-9 ...... 200

Figure A 31 1H NMR of 2-10 ...... 201

Figure A 32 13C NMR of 2-10 ...... 201

Figure A 33 1H NMR of 2-11 ...... 202

Figure A 34 13C NMR of 2-11 ...... 202

Figure A 35 1H NMR of 2-12 ...... 203

Figure A 36 13C NMR of 2-12 ...... 203

Figure A 37 1H NMR of 2-24 ...... 204

Figure A 38 13C NMR of 2-24 ...... 204

Figure A 39 1H NMR of 2-25 ...... 205

Figure A 40 13C NMR of 2-25 ...... 205

Figure A 41 1H NMR of 2-14 ...... 206

Figure A 42 13C NMR of 2-14 ...... 206

Figure A 43 1H NMR of 2-15 ...... 207

xix

Figure A 44 13C NMR of 2-15 ...... 207

Figure A 45 1H NMR of 2-17 ...... 208

Figure A 46 13C NMR of 2-17 ...... 208

Figure A 47 1H NMR of 2-16 ...... 209

Figure A 48 13C NMR of 2-16 ...... 209

Figure B 1 1H NMR of 3-5...... 210

Figure B 2 13C NMR of 3-5 ...... 210

Figure B 3 1H NMR of 3-6...... 211

Figure B 4 13C NMR of 3-6 ...... 211

Figure B 5 1H NMR of 3-7...... 212

Figure B 6 13C NMR of 3-7 ...... 212

Figure B 7 1H NMR of 3-8...... 213

Figure B 8 13C NMR of 3-8 ...... 213

Figure B 9 1H NMR of 3-9...... 214

Figure B 10 13C NMR of 3-9 ...... 214

Figure B 11 1H NMR of 3-10...... 215

Figure B 12 13C NMR of 3-10 ...... 215

Figure B 13 1H NMR of 3-11...... 216

Figure B 14 13C NMR of 3-11 ...... 216

Figure B 15 1H NMR of 3-12...... 217

Figure B 16 13C NMR of 3-12 ...... 217

Figure B 17 1H NMR of 3-13...... 218

Figure B 18 13C NMR of 3-13 ...... 218

Figure B 19 1H NMR of 3-14...... 219

Figure B 20 13C NMR of 3-14 ...... 219

Figure B 21 1H NMR of 3-17...... 220

Figure B 22 13C NMR of 3-17 ...... 220

Figure B 23 1H NMR of 3-18...... 221

Figure B 24 13C NMR of 3-18 ...... 221

Figure B 25 1H NMR of 3-19...... 222

Figure B 26 13C NMR of 3-19 ...... 222

Figure B 27 1H NMR of 3-20...... 223

Figure B 28 13C NMR of 3-20 ...... 223

Figure B 29 1H NMR of 3-21...... 224

Figure B 30 13C NMR of 3-21 ...... 224

Figure B 31 1H NMR of 3-22...... 225

Figure B 32 13C NMR of 3-22 ...... 225

Figure B 33 1H NMR of 3-23...... 226

Figure B 34 13C NMR of 3-23 ...... 226

Figure B 35 1H NMR of 3-24...... 227

Figure B 36 13C NMR of 3-24 ...... 227

Figure B 37 1H NMR of 3-26...... 228

Figure B 38 13C NMR of 3-26 ...... 228

Figure B 39 1H NMR of 3-27...... 229

Figure B 40 13C NMR of 3-27 ...... 229

Figure B 41 1H NMR of 3-29...... 230

xxi

Figure B 42 13C NMR of 3-29 ...... 230

Figure B 43 1H NMR of 3-30...... 231

Figure B 44 13C NMR of 3-30 ...... 231

Figure B 45 1H NMR of 3-31...... 232

Figure B 46 13C NMR of 3-31 ...... 232

Figure B 47 1H NMR of 3-33...... 233

Figure B 48 13C NMR of 3-33 ...... 233

Figure B 49 1H NMR of 3-32...... 234

Figure B 50 13C NMR of 3-32 ...... 234

Figure C 1 1H NMR of 4-1 ...... 235

Figure C 2 1H NMR of 4-3 ...... 235

Figure C 3 1H NMR of 4-6 ...... 236

Figure C 4 13C NMR of 4-6 ...... 236

Figure C 5 1H NMR of 4-9 ...... 237

Figure C 6 13C NMR of 4-9 ...... 237

Figure C 7 1H NMR of 4-4 ...... 238

Figure C 8 13C NMR of 4-4 ...... 238

Figure C 9 2D (1H-1H) COSY of 4-4 ...... 239

Figure C 10 2D (1H-13C) HSQC of 4-4...... 240

Figure C 11 2D (1H-13C) HMBC of 4-4 ...... 240

Figure C 12 1H NMR of 4-10 ...... 241

Figure C 13 13C NMR of 4-10 ...... 241

Figure C 14 2D (1H-1H) COSY of 4-10 ...... 242

xxii

Figure C 15 2D (1H-13C) HSQC of 4-10...... 243

Figure C 16 2D (1H-13C) HMBC of 4-10 ...... 244

Figure C 17 1H NMR of 4-11 ...... 245

Figure C 18 13C NMR of 4-11 ...... 245

Figure D 1 HPLC chromatogram of 7’-bromo DMIC borylation ...... 246

Figure D 2 HPLC chromatogram of 3’-bromo DMIC borylation optimization ...... 247

Figure E 1 UV/Vis spectra of 5,6-DMIC, Benzothiazole, and mixed melanoid ...... 248

Figure E 2 Luminescence spectra of mixed melanoid (4-4) ...... 248

Figure E 3 UV/Vis spectrum of mixed melanoid (4-11) ...... 249

Figure E 4 Luminescence spectra mixed melanoid (4-11) ...... 249

Figure F 1 1H NMR of 5-2 ...... 250

Figure F 2 1H NMR of 5-3 ...... 251

Figure F 3 1H NMR of 5-4 ...... 252

Figure F 4 1H NMR of 5-9 ...... 253

Figure F 5 1H NMR of 5-5 ...... 254

Figure F 6 13C NMR of 5-5 ...... 254

Figure F 7 1H NMR of 5-9 ...... 255

Figure F 8 13C NMR of 5-9 ...... 255

Figure F 9 1H NMR of 5-11 ...... 256

Figure F 10 13C NMR of 5-11 ...... 256

Figure F 11 1H NMR of 5-12 ...... 257

Figure F 12 13C NMR of 5-12 ...... 257

Figure F 13 1H NMR of 5-13 ...... 258

xxiii

Figure F 14 13C NMR of 5-13 ...... 258

Figure G 1 Full IR spectrum of microcrystalline Au heli-viologen at 298 K ...... 259

Figure I 1 DFT M06/CEP-31G(d) calculated ground state of

- -2 2+ [Au(CN)2] /[Au(CN)2]2 coordinated heli-viologen ...... 262

Figure I 2 TD-DFT M06/CEP-31G(d) energy diagram calculated for

Au heli-viologen at 399 nm excitation for top 3 transitions ...... 264

Figure I 3 TD-DFT M06/CEP-31G(d) energy diagram calculated for

Au heli-viologen at 399 nm excitation for top 3 transitions ...... 265

Figure J 1 TD-DFT M06/CEP-31G calculated UV-vis of Au heli-viologen compared to experimental UV-Vis ...... 266

Figure J 2 Isodensity representations of MO transitions of excited state at 399 nm for Au heli-viologen with percent contribution ...... 269

xxiv

LIST OF SCHEMES

Scheme 2-1 Synthesis of 4’-bromo DMIC (2-8) from isovanillin ...... 45

Scheme 2-2 Synthesis of 3’-bromo DMIC and 7’-bromo DMIC...... 49

Scheme 3-1 Synthesis of ortho-methoxyaniline (CH2CN sidechain) ...... 75

Scheme 3-2 Smiles rearrangement of homovanillonitrile ...... 76

Scheme 3-3 Synthesis of benzothiazoles ...... 78

Scheme 3-4 Reduction of aminobenzothiazole ...... 81

Scheme 3-5 Protection-thiocyanation-reduction scheme for o-aminothiophenol synthesis ...... 81

Scheme 3-6 Halogenation-Ullman type thiolation ...... 83

Scheme 3-7 Synthesis of benzothiazines by alkylate condensation ...... 86

Scheme 4-1 Reaction pathways for mixed melanoid (4-4) synthesis...... 113

Scheme 5-1 Retrosynthetic scheme for viologen synthesis ...... 138

Scheme 5-2 Step-wise Wittig route to access quinolyl-E-stilbene (5-5) ...... 139

Scheme 5-3 Gold-heli viologen(5-13) synthesis ...... 143

xxv

1 INTRODUCTION 1.1 Nitrogen heterocycles Nitrogen heterocycles (N-heterocycles) are one of the most significant chemical class of organic molecules, as they are widely found in natural products and active pharmaceuticals.1 N- heteocycles are present in building blocks of life- DNA, RNA, ribosomes and conenzymes.2

Over 75% of pharmaceuticals approved by the FDA contain nitrogen-heterocycles.3 Most common structural classes of five and six membered nitrogen heterocycles present in pharmaceuticals have been shown in Figure 1-1.4

Figure 1-1- Pharmaceuticals containing nitrogen Heterocycles4 (Adapted with permission from Njardson et al.).

Four membered β-lactams are of importance due to their activity in antibiotics (e.g. Penicillin) among other bioactive heterocycles.3 Five membered N-heterocycles-indole, thiazole, and imidazole, and six membered N-heterocycles-quinoline, pyridine, and thiazine have been of interest in organic, medicinal and materials chemistry. N-heterocycles exhibit hydrogen bonding, dipole-dipoe interactions, π-π stacking interactions in addition to electron acceptor and donor properties, which are important in biological functions as well as materials development.1,3,4 N- heterocycles can be synthesized via biosynthesis and chemoenzymatic synthesis, or chemical synthesis methods such as transition metal catalysis, cycloaddition, photocatalysis, nitrene

1 insertion and other radical reactions. Subclasses of N-heterocycles such as indole, thiazole, thiazine and quinoline (Figure 1-2) are of interest to the research discussed in this work. Indole compounds are most commonly synthesized by intramolecular reaction of two ortho-substituents on an aromatic ring or by internal cyclization of a single substituent onto the aromatic ring.

Thiazoles are synthesized by metal catalyzed thiocyanation. Quinolines are generally synthesized by Skraup synthesis or Michael type reactions.5 Our interest in these particular heterocycles stem from their presence and role in biological systems as well as potential material applications of these conjugated systems.

Figure 1-2 N-heterocycles of interest.

Indoles, thiazoles and thiazines are widely present in melanin which are natural biopolymers found in skin, hair pigments and brain of humans and other animals. They exhibit interesting photoprotective and phototoxic behavior due to their unique photophysical properties. Pyridines and quinolones are present in viologens (diquaternary salts of Bipyridine), which have applications in optical memory storage and electrochromic devices. Detailed background on melanins and viologens is discussed in the following sections.

1.2 Melanin Melanin pigments are one of the most widely present biological molecules in the animal kingdom as well as fungi, plants and bacteria.6,7 These compounds are involved in multiple biological functions such as thermoregulation, virulence factor where it protects organism from host immune response.8 They also possess interesting material, photophysical and chemical properties such as broadband radiation absorption (UV-Vis and Infrared), metal chelation, and free radical 2 scavenging.9 There are a few different types of melanin: eumelanin, pheomelanin, neuromelanin and allomelanin. Allomelanin is the only type of melanin which does not contain nitrogen or sulfur in the heterocycle as they are derived by polymerization of catechol and dihydroxynapthalene type monomers.9 Two of the most commonly found melanin in nature are Eumelanin and Pheomelanin.

Eumelanin is an insoluble brown to black pigment, whereas pheomelanin is a red to yellow pigment as shown in Figure 1-3.10 The arrows in the chemical structure indicate branching points or chain proliferation sites of these bio-polymers.

Figure 1-3 Melanin pigments responsible for hair color.

Both of these melanins are derived from dopaquinone which is formed from oxidation of amino acid L-tyrosine by the enzyme tyrosinase.11 The structural difference between the two is the presence of sulfur it in pheomelanin due to the role of cysteine in formation of pheomelanin.9,12

Neuromelanin is a dark polymer pigment present in substantia nigra and locus coereleus of the brain, and it is composed of mixed melanins (conjugated eumelanins and pheomelanins), metals,

3 and proteins.13 In order to understand the structure and the function of these biomolecules, discussion of melanogenesis is crucial.

1.2.1 Melanogenesis 1.2.1.1 Rapor-Mason-Prota pathway of Melanogenesis: Basic understanding of melanogenesis (Figure 1-4) was derived by Ito in 2003 from the works of

Raper14 who isolated dopa, 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole carboxylic acid

(DHICA) from tyrosine oxidation by tyrosinase; Mason15, who spectroscopically characterized dopachrome; and Prota16 who proved cysteinyldopa as a pheomelanin precursor leading to the understanding of late stage melanogenesis.17 As mentioned above both types of melanins found in animals, eumelanin and pheomelanin are formed from dopaquinone, which is highly reactive. It was believed previously that DOPA (3,4-dihydroxyphenylalanine) is formed from L-tyrosine which then oxidizes to dopaquinone. However, it was proved by Cooksey et al.18 using N,N- dimethyldopamine as a substrate for tyrosinase enzyme that dopaquinone is formed directly from tyrosine during the initial stage of melanogenesis.18–20 In the absence of cysteine (sulfuryl compound), dopaquinone, due to its high reactivity undergoes intramolecular amine addition by the L-dopa sidechain amine group forming cyclodopa (leucodopachrome). Redox exchange between dopaquinone and cyclodopa results in formation of dopachrome and dopa. As described in Figure 1-4, dopa can convert back to dopaquinone by tyrosinase-catalyzed oxidation.

Figure 1-4 Rapor-Mason-Prota pathway of Mixed melanogenesis.17

Dopachrome is a red-orange intermediate undergoes intramolecular rearrangement to form natural eumelanoid monomers, 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole

(DHI) if it decarboxylates during rearrangement.14,17,20 These eumelanoid monomers further oxidize and polymerize to form the brown-black insoluble polymer pigment, eumelanin. When sulfuryl compounds such as cysteine are present, it converts dopaquinone to one of the two isomers: 5-S-cysteinyldopa (5-S-CD) and 2-S-cysteinyldopa (2-S-CD), among which 5-S-CD is the major isomer.21 Redox exchange between dopaquinone and cysteinyldopa leads to formation of cysteinyldopaquinones (CD-quinones) and DOPA. CD-quinones further oxidize to form benzothiazine intermediates, which can convert to benzothiazoles via rearrangement.

Benzothiazines and benzothiazoles are the natural pheomelanoid monomers. These small

5 molecules further oxidize and polymerize to form pheomelanin pigment.17,20,21 Several enzymes are involved in melanogenesis regulation. Tyrosinase, a class of proteins containing copper with ability to bind dioxygen, regulates formation of dopaquinones.22 Dopachrome tautaomerase (Dct), a melanogenic Tyrosinase-related protein-2 (Tyrp-2) catalyzes the tautomerization of dopachrome to DHICA.19,23,24 This isomerization involving hydrogen shift is also facilitated by divalent metal ions.25 DHI oxidative polymerization was previously attributed to tyrosinase activity, but it was proved through pulse radiolysis study that this process can be facilitated by oxidation of dopaquinone to DOPA.26,27 DHICA oxidative polymerization is catalyzed by Tyrosinase-related protein-1 (Tyrp-1).17

1.2.1.2 Kinetics of melanogenesis Dopaquinone is a type of orthoquinone, which are very reactive and unstable compounds. Due to their low stability, to study kinetics of orthoquinone, pulse radiolysis is used. In this technique, pulsed ionized radiation (through a linear accelerator) is used to irradiate the sample and an analyzing beam (UV-Vis) is delivered perpendicularly (to the electron beam).28 Induced absorbance change is measured through this fast spectrophotometer and analyzed.22 Hydoxyl radicals produced from water initially and upon addition of halide such as bromine under saturated

20,22 N2O, dibromide radicals are produced in buffer with KBr. Dibromide radicals oxidize DOPA to form dopaquinone upon addition of catechol under pulse radiolysis conditions. Rate constants can be measured and quantified by observing subsequent changes in absorption wavelengths for different reactions.22 Figure 1-5 details the rate constants of reactions leading to formation of dopachrome and cysteinyldopaquinone from dopaquinone.

Figure 1-5 Kinetics of melanogenesis.22,29

7 -1 -1 In the presence of cysteine dopaquinone is rapidly (k3=3 x 10 M s ) conjugated to cysteinyldopa with the major isomer being 5-S-CD.22 Redox exchange step to form 5-S-cysteinyldopaquinone however is the rate determining step with the rate constant of 8.8 x 105 M-1s-1. It also produces

DOPA, which is dearmoatized to dopaquinone. The rate of 5-S-CD formation depends on the rate of formation of dopaquinone.30 Through stages of pheomelanogenesis, Cysteinyldopaquinone produces pheomelanin biopolymer. In the absence of the sulphydryl (SH) compound cysteine,

-1 dopaquinone cyclizes to form cyclodopa. This step is very slow (k1=3.8 s ) and is possible due to the process being intramolecular at pH 8.6. This cyclization is also pH dependent as the rate is 100 fold slower when pH is reduced to 5.6.29,30 Through redox exchange, (dopaquinone to DOPA) cyclodopa converts to dopachrome. This reaction is faster compared to the cyclization step with a

7 rate constant of 5.3 x 106 M-1s-1. Dopachrome through stages of eumelanogenesis produces eumelanin biopolymer.

1.2.1.3 Eumelanogenesis In the absence of cysteine, dopaquinone (DQ) cyclizes to form cyclodopa (CD). Figure 1-6 describes formation of DHI and DHICA quinones from cyclodopa, which with further oxidation and polymerization form eumelanin. Through redox exchange with dopaquinone, Cyclodopa converts to dopachrome, which has a half-life of ~30 minutes.

Figure 1-6 Kinetics of Eumelanogenesis: formation of DHI and DHICA quinones.

Dopachrome undergoes spontaneous decomposition under physiological conditions resulting in formation of DHICA and as it decarboxylates it forms DHI. This process is non-enzymatic and it forms decarboxylated DHI as the major product with DHI:DHICA being 70:1.31 Through redox exchange with dopaquinone, DHI oxidizes to form DHI quinone with a slow reaction rate with rate constant of 1.4 x 106 M-1s-1 as this reaction does not go to completion.20,27 In the presence of dopaquinone tautomerase (Dct or Tyrp2), dopachrome is converted to DHICA through this enzymatic tautomerization where Cu(II) plays a key role.25,31,32 DHICA, through redox exchange with dopaquinone oxidizes to form DHICA quinone, which has a much slower rate of 1.6 x 105

M-1s-1. As this process will not go to completion, involvement of enzymes such as tyrosinase speeds up oxidation of DHICA to DHICA quinone.27,33 Chemical or enzymatic oxidation of DHI and DHICA quinones leads to oligomerization and polymerization as rapid deposition of a black solid is observed leading to formation of eumelanin polymer.34,35 Oxidation of these quinones was studied by pulse radiolysis, which showed that it tautomerizes to imine and methide form. These tautomers undergo nucleophilic addition in order to make dimers, trimers and oligomers.29

1.2.1.3.1 Eumelanin structure elucidation: The structure of eumelanin was elucidated by chemical degradation of this natural polymeric pigment.36 Through degradation studies using chemical oxidizing agents such as potassium permanganate, hydrogen peroxide or via reductive hydrolysis using hydroiodic acid. HPLC methods have been developed to detect the degradation products and the quantification of these products elucidates composition of Eumelanin.36,37

Figure 1-7 Oxidative degradation of eumelanin by Alkaline H2O2. (Arrows indicate conjugation/chain proliferation sites of the polymer)

As shown in Figure 1-7, degradation of different types of melanin using hydrogen peroxide under alkaline conditions results in simple degradation products, which can be detected and quantified by HPLC. Eumelanin containing dihydroxyindole (DHI) subunit results in pyrrole-

2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA) upon oxidative degradation. Eumelanin containing dihydroxyindole carboxylic acid (DHICA) results in PTCA upon oxidative degradation whereas cross-linked eumelanin results in pyrrole-2,3,4,5- tetracarboxylic acid (PTeCA). These degradation products are used as bio-markers for analysis of eumelanin in biological melanin samples.

1.2.1.4 Pheomelanogenesis In presence of cysteine formation of cysteinyldopas (5-S-CD and 2-S-CD) and cysteinyldopaquinones (5-S-CysQ and 2-S-CysQ) from dopaquinone is favored.

Figure 1-8 Nucleophilic addition of cysteine.

Ito and Prota showed (Figure 1-8) that through enzymatic reaction catalyzed by mushroom tyrosinase producing 5-S-cysteinyldopa as a major (74%) product, and 2,S-cysteinyldopa (14%),

2,5-S,S’-dicysteinyldopa (5%), 6-S-cysteinyldopa (1%) as minor products.21 Due to this reaction major pheomelanoids observed in nature, in pheomelanin or neuromelanin are functinalized with

Sulphur at 5’ position. As 5-S-CD is the major product it leads to the formation of the key intermediate 1,4-benzothiazine and other benzothiazine derivatives as described in Figure 1-9.

During the late stage of pheomelanogenesis, 5-S-CD is converted to 5-S-cysteinyldopaquinone (5-

S-CysQ) with redox exchange with DOPA as it converts to dopaquinone. Cysteinyldopaquinone cyclizes to form 2H-1,4-benzothiazine-o-quinonimine(QI) as amine nucleophile attacks the neighboring carbonyl carbon. The rate of this reaction was measured with pulse radiolysis and was found to be fast with a value of 10 s-1.27 The competing reaction in presence of higher cysteine

11 content is addition of an extra cysteine to the 5-S-CD-Q which forms, 2,5-S,S’-dicysteinyldopa

(2,5-S,S’-diCD). The rate of this reaction is much lower, 1 x 104 M-1s-1 and it only becomes a competitive process when cysteine concentration is higher than 1 mM in-vivo.17,30

Figure 1-9 Pheomelanogenesis: formation of 1,4-benzothiazines.38

Oxidation of cysteinyldopa and 1,4-benzothiazines was studied by enzymatic and other catalytic reaction using reagents such as peroxidase/H2O2, tyrosinase, periodate and ferricyanide under physiological pH.38 2H-1,4-benzothiazine-o-quinonimine (QI) is the key benzothiazine intermediate which leads to the formation of pheomelanoid monomers. QI undergoes

12 rearrangement with the reaction rate (measured by pulse radiolysis) of 6.0 s-1 in order to form 1,4- benzothiazine carboxylic acid (BTZCA) as a major product (85%) and part of it decarboxylates to form 1,4-benzothiaine (BTZ).20,38–40 QI undergoes redox exchange with 5-S-cysteinyldopa as it is converted to 5-S-cysteinyldopaquinone resulting in formation of dihydro-1,4-benzothiazine carboxylic acid (DHBCA).20,41 Reduction of benzothiazine intermediate (BTZ) forms dihydro-1,4- benzothiazine (DHB), and hydrolysis of this unstable DHB leads to the formation of dihydroxy- dihydro-1,4-benzothiazine (DHDHB).38

Cysteinyldopa and its 1,4-BTZ derivatives have shown to chelate strongly with metal ions such as

Zn2+, Fe3+, Cu2+, Al3+, Co2+, and Cd2+. Zinc catalyzes oxidation of BTZCA and forms BTZCA- dimers and further oxidation leads to formation of trichochrome C.38 Iron and copper ions chelate with BTZ and form a complex which assists in decarboxylation and ring contraction resulting in formation of 1,3-benzothiazole (BTZL) as described in Figure 1-10.38

Figure 1-10 Oxidation of benzothiazine to benzothiazole.38

Cysteinyldopa and its 1,4-BTZ derivatives under enzymatic and metal oxidative conditions form pheomelanin polymer through oxidative polymerization. Through biomimietic conditions using enzymatic systems (peroxidase, tyrosinase), chemical agents (H2O2) and aerial oxidation, oxidative polymerization of cysteinyldopa and its derviatives have been studied.38,42 Different oligomers were made under different conditions and concentrations of different pheomelanoids, which implied that the make up of the final pheomelanin polymer is highly dependent on oxidative

13 conditions and the nature of polymerization. Degradation studies of natural pheomelanin polymers discussed below sheds light on the nature of polymerization and conditions in-vivo.

1.2.1.4.1 Pheomelanin structre elucidation: Structure of Pheomelanin in natural melanin pigment is also determined by degradation experiments. Oxidative degradation using hydrogen peroxide or reductive hydrolysis by hydroiodic acid is used. Products are analyzed and quantified by HPLC to study the composition of pheomelanins. Benzothiazine and its oxidation product benzothiazole are the most commonly found subunits in natural pheomelanin.36,37,43

Figure 1-11 Pheomelanin degradation by oxidation and reductive hydrolysis.

As shown in Figure 1-11, reductive hydrolysis of pheomelanin with benzothiazine unit using hydroiodic acid resuls in generation of 4-amino-3-hydroxyphenylalanine (4-AHP). Its isomer, 3-

AHP is also used as a biomarker for pheomelanin. Oxidative degradation of benzothiazine- pheomelanin with alkaline hydrogen peroxide results in Pheomelanin containing benzothiazole unit results in benzothiazole carboxylic acid (BTCA isomers- BTCA-5 and BTCA-2), which can further degrade to yield thiazole-4,5-dicarboxylic acid (TDCA) and thiazole -2,4,5-tricarboxylic

14 acid (TTCA). Pheomelanin containing benzothiazole subunit also oxidizes to yield TDCA and

TTCA. These degradation prodcts can be analysed and quantified by HPLC and are used as biomarkers to understand pheomelanin strcture.36,37,43

1.2.1.5 Mixed melanogenesis Three step pathway (Figure 1-12) proposed by Ito shows that melnogenesis has three stages of production, which depend on presence and concentration of cysteine. Initially when tyrosinase activity is low, dopaquinone is being produced at a low rate. When tyrosinase activity goes up resulting in increased dopaquinone production, the first stage of melanogenesis occurs as cysteinyldopas are being produced with presence of cysteine. This production occurs until cysteine concentration falls below 0.13 μM, after which the second stage begins where cysteinyldopas oxidize to produced 1,4-benzothiazine intermediates, which further oxidize to produce pheomelanin polymer. The second stage continues as long as cysteinyldopa concentration is above

9 μM.12,17,19,20 Once cysteine is depleted and cysteinyldopa concentration has lowered, dopaquinone produces cyclodopa as the third stage begins. At this stage, cyclodopa produces DHI and DHICA via dopachrome, which further oxidize to form eumelanin polymer.

Figure 1-12 Cysteine dependent mixed melanogenesis. (Adapted with permission from Ito and Wakamatsu, 2008)20 15

This phenomenon of pheomelanin formation followed by eumelanin leads to casing model, which suggests that pheomelanin polymer forms the core, as its production is favored in the initial stage when cysteine concentration is high. Once cysteine is depleted, eumelanin production occurs and eumelanin forms the shell. This model supports structure and formation of neuromelanin, a mixed melanin polymer.11,44,45 As shown in Figure 1-12 melanogenesis depends on tyrosinase activity, which dictates dopamine production, as well as presence and concentration of cysteine. When cysteine levels are low, cysteinyldopaquinone production is low and as a result, pheomelanogenesis stage is short. In this case, eumelaninogenesis predominates. When cysteine concentration is high, cysteinyldopa-quinone production is high resulting in longer pheomelanogenesis and increased pheomelanin production.12,17,19,20

1.2.1.5.1 Neuromelanin structure elucidation: Neuromelanin is a biopolymer structure consisting of pheomelanin and eumelanin as its major components. Degradative studies have suggested the benzothiazine and indole structures as subunits of this biopolymer. However, in contrast to pheomelanin and eumelanin found in skin and hair pigments, neuromelanin is derived from dopamine. In order to study the structure of neuromelanin, Ito and coworkers synthesized dopamine-derived eumelanin and cysteinyl dopamine derived pheomelanin via chemo enzymatic synthesis using mushroom tyrosinase enzyme.17 Degradation products of synthetic melanin and neuromelanin extracted from substantia nigra were generated from oxidative degradation and reductive hydrolysis as shown in Figure 1-13.

Figure 1-13 Degradation products of dopamine and cys-dopamine derived melanin.

Upon oxidation with alkaline hydrogen peroxide, DHI melanin resulted in PDCA and PTCA

Cysteinyl dopamine derived melanin (benzothiazole and benzothiazine subunit) upon oxidative degradation generated TDCA and TTCA and upon reductive hydrolysis generated 4-amino-3- hydroxyphenylethylamine (4-AHPEA). Comparison of the neuromelanin reductive degradation products to synthetic melanin degradation products showed that neuromelanin contains nearly

6% of DOPA sidechain compared to dopamine sidechain indicating that neuromelanin is mostly composed of dopamine derived melanins. Comparison of oxidative degradation products of neuromelanin with that of synthetic melanin showed that ~25% of melanin content in neuromelanin is cysteinyl dopamine derived in form of benzothiazine structure.36,46,47

1.2.2 Sources and synthesis of Melanins: 1.2.2.1 Extraction: Natural melanin can be extracted from bacterial, fungal, animals or humans (hair, brain tissue).

Method of extraction and purification depend on the source, but the commonality between the methods is use of alkaline solutions (1 M NaOH or KOH) followed by autoclavation.7 Fungal melanin can be extracted by incubation with 1 M NaOH at 60 °C followed by precipitation using

HCl. Precipitate is freeze-dried and resolubilized in alkaline solution where the above steps are repeated several times to purify the melanin.48–51 Extraction of human hair melanin involves washing with phosphate buffer and DTT (dithiothreitol), followed by incubation with proteinase

K enzyme which is used for keratin digestion as it breaks the peptide bond at carboxyl group adjacent to alpha amino groups. Repeated treatment with proteinase K and successive washing with buffer and DTT is utilized to purify human hair melanin.52

Figure 1-14 (A-D)SEM images (scale bar-200 nm) of neuromelanin from A) putamen B) premotor cortex C) cerebellum and D) substantia nigra. E) AFM of neuromelanin pigment from premotor cortex (scale bar-100 nm). F) Wavelength dependent photoelectron emission microscopy data. (Adapted from Zecca et al., 2008)53

However acid/base extraction changes the molecular structure of melanin by changing concentration and distribution of metals coordinated to melanoids, as observed by morphology and spectroscopy.13,51,54 Human neuromelanin is extracted from one of the following organelles: substantia nigra, putamen, premotor cortex, and cerebellum. The process involves multiple steps of gradient centrifugation. Figure 1-14 (A-D) shows morphology of the extracted melanins visualized through electron microscope. Morphology and particle size of neuromelanin differ based on the source. However, as seen in the plot F in Figure 1-14; of wavelength dependent UV free electron laser photoelectron emission microscopy, all these melanins have common surface

19 oxidation potential.53 This observation provides additional evidence indicating that the process of melanogenesis involves synthesis of pheomelanins until cysteine is depleted, which forms the core.

Once cysteine is depleted, eumelanins form creating the outer shell of the neuromelanin resulting in common surface oxidation potential among natural melanin samples.

1.2.2.2 Chemoenzymatic synthesis: Synthetic melanins are obtained using tyrosinase enzyme to oxidize dopamine in order to get eumelanin and dopamine in presence of cysteine in order to get pheomelanin as shown in Figure

1-15. Tyrosinase is extracted from plants (e.g. campanulatus) or mushrooms (e.g. Agaricus

Bisporus).10,52,55,56 For pheomelanin synthesis, DOPA or dopamine and cysteine are oxidized using tyrosinase in sodium phosphate buffer as pheomelanin is precipitated by lowering the pH to 3-4 using acetic acid or hydrochloric acid.13,57

Figure 1-15 Chemoenzymatic synthesis of melanins.

Eumelanin can be synthesized by following similar procedure without the presence of cysteine, using DOPA, dopamine, DHI or DHICA, where enzymatic oxidation results in rapid deposition

34 of black-brown pigment: Eumelanin. Enzymes such as peroxidase with H2O2 can also be used for controlled oligomerization of DHI and DHICA as studied by d’Ischia to synthesize dimers and tetramers of DHI to study the mechanism behind oxidative polymerization.10,55,58 Horseradish peroxidase can also be used to yield synthetic melanin from 5,6-dihydroxyindole substrate.59

1.2.2.3 Oxidative polymerization: Oxidative polymerization or autooxidation of dopamine, DOPA, DHI or DHICA results in formation of pheomelanin, eumelanin or neuromelanin.60–64 e.g. Melanin can be synthesized via oxidative polymerization of dopamine hydrochloride in alkaline solution. Nanoparticles of melanin were synthesized using dopamine hydrochloride through oxidation and self- polymerization of dopamine in aqueous ammonia in ethanol at room temperature.65

Figure 1-16 Melanin (polydopamine) synthesis via oxidative polymerization (Adapted with permission from Ju et al., 2011)88

Water-dispersible eumelanin nanoparticles were synthesized (Figure 1-16) by reaction of dopamine hydrochloride in deionized water with sodium hydroxide, which produced a pale yellow

21 basic solution at 50 °C and through spontaneous oxidation nanoparticles of melanin formed as the color change to dark-brown was observed.66

1.2.2.4 Chemical synthesis of melanin subunits: 1.2.2.4.1 Synthesis of 5,6-dihydroxyindole carboxylate compounds: Indoles are one of the most commonly studied nitrogen heterocycles, and several synthetic pathways are available in literature. However, methods to synthesize 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole carboxylate (DHICA) are limited. Figure 1-17 shows commonly used methods to synthesize 5,6-dimethoxyindole carboxylate (DMIC). Methyl/ethyl azidoacetate sidechain condensation on aldehyde followed by thermal cyclization following Hemetsberger indole synthesis is one of the first methods used to synthesize DMIC (Figure 1-17a).67,68

Figure 1-17 Methods to synthesize 5,6-dimethoxyindole carboxylate:(a) Hemetsberger indole synthesis67 (b) Copper catalyzed domino coupling-cyclization69 (c) Horner-Wadsworth-Emmons synthesis-Cadogan type cyclization.70

Copper-catalyzed domino coupling followed by cyclization of aldehyde with isocyanoacetate

(Figure 1-17b) was used by Fang and coworkers to synthesize DMIC and its bromo derivatives in order to investigate their agonist activity for GRP35 (a G protein coupled receptor involved in pain, inflammation, cardiovascular diseases).69 Another method to synthesize DMICA was proposed by Chai, Huleatt and co-workers, where Horner-Wadsworth-Emmons method was used to more efficiently synthesize cinnamate sidechain from the corresponding nitroveratraldehyde followed by Cadogen type cyclization. This method used excess of triphenylphosphine in toluene under microwave conditions to achieve the DMICA product.70

1.2.2.4.2 Synthesis of Eumelanoid oligomers Chemical synthesis of oligomeric dihydroxyindole (DHI), dihydroxyindole carboxylate

(DHICA), and benzothiazine have been pursued by melanin chemists in last 20 years.35,59,64,71,72

Chemical synthesis of these molecules would help understand complex biosynthesis of melangenesis. In addition, these oligomers and polymers have shown to be significant in developing bio-materials with applications in light-harvesting, bio-adhesion, and drug delivery.

One of the synthetic strategy to access 5,6-DHI dimers and trimers is shown in Figure 1-18 using which dimers and trimers were synthesized from o-ethynylaniline derivatives.71,72 2,3’-biindolyl and 2,7’-biindolyl were synthesized from 3-iodoindole derivative and 7-iodoindole derivatives respectively using Pd catalysed Sonogashira coupling with o-ethynylaniline compound followed by intramolecular cyclization. 2,2-biindolyl compound was synthesized from oxidative dimerization followed by double concerted intramolecular cyclization of o-ethynylaniline compound.71

Figure 1-18 Strategy to synthesize biindolyls.71,72

This work was further extended by synthesizing trimers of 5,6-dihydroxyindoles using the same strategy. After deprotection, the dimers and trimers compound matched minor intermediates recovered during 5,6-dihydroxyindole polymerization to eumelanin.72

Figure 1-19 Suzuki coupling to conjugate indole to a 5,6-Dimethoxyindole carboxylate (DMICA) compound73

Palladium catalyzed couplings are commonly used for indole compounds, however only a few studies using Pd catalyzed couplings were found for 5,6-dihydroxyindole compounds.68,71,72,74,75

Recently, palladium catalyzed Suzuki coupling was used to conjugate DMIC to simple indole compounds in order to synthesize dimers and trimers of eumelanoid type small molecules.

Commercially available DMIC ethyl ester derivatives were mono and dibrominated, and conjugated with borylated simple aromatic compounds in order to synthsize these derivatives.73

1.2.2.4.3 Synthesis of aminobenzothiazole and 1,4-benzothiazine: Methods to synthesize benzothiazole include Hoffman method, Jacobson cyclization or oxidation by bromine under acidic condition or copper/palladium catalyst.76 One of the first methods utilized to synthesize aminobenzothiazole was to thiocyanate o-aminothiol followed by cyclization.77 Thiocyanation using potassium thiocyanate or ammonium thiocyanate with bromine followed by oxidative cyclization are common conditions in order to synthesize benzothiazoles.76,78 A method to synthesize aminobenzothiazoles from arylthiourea under mild conditions is by using benzyltrimethylammonium tribromide as bromine source.79

Figure 1-20 Methods for aminobenzothiazole and 1,4-benzothiazine synthesis.

Most commonly used method to synthesize benzothiazine compound is alkylate condensation of dihalogenated alkylating reagent to ortho-aminothiol. ortho-halogenated thiol can also be used as a substrate with halogenated primary amine alkylating reagent.80–82 Similar to alkylate condensation, benzothiazine synthesis has also been doen by intramolecular cyclization with ethylamine tethered to the ring with thioether linkage.83,84 This method is similar to the conventional method to synthesize 1,4-benzothiazine from 5-S-cysteinyldopa using potassium ferricyanide as catalyst.40 Cho and coworkers developed a ring expansion reaction of cyclic ketones and ketoximes using DIBAL-H in order to access five to eight membered heterocycles one of which was benzothiazine. Reaction proceeds through a reductive rearrangement of oximes which provide cyclic secondary amines.85,86

1.2.2.4.4 Synthesis of pheomelanoid oligomers:

Figure 1-21 Synthesis of ∆2,2’-bibenzothiazine via oxidative coupling of 2-phenyl-1,4- benzothiazine. 87

Trichochromes-present in red hair pigment are consisted of dimeric units of benzothiazines. A strategy to synthesize ∆2,2’-bibenzothiazine from 3-phenyl-1,4-benzothiazine was done as shown in Figure 1-21. Oxidative coupling of 3-pheyl-1,4-benothiazine was performed using hydrogen peroxide in presence of oxygen under strong acidic condition (methanol:36%HCl-3:1). The reaction goes through a 2,2’-bi(2H-1,4-benzothiazine) intermediate as indicated by DFT calculations.87

1.2.3 Properties of Melanins: 1.2.3.1 Physiochemical properties Melanin pigments exhibit low solubility in water, most common organic solvents such as ethanol, methanol, chloroform, acetone, benzene, hexane, diethyl ether. Synthetic and natural melanins are soluble in alkaline solutions such as 1 M sodium or potassium hydroxide and can be precipitated from alkaline solution at pH below 3 (commonly with 3 M HCl).7,49,61,88 Some common qualitative chemical tests and extraction protocols are used to identify, extract and analyze melanins.

Precipitated melanin pigment can be decolorized/bleached by oxidizing agents such as KMnO4,

45,48,49,61,88–90 K2Cr2O7, H2O2 and NaClO or by reducing agents such as H2S and Na2S2O4. Melanins give positive reaction to the polyphenol test as it produces colloidal brown precipitate with FeCl3, and it produces gray precipitate with ammoniacal silver nitrate solution.7,48,61 Melanin pigment exhibits high resistance to thermal degradation as observed in the thermogravimetric (TGA) and derivative thermogravimetricanalysis (DTG).

Figure 1-22 DTG (dashed line) and TGA (solid line) of sepia ink melanin. (Adapted from Praleaet al., 2019)7

This analysis was done with synthetic (DOPA-melanin) and natural melanin (from fungal and bacterial organisms) samples, of which data of melanin extracted from sepia ink has been shown in Figure 1-22. The first endothermal peak around 71 °C is attributed to evaporation of bound water. The exothermal peak at 306 °C is attributed to loss of carbon dioxide. Major thermal decomposition of melanin occurs at a much higher temperature around 998 °C, due to decarboxylation. It was observed that synthetic melanin are more resistant to decomposition than natural melanin. Morphology and melanin particle size has been studied with Scanning Electron

Microscopy (SEM)91–100, Transmission Electron Microscopy (TEM)101–103, and Atomic Force

Microscopy (AFM)54,104–107 of synthetic and natural melanins. Elemental analysis is used to identify and classify the subtype of melanins as the C:N:S, C:N, and S:N ratios differ between pheomelanin, eumelanin and allomelanin.

1.2.3.2 Spectral and Photophysical properties Structure of melanin contains highly conjugated network of phenol, benzothiazole, benzothiazine, indole functionalities, and as a result, they exhibit high UV absorption. The UV absorption range of melanin in alkaline solution is 196-300 nm and varies between synthetic and natural melanins. Light absorption in visible region decreases as the wavelength increases. Plot of logarithmic absorption of melanin with wavelength gives straight line with negative slope, and is unique to the type of melanin.48,49 This method can be used to characterize and differentiate between the types of melanins. An example of UV-Vis absorption spectra of synthetic (DOPA) melanin and sepia melanin along with their logarithmic plots has been shown in Figure 1-23.

Both samples were dissolved in 0.1 M NaOH and sonicated, followed by spectral scan of 220-

800 nm wavelength range. Both the samples have absorption maxima at 220 nm and show strong absorption in UV region (plot a). However they product unique absorption pattern as observed in logarithmic plot (b) with different slopes.

Figure 1-23 A) UV-vis absorption spectra of sepia ink melanin (blue) and synthetic melanin (green), B) Linear plot of logarithmic absorbance vs wavelength. (Adapted from Pralea et al., 2019)7

Spectral analysis of human melanin has been done through in-vitro (400-720 nm) and in-vivo measurements (620-720 nm).108,109 Melanin quantity can be approximated by slope of the absorption spectra from the in-vivo experiment results (Figure 1-23).109 Epidermal melanin and neuromelanin exhibit similar broadband spectrum showing increasing absorbance at higher energies.53,108–110 Spectral analysis is also useful in differentiating the types of melanin as they exhibit different behaviors in-vitro and in-vivo. UVA induced absorption of pheomelanin and eumelanin was studied in-vivo and in-vitro, which showed difference in spectra and dose dependency on photo-oxidation.111

Figure 1-24 IR absorption spectra of synthetic melanin and sepia ink melanin. (Adapted from Pralea et al., 2019)7

Fourier-transform infrared spectroscopy is also used to identify melanin pigment. IR of melanin is dependent on the source of the melanin and extraction process. An example spectrum of synthetic melanin and melanin from sepia officinalis has been shown in Figure 1-24.The scan was done on sample powder (synthetic or sepia) with KBR discs in the range of 400-4000 cm-1. The broad absorption between 3000 and 3600 cm-1 is characteristic of -OH and -NH stretching vibrations as melanin contains amine, amide, phenol, carboxylate and aromatic functionalities in indoles, benzothiazole, benzothiazine core structures as well as different sidechains. C=C and C=O stretches are observed between 1600-1750 cm-1. Characteristic melanin stretches however are observed between 1400-1500 cm-1 that are attributed to bending of N-H and stretching modes of secondary amine (C-N) of indoles, in addition to phenolic stretching between 1100-1150 cm-1.

49,112–114 In addition to UV-Vis and FTIR, NMR (1H and 13C) is also used to characterize melanin pigments. Proton NMR showed peaks for aromatic hydrogens of phenyl, indole and pyrrole rings, methylene groups (sidechain), and N-H of indoles, and carbon NMR showed characteristic peaks

30 for carbonyl carbons in carboxyl, amide, and carbon atoms linked to heteroatoms such as nitrogen and sulfur.115–117 Mass spectrometry techniques such as (electrospray ionization) ESI-MS, matrix assisted laser desoption/ionization (MALDI), secondary ion mass spectrometry (SIMS), electron impact (EI) have been used to characterize structures of synthetic and natural melanins.50,118,119

1.2.4 Significance and applications: 1.2.4.1 Biological significance: Melanin has a protective role due to its ability to scatter photons, scavenge free-radicals, and dissipation of absorbed photons in non-radiative manner.120 It is present in hair and skin in different forms in humans, and is responsible for the color. Neuromelanin is a dark pigment, which accumulates in brain primarily in substantia nigra and locus coerleus. Formation of neuromelanin prevents accumulation of reactive quinones, which are generated from oxidation of catecholamine neurotransmitters. In addition, it has ability to sequester and bind metal ions, which reduces the metal toxicity. However, due to its affinity towards iron, it accumulates high level of iron (III) in form of high-spin, pseudo-octahedral centers with oxo and hydroxo bridges at the binding sites and its concentration can go up to 10 μg/mg of pigment.47,121,122

Figure 1-25 MRI images of substantia nigra and locus coeruleus of healthy individual. (NM present-white) and patient with PD (depleted NM). (Adapted from Isaias et al., 2016)123

This high accumulation of metals may be related to role of neuromelanin in neurodegenerative diseases such as Parkinson’s Disease (PD). Substantia nigra and locus coeruleus are the most affected areas in this disorder as shown in MRI of PD patient brain vs healthy brain in Figure

1-25.123 In the affected areas, accumulation and presence of neuromelnain has reduced significantly in patients with PD whereas significant presence of neuromelanin can be seen in healthy individual brain. Structural and redox properties of mixed melanin need to be studied in order to further understand the structure and function of neuromelanin and its role in Parkinson’s disease.

1.2.4.2 Materials applications: Despite issues such as insolubility in organic solvents and certain material properties (amorphous polymer), melanins have been of interest to material chemists due to its desired physiochemical and photophysical properties.62 Melanins have photoconductivity (UV-Vis) and electrical conductivity (AC and DC), and have confirmed to be a natural amorphous semiconductor due to

32 its ability to do bistable electrical switching.124,125 These properties can be utilized in organic electronics. Figure 1-26 describes applications (current and potential) of dopamine and polydopamine derived melanins.62

Figure 1-26 Applications of DOPA and dopamine derived melanins. (Adapted with permission from d’Ischia et al., 2014)62

Metal and drug binding properties of melanin can be utilized for water purification, and free radical scavenging property of melanin has applications in polymer stabilization.126,127 UV and photoprotective properties of melanin has applications in materials for UV screens, and its adhesion properties can be used in surface coating and bio-adhesion.62,128 Recent applications of polydopamine type melanin materials include biosensing, nano particle and nanocapsule preparation for drug delivery, light harvesting and cell interfacing.62,66,125,129–135

1.3 Viologens Viologens are redox active, strong electron accepting diquaternary salts of 4,4’-bipyridine

(MV2+). Their photoreduction properties have been known for several years and their common applications are as herbicides, photocleaving agents, electronic and electrochromic devices.136

Viologens (MV2+) can abstract an electron to generate radical cation (MV+), and if it two electrons are abstracted it generates neutral species (MV). These species exhibit unique electronic and photophysical properties.

1.3.1 Viologen Applications:

Figure 1-27 Viologen as (a) electron acceptor (b) electron donor (c) Donor-acceptor tandem. (Adapted with permission from Kiriya et al., 2014 and Xu et al., 2014)137,138

Viologens are good electron acceptors in their dicationic (MV2+) state. e.g. Paraquat is a widely used herbicide which is a dichloride salt of 4,4-bipyridine. During photosynthesis, it abstracts an electron from mitochondria generated by oxidation of NADH to NAD+. As a result, it oxidizes to radical cation of paraquat (Figure 1-27a).139 This unstable species abstracts and electron from oxygen (O2) generating reactive oxygen species (ROS) which leads to degradation of mitochondria and other organelles. Electron donating property of viologens can be utilized in conductors as shown in Figure 1-27b. Benzyl viologen (BV) can be used as dopant MoS2 transition metal dichalcogenide semiconductor to form air stable electron transfer complex. It can also be used as surface dopant at metal junctions in electronic devices which reduces contact resistance.137 As indicated in Figure 1-27c, triethanolamine (TEOA) donates an electron to the

Ir(III) complex. Photoexcitation of this electron leads to electron transfer to an electron relay viologen-1,1-ethylene-2,2-bipyridyldiylium (DQ2+). Generated radical cation of this viologen reduces the acceptor viologen-propyl viologen sulfonate (PVS) by electron transfer through zeolite solution interface resulting in charge separation. This system works as an artificial photosynthesis system.138

Figure 1-28 Electochromic switching in tri-cat viologen system. (Adapted with permission from Sun et al., 2015- https://pubs.acs.org/doi/10.1021/jacs.5b09274, further permissions related to the material excerpted should be directed to the ACS)140

Viologens can be used in electrochromic molecular switches. e.g. Cyclobis(paraquatp- phenylene) (CBPQT4+) ring can be switched among different units reversibly as shown in Figure

1-28.140 Each unit responds differently to external redox potentials, donating the electrons to the viologen ring. Each of these oxidation states and bound-unbound states exhibit different colors.

These electrochromic properties can be utilized in electronic and optical devices.

1.3.2 Ion-Pair Charge Transfer (IPCT) complex: Ion-Pair Charge Transfer (IPCT) metal-viologen complexes are made of dianionic transition metals and dicationic methyl viologen. These structures exhibit photoinduced electron transfer between the donor and the acceptor. IPCT complexes with metals such as Au, Ag, Sm, Eu, Fe have been studied.137,141–145 With donors such as dicyanoaurate, transfer properties of IPCT can be applied in photochromic and electrochromic devices. Phenyl-viologens such as DNP2+ have shown desirable optical and electronic properties due to extended π conjugation.146 Their π-π stacking interactions are being explored in chemistry and biology due to its effect on photophysical properties of supramolecular complexes.

1.3.2.1 Au(CN)2-DNP viologen complex: Dicyanoaurate (I) complexes are of interest as they form one of the most stable two-coordinate complexes due to their high stability constant as well as potential applications in semiconductor materials and photonic devices147,148. Dicyanoaurate(I) has unique geometric and charge-transfer

10 - properties as d Au(I) metal center favors aurophilic interactions and aggregation of [Au(CN)2

149,150 ]n, which would allow tuning of the optical properties Due to their strong electron accepting potential, viologens are fluorescence quenchers as they can quench triplet MLCT luminescence in a complex with metals such as gold (I). {DNP-[Au(CN)2]2}.4H2O exhibit dual luminescence in visible region as shown in temperature dependent excitation-emission spectra Figure 1-29.150

DNP2+

2+ 2- Figure 1-29 Temperature dependent luminescence of DNP -[Au(CN)2]2 . (Adapted with permission from Abouelwafa et al., 2012)150

Excitation at 355 nm results in gold-dimer broad emission at 580 nm.As the temperature increases, the gold dicyanoaurate dimer luminescence (ex-355 nm) is quenched by DNP2+ cations. Oxidative electron transfer results in quenching of the excited state gold-dimer luminescence at higher temperatures. Viologen luminescence (ex-410 nm) is not quenched at

- higher temperatures as oxidation of ground state [Au(CN)2] is not observed. These observations have been shown in the energy diagram in Figure 1-30.150

Figure 1-30 Energy diagram of the {DNP-[Au(CN)2]2} complex. (Adapted with permission from Abouelwafa et al., 2012)150

At lower temperatures (10 K) fluorescence and phosphorenscence of excited state, ground state gold-dicyanide, and phosphorescence of excited state DNP2+ are observed as confirmed by the lifetimes measurements. At higher temperatures (>50 K), after intersystem crossing (ISC) from singlet to excited state triplet state, luminescence of gold-dicyanide is quenched by the DNP- viologen via electron transfer quenching. Findings of the photophysical study of this metal-

- viologen complex indicated that low energy phosphorescence of [Au(CN)2] and DNP despite

DNP being non-luminescent in its free state. This observation was attributed to heavy atom effect due to gold resulting in gold-viologen orbital overlap.150

1.3.2.2 Ag(CN)2-DNP viologen complex: It was observed that DNP, which is non-luminescent in its free state, exhibits phosphorescence when coupled with dimeric gold-dicyanoaurate, the DNP viologen was complexed with

- 146 dicyanoargentate which is known form [Ag(CN)2 ]n. As shown in Figure 1-31, excitation spectra at 10 K and 78 K are similar(285 nm and 298 nm respectively). Emission of 412 nm was observed

38 at 78 K with excitation at 265 K. Low energy excitation of DNP results in excited singlet state(S1)

2+ of DNP with radiationless decay to triplet excited state (T1) followed by intersystem crossing to

- - Ag(CN)2 lowest triplet excited state (T1). Phosphorescence is observed due to Ag(CN)2 emission

- from T1 to S0. This emission band is high energy due to direct excitation of Ag(CN)2 resulting in

146 T1 to S0 emission via charge transfer transition.

2+ 2- Figure 1-31 Luminescence of DNP -[Ag(CN)2]2 . (Adapted with permission from Ahern et al., 2014)146

Luminescence of Ag-DNP complex is different from Au-DNP complex not only due to metal identity, but due to lack of argentophilic interaction (Ag-Ag) as compared to aurophilic interactions (Au-Au) observed in Au-DNP complex. Crystallographic analysis along with optical data suggest that there is little to no interaction between DNP2+ cation and dicyanoargentate as

146 the excitation-emission spectra of metal-viologen complex is similar to spectra of KAg(CN)2.

1.3.3 Helicene viologen Helicene compounds are nitrogen heterocycles which possess extended armoaticity, chirality, solid state structures, and ability to form transition metal complexes which can be applied to development of chemosensers, photovoltaics, and electrochromic devices.151 5,10- diaza[5]helicene is one of the helicene compounds which exhbit helical chirality and partially electron rich and poor molecular regions as indicated by electrostatic potential energy surface in

Figure 1-32.

Figure 1-32 Electrostatic potential energy surface and crystal motif of 5,10-diaza[5]helicene. (Adapted with permission from Zhang et al., 2016)141

5,10-diaza[5]helicene viologen has C2 symmetry and shows π-π stacking due to extended aromaticity in the crystal structure, however stacking is not sandwich like due to non-planar topography as shown in Figure 1-32.141 It shows fluorescence at 504 nm and phosphorescence at

623 nm in its free state. DFT calculations indicate that it exhibits charge transfer character ( S0 to

S1 electronic transition). This molecule when coupled with anionic metal donors could have potential applications in electrochromic switches, molecular electronics and sensors.141

1.4 Thesis overview: Enzymatic and chemical synthesis of melanin from dopamine, DOPA, cysteine, DHI and DHICA has been widely studied. However, the nature and mechanism of oligomerization and polymerization have been challenging. Eumelanin has been widely studied ranging from dimerization, oligomerization (through enzymatic process) to oxidative polymerization involving development of materials for applications, pheomelanin and its oligomerization mechanisms still need to be explored. Access to monomeric units of pheomelanin would help bridge this gap. In addition, it will provide an opportunity for controlled oligomerization using different types of monomeric units including synthesis of mixed melanoids.

Melanin-based materials have been developed through empirical approaches rather than structure- activity relationships.94 These developments are based on simple eumelanin monomers such as polydopamine. Due to difficulties in synthesis and properties of pheomelanin, such as benzothiazine and benzothiazole there is a lack of knowledge in bottom-up synthesis of these compounds. In addition, materials research related to melanin other than eumelanin is limited. This research work discusses regioselective synthesis and modification of eumelanoids (eumelanin monomeric units). Chemical synthesis of pheomelanoids (pheomelanin monomeric units) benzothiazole and benzothiazine was accomplished from ortho-methoxyaniline (via thiocyanation) and o-aminothiol (via alkylate condensation) respectively. Following the synthesis of monomeric melanoids, mixed melanoids (conjugates of eumelanoid and pheomelanoid) were synthesized via Miyaura borylation and Suzuki coupling. These melanoids may have applications in biomaterials, organic electronics, nanomedicine, bioadhesion, and photoprotection. In addition,

41 preliminary UV-Vis and Fluorescence spectroscopy analysis of the mixed melanoids material were performed in order to understand its photophysical properties.

Figure 1-33 Overview of the thesis.

DNP-viologen does not exhibit luminescence in its free state, however when complexed with gold-dicyanoarate it exhbits phosphorescence due to orbital overlap with dicyanoaurate dimer.

Helicine viologen exhibits luminescence in its free state. In addition it is able to form 2D solid- state architectures due to π-π stacking interactions. These propeties can be harvested by coupling this ligand with an anionic metal salt. It would be interesting to study the photophysical properties of a helicene viologen when coupled with dicyanoaurate. As shown in Figure 1-33, helicene viologen was synthesized from bromoquinoline starting material. This was accomplished by Pd catalyzed stille coupling or Hiyama-Heck coupling of bromoquinoline, followed by photocyclization of the resulting stillbene. Product of photocyclization was methylated and coupled with gold-dicyanoaurate in order to yield the Gold-heli viologen. Crystal structure of gold-heli viologen was determined and the photophysical properties were studied and reported.

2 SYNTHESIS, AND PHOTOPHYSICAL STUDIES OF REGIOSELECTIVELY FUNCTIONALIZED EUMELANOIDS 2.1 Introduction Eumelanin is an insoluble black-brown polymeric pigment which is derived from DHI (5,6- dihydroxyindole) and DHICA (5,6-dihydroxyindolecarboxylic acid). These polymers possess UV and photoprotective properties, free radical scavenging, and adhesion; and have applications in polymer stabilization, photoprotection, organic electronics, biosensing, nanomaterial drug delivery, and bioadhesion.62,124,133–135,152–154,125–132 In addition, eumelanin has biological significance due to its presence in human skin, hair and brain. Eumelanin can be synthesized chemoenzymatically, via oxidative polymerization from monomeric DHI or DHICA units.

Intermediates of DHI and DHICA polymerization are an area of interest due to their structure and properties. Chemical synthesis of subunits of eumelanin biopolymer has been explored in literature.67–70,73 5,6-dimethoxyindole carboxylate (DMIC) and its 3’-bromo derivative were synthesized using Hemetsberger-Knittel indole synthesis.67 Huleatt et al. described synthetic strategies to access a small library functionalized DHI and DHICA derivatives via expedient routes using similar methods.68 A more efficient assembly of these functionalized derivatives was done which involved Cadogen indole synthesis using ortho-nitroveratraldehyde derivatives.70 DHICA derivatives were synthesized using copper catalyzed domino four component coupling and cyclization in order to study their GPR35 agonist activity (GPR35- A G-protein coupled receptor involved in inflammation, pain, metabolic disorders).69

The focus of this research was chemical synthesis of small DMIC eumelanoids and their regioselective functionalization to utilize these functionalized derivatives for mixed melanoid synthesis via Miyaura-Suzuki coupling with pheomelanoid subunits (Chapter-3). Figure 2-1 shows the desired DMIC derivatives functionalized with bromine. Despite Cadogen type indole synthesis

43 considered more efficient, Hemetsberger indole synthesis was utilized for this work, as the

Cadogen synthesis requires access to nitroveratraldehydes involving an extra synthetic step, and it uses a molybdenum catalyst for cyclization, which is not easily accessible. In addition, variety of reaction conditions are required for cyclization step for different substrates.70

Figure 2-1 Targeted 5,6-dimethoxyindole carboxylate (DMIC) derivatives.

Research goal of this project included synthesis of monomeric 5,6-dimethoxyindole carboxylate

(DMIC) from simple starting material like vanillin or isovanillin. Targeted DMIC derivatives were a) mono-brominated derivatives-3’-bromo, 4’-bromo and 7’-bromo DMIC; b) dibrminated 3,4’- dibromo, 3,7’-dibromo and 4,7’-dibromo DMIC; and c) 3,4,7’-tribromo DMIC. Synthesis of brominated variants have been explored, however synthesis of multibrominated eumelanoids and synthesis and stability of brominated DHICA has not been studied. Following the synthesis of the desired regioselectrively brominated DMIC derivatives, deprotection was done on mono- brominated compounds to establish deprotection procedures and synthesize deprotected natural analogues of DHICA (5,6-dihydroxyindole carboxylic acid).

2.2 Results and discussion Expedient pathway suggested by Huleatt et al. was followed for this work for synthesis of mono- brominated eumelanoids.18 Regioselective bromination was done at different steps in the sequence to get brominated indole products at 3’, 4’ and 7’ positions. These bromo DMIC eumelanoids were synthesized from simple starting material like vanillin or isovanillin.

Experiment procedures were altered and optimized in order to fill the gap in experimental methods, results and characterization data.

Scheme 2-1 Synthesis of 4’-bromo DMIC (2-8) from isovanillin.

Synthesis of 4’-bromo DHICA has been shown in Scheme 2-1. Isovanillin was brominated using

N-bromosuccinimide (NBS) to get the bromination at the 4’-position of the final indole compound. Recrystallized product was methylated using methyl iodide and purified with silica gel flash chromatography. In the following step, methyl azidoacetate was condensed on the sidechain aldehyde using sodium methoxide (1M in methanol) as base.

Figure 2-2 Mechanism of methyl azidoacetate condensation over sidechain aldehyde.

This reaction follows Knoevenagel condensation type mechanism as shown in Figure 2-2. Sodium methoxide activates α-carbon of methylazioacetate, which attacks the electrophilic carbonyl carbon. Mechanism proceeds via several proton transfer steps, and deprotonation and dehydration of the final intermediate results in formation of α-azido β-arylacrylate as the condensation product.155 Condensation product was purified by silica gel flash chromatography and characterized by NMR as well as IR. As observed in Figure 2-3, the condensation product’s infrared spectrum shows a strong asymmetric stretch at 2124 cm-1 and symmetric stretch around

1250 cm-1, which are characteristic stretching modes of azides.

Azide (N3)

Figure 2-3 Infrared spectrum of azidoacetate condensation product.

The final step to synthesize the 4’-bromo DMICA was cyclization via nitrene insertion. Azide activation is required to create highly electrophilic nitrene intermediate, which would insert into the aromatic ring resulting in cyclization and indole formation, which could be a light or heat induced process. Cyclization via Hemetsberger indole synthesis was utilized as heat was used to generate nitrene.156–158 A tentative reaction mechanism is described in Figure 2-4. Due to heat nitrogen is evolved as nitrene is generated. Nitrene forms an unstable tricyclic intermediate with the neighboring unsaturated carbons. Due to low ring stability electrons of the aromatic phenyl ring opens the tricyclic intermediate as the phenyl ring is dearomatized and nitrogen attaches to the electrophilic position at the ring.

Figure 2-4 Mechanism of cyclization via nitrene insertion.

In alternate mechanism, 1,3-dipolar cycloaddition occurs creating the same intermediate.157 This final intermediate undergoes [1,5]-H shift to generate the desired indole product. Overall yield of

4’-bromo DMIC (2-8) was 54% over four steps. As described in Scheme 2-2, 7’-bromo DMIC (2-

12) and 3’-bromo DMIC (2-4) can be synthesized from vanillin. Similar reaction conditions, purification and analytical techniques were used as 4’-bromo DMIC(2-8) synthesis (Scheme 2-1).

Scheme 2-2 Synthesis of 3’-bromo DMIC and 7’-bromo DMIC. Bromination- NBS, DCM, RT, 8 hrs; Methylation-MeI, K2CO3, Acetone, 60 °C, 12 hrs; Sidechain condensation- Methyl azidoacetate, NaOMe, MeOH, -15 °C-RT, 18 hrs; Nitrene insertion-Toluene, 110 °C, 24 hrs

3’-bromo DMIC (2-4) was synthesized in four steps by the following reaction sequence- methylation (compound 2-1), sidechain condensation (compound 2-2), nitrene insertion

(compound 2-3) and bromination of vanillin. The overall yield of 3’-bromo DHICA (2-4) from vanillin was 48% in four steps. Bromination of vanillin results in selective bromination at ortho- position to the phenolic OH.

Figure 2-5 7’-bromo DMIC (2-12) synthesis via Hemetsberger-Knittel indole cyclization.

7’-bromo DMIC (2-12) was synthesized from bromination product (2-9) by methylation

(compound 2-10), sidechain condensation (compound 2-11) and nitrene insertion. The overall yield for 7’-bromo DMIC (2-12) with the above synthetic sequence from vanillin was ~31%.

Overall yield was lower due to the nitrene insertion step, where significant amount of side product was recovered (Figure 2-5), which required prep-HPLC purification in addition to silica gel purification.

Figure 2-6 Synthesis of 3,4’-dibromo DMIC (2-14) and 3,7’-dibromo DMIC (2-15).

Once monobrominated compounds were accessed, different combinations of bromination- selective debromination were utilized to synthesize di and tri-brominated DMIC derivatives.

Figure 2-6 describes synthetic methods to synthesize 3,4’-dibromo (2-14) and 3,7’-dibromo DMIC

(2-15) from 4’-bromo DMIC (2-8) and DMIC (2-3) respectively. Using stoichiometric amount of

N-bromosuccinimide (NBS) to brominate 4’-bromo DMIC (2-8) results in selective bromination at 3’-position resulting in 3,4’-dibromo DMIC (2-14). 3,7’-dibromo DMIC was synthesized by controlled double bromination of DMIC with NBS(4 eq.) by heating it at 65 °C for 3 hours. Limited generation of tribromo DMIC was observed. However, if this reaction is ran for 24 hours at 65

°C, it generates 3,4,7’-tribromo DMIC (2-16), which was recovered in 80% yield as shown in

Figure 2-7.

Figure 2-7 Synthesis of 4,7’-dibromo DMIC (2-17) and 3,4,7’-tribromo DMIC (2-16).

This tribromo derivative (2-16) undergoes regioselective debromination of 3’-bromo under acidic conditions in presence of lithium bromide and m-dimethoxybenzene (used as a trapping agent) to generate 4’, 7’-dibromo eumelanoid compound (methyl 4,7-dibromo-5,6-dimethoxy-1H-indole-2- carboxylate).9,10 Reaction was monitored with HPLC and TLC.

Figure 2-8 Analytical HPLC chromatogram of regioselective debromination reaction.

Figure 2-8 shows analytical HPLC (C-18) chromatogram of reaction mixture at 75 minutes into the reaction where most of the SM has been consumed (retention time-6.08 minutes) and m- dimethoxybenzene is present at 3.21 minutes. Products were purified with prep-reverse phase chromatography and fractions were analyzed with NMR. Desired 4’-7’-dibromo product is the major trace in the chromatogram at 5.46 minutes. The small traces could be impurities such as brominated m-dimethoxybenzene.

Figure 2-9 Mechanism of regioselective debromination of tribromo eumelanoid.

Mechanism of regioselective 3’-debromination is shown in Figure 2-9. Protonation occurs at C3 position resulting in the carbocation intermediate, which is attacked by bromide resulting in bromine and the desired 4’,7’-dibromo DMIC (2-17). However, this compound can re-brominate in presence of bromine resulting in the tribromo starting material, which is thermodynamically more stable.73,159 In order to prevent this, o-methoxybenzene was added as a trapping agent, which is more nucleophilic than the indole. It reacts with bromine and brominates resulting in bromine trapping, and as a result 4’,7’-bromo DMIC is recovered as the major product after purification.

Monobrominated DMIC synthetic eumelanoids were deprotected in order to get the natural analogues of DHICA eumelanoids. Deprotection was done by saponification of respective monobrominated DMICA by lithium or sodium hydroxide followed by demethylation of methoxy protecting groups by boron tribromide.

Figure 2-10 Synthesis of mono-brominated 5,6-dihydroxyindole carboxylic acid (DHICA)- A) NaOH (5 eq.), ethanol:water(1:1), RT, B) BBr3, DCM, -78 °C-RT, C) LiOH, ethanol:water (1:1), RT

As shown in Figure 2-10, DHICA and its three-monobrominated derivatives were synthesized by deprotection of respective DMIC derivatives. DHICA (2-19) was synthesized by saponification of DMIC with sodium hydroxide followed by demethylation. Following similar conditions, 3’- bromo, 4’-bromo and 7’-bromo DMIC were deprotected by saponification with lithium hydroxide and demethylation with boron tribromide. Final products were purified by prep-

HPLC. These deprotections were done in order to access DHICA and mono-brominated derivatives of this natural eumelanin subunit.

Figure 2-11 UV-Vis spectra of 4’-bromo DMIC (2-8) and 4’-bromo DHICA (2-21).

UV-Vis absorption properties of protected (DMIC) and deprotected (DHICA) monomeric eumelanoids were studied. As shown in Figure 2-11 λmax of the protected derivative 4’-bromo

DMIC (2-8) and the deprotected derivative 4’-bromo DHICA (2-21) are 320 nm and 325 nm respectively. Despite this similar UV-absorption behavior, 2-21 exhibits broad absorption (with exponential fit) compared to 2-8 from ~400-650 nm in the visible region. This behavior is due to aggregation of 4’-bromo DHICA molecules and is similar to broad band absorption observed in synthetic eumelanin (derived from non-enzymatic oxidation of DOPA) UV-Vis spectrum where no chromophore is present.160,161

Figure 2-12 UV-Vis absorption spectra of 5,6-dihydroxyindole (DHICA) and its bromo derivatives.

After deprotection of Methyl-5,6-dimethoxyindole carboxylate (DMIC) and its monobrominated derivatives, UV-Vis spectra of DHICA (2-19), 3’-bromo DHICA (2-23), 4’-bromo DHICA (2-

21), and 7'-bromo DHICA (2-25) were recorded. In literature DHICA has been shown to absorb in UV region at ~330 nm and it generates violet chromophore as a result of non-enzymatic oxidation in which shows a broad absorption band in visible 500-550 nm.162 Similar to the literature, Figure 2-12 shows absorption at 330 nm followed by a broad absorption band around

550 nm indicating aggregation of DHICA molecules. Interestingly, the mono-brominated

DHICA derivatives show absorption at 320 nm in UV region with low intensity broad absorbance in the visible region. However, the absence of absorption band at 550 nm indicates inhibition of oxidation in brominated DHICA compounds as the chromophore are not generated.

Figure 2-13 UV-Vis spectra of DHICA (2-19) and 4’-bromo DHICA (2-21) at t=5 mins, 1 hr, and 24 hrs.

As shown in Figure 2-13, at 5 mins and 1 hour, the absorbance of 5,6-DHICA does not show significant change with ~330 nm λmax and absorption ~550 nm due to chromophore generation due to oxidation of DHICA. UV-Vis of 2-19 at 24 hours show the broaderning of the chromophore absorbance and scattering due to precipitation which could also be visibly observed by darkening solution with precipitates present. 4’-bromo DHICA showed similar absorbance behaviour at 5 minutes, 1 hour and 24 hours with maximum absorbance at 320 nm and weak broad absorbance attributed to slow aggregation of 4’-bromo DHICA molecules. This solution was observed to darken very little over the course of 24 hours and showed no precipitation. This indicates that chromophore generation resulting from DHICA oxidation is may be slow as seen by lack of absorption peak ~550 nm.

2.3 Conclusions Methyl-5,6-dimethoxyindole carboxylate (DMIC) and its regioselectively functionalized derivatives were synthesized using Hemetsberger-Knittel indole synthesis. Indole synthesis was done from vanillin or isovanillin by using bromination, methylation, sidechain condensation of azidoacetate, and nitrene insertion in different orders to access DMIC, 3’-bromo DMIC, 4’- bromo DMIC, and 7’-bromo DMIC. These DMIC derivatives were further brominated to access di and tribromo DMIC derivatives. 4’-bromo DMIC was further brominated to access 3,4’- dibromo DMIC. Controlled bromination of DMIC resulted in formation of 3,7’-dibromo DMIC.

Bromination of DMIC with excess NBS was done to synthesize 3,4,7’-tribromo DMIC.

Regioselective debromiation of tribromo DMIC resulted in selective 3’-debromination forming

4,7’-dibromo DMIC. DHICA and Mono-brominated DHICA were synthesized from respective

DMIC derivatives. Deprotection of DMIC, 3’-bromo DMIC, 4’-bromo DMIC, and 7’-bromo

DMIC resulted in formation of DHICA, 3’-bromo-DHICA, 4-bromo DHICA, and 7’-bromo

DHICA respectively. UV-Vis spectra of DHICA and its mono-brominated derivatives indicated that bromination slows down aggregation and chromophore generation via oxidation of DHICA molecules as indicated by absence of broad absorption band at 550 nm.

2.4 Synthesis 2.4.1 General information All reagents were purchased from Sigma-Aldrich and Fisher Scientific and used directly without further purification. Solvents were dried with activated 4 Å molecular sieves or anhydrous solvents were dispensed from Solvent system. Reactions were monitored by TLC or analytical HPLC. TLC analysis was performed with silica gel 60 and visualized with UV light (254 nm and 365 nm). All products were purified with silica-gel flash chromatography or preparative HPLC. Silica gel flash chromatography was performed with SiliaFlash® F60 (Silicycle, 40-60 μm). HPLC was

57 performed on a Gilson® and Agilent® analytical and Gilson® semi-preparative system with UV-

Vis spectrophotometer, using Kinetex 5 µm EVO C18 100Å 4.6 x 150 mm for analytical and

Kinetex 5 µm EVO C18 100Å 21.1 x 150 mm for preparative purpose. Products were characterized by 1H and 13C NMR and mass spectrometry. NMR characterization results and J values were compared with published results. 1H and 13C NMR spectra were recorded on Agilent/Varian 400

MHz or Bruker Neo 500 MHz and 101 MHz NMR spectrometer respectively. 1H NMR multiplicities are defined as ‘s’-singlet, ‘d’-doublet, ‘t’-triplet, ‘dd’-doublet of doublets, and ‘m’- multiplet. ~1 mg of dried sample was sent out to University of Illinois Mass Spectrometry lab where Mass spectra were recorded using a Synapt G2 Q-TOF ESI mass spectrometer. UV-Vis absorption spectra were recorded on the SpectraMax i3x multi-mode detection device.

Abbreviations: TLC-Thin layer chromatography, HPLC-High Performance Liquid

Chromatography, NMR-Nuclear Magnetic Resonance, Q-TOF-Quadrupole time of flight, ESI-

Electrospray ionization

2.4.2 Synthesis of DMIC(2-3) and 3’-bromo DMIC (2-4) 3,4-dimethoxybenzaldehyde (2-1)

To a stirred solution of vanillin (600 mg, 3.94 mmol) in acetone (15 mL) in a scintillation vial was added potassium carbonate (1.089 g, 7.88 mmol, 2.0 eq.) followed by addition of methyl iodide

(1.399 g, 9.85 mmol, 2.5 eq.) at RT. The mixture was heated at 60 °C for 12 hrs. The mixture was cooled to RT and solvent was evaporated under reduced pressure. The crude product was dissolved in ethyl acetate (100 mL) and washed with water, brine and dried over sodium sulfate. The crude

58 product was purified with silica gel flash chromatography (30%:70% EtOAc:Hexanes). Product

1 1 was confirmed with H NMR. Yield: 583 mg, 89% H NMR (400 MHz, CDCl3) δ 9.85 (s, 1H),

7.46 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 7.41 (d, 1H, J = 1.6 Hz), 6.98 (d, 1H, J = 8.0 Hz), 3.97 (s, 3H),

13 3.95 (s, 3H) ppm. C NMR (125 MHz, CDCl3) δ 191.05, 154.66, 149.80, 130.31, 127.02, 110.54,

109.11, 56.33, 56.17 ppm. Spectral data matched with those in literature.163

Methyl (Z)-2-azido-3-(3,4-dimethoxyphenyl)acrylate (2-2)

To a solution of 3,4-dimethoxybenzaldehyde (250 mg, 1.5 mmol), and methyl 2-azidoacetate (693 mg, 6 mmol, 4 eq.) in methanol (0.5 mL) was added sodium Methoxide (1M in methanol, 4.25 mL, 2.5 eq.) at -15 °C. The mixture was stirred at -15 °C for 2 hours and RT for 18 hrs. TLC confirmed the consumption of benzaldehyde and formation of the product. The mixture was washed with water (25 mL) and extracted with diethyl ether (3x25 mL). Combined organic layers were washed with brine and dried over sodium sulfate. Solvent was evaporated and crude was

1 purified with silica gel (10:90 EtOAc:Hex). 328 mg, 83% Yield H NMR (400 MHz, CDCl3) δ

7.51 (d, 1H, J = 2.0 Hz), 7.34 (dd, 1H, J = 2.0 Hz, 8.8 Hz), 6.88 (s, 1H), 6.87 (d, 1H, J = 8.8 Hz),

13 3.92 (s, 3H), 3.91 (s, 3H), 3.90 (s, 3H) ppm. C NMR (125 MHz, CDCl3) δ 164.37, `150.42,

148.75, 126.37, 126.03, 125.06, 123.36, 113.13, 110.90, 56.08, 56.06, 52.94 ppm. Spectral data matched with those in literature.164

Methyl 5,6-dimethoxy-1H-indole-2-carboxylate (DMIC) (2-3)

3,4-dimethoxy7-ene azidoacetate (150 mg, 0.57 mmol) was dissolved in toluene (5 mL). The vial was capped with teflon heat resistant cap and tape, and refluxed for 24 hours. Crude was purified with p rep-HPLC. Solvent was evaporated under reduced pressure yielding the desired product.

1 Yield-94 mg, 71% H NMR (400 MHz, CDCl3) δ 8.88 (N-H), 7.11 (d, 1H, J=2.4 Hz), 7.04 (s,

13 1H), 6.85 (s, 1H), 3.93 (s, 3H), 3.92 (s, 6H), ppm. C NMR (125 MHz, CDCl3) δ 162.22, 150.17,

146.25, 132.00, 125.63, 120.47, 108.84, 102.59, 93.74, 56.16, 51.78 ppm. Spectral data matched with those in literature.165

Methyl 3-bromo-5,6-dimethoxy-1H-indole-2-carboxylate (2-4)

To a scintillation vial equipped with a stir bar was added Methyl 5,6-dimethoxy-1H-indole-2- carboxylate (86 mg, 0.37 mmol), followed by addition of methylene chloride (1.5 mL), N- bromosuccinimide (66 mg, 0.37 mmol, 1 eq.) at RT. The mixture was stirred at RT for 3 hrs.

Yellow precipitate were filtered and dried to get Methyl 3-bromo-5,6-dimethoxy-1H-indole-2-

1 carboxylate. Yield- 106 mg, 92%. H NMR (400 MHz, CDCl3) δ 8.91 (N-H), 6.97 (s, 1H), 6.81

13 (s, 1H), 3.97 (s, 3H), 3.97 (s, 3H), 3.94 (s, 3H) ppm. C NMR (125 MHz, CDCl3) δ 161.43,

151.30, 147.15, 130.50, 122.48, 121.34, 101.05, 98.27, 93.79, 56.35, 56.34, 52.11 ppm. Spectral data matched with those in literature.70

2.4.3 Synthesis of 4’-bromo DMIC (2-8) 2-bromo-3-hydroxy-4-methoxybenzaldehyde (2-5)

To a scintillation vial equipped with a stir bar was added 3-hydroxy 4-methoxybenzaldehyde/ isovanillin (500 mg, 3.29 mmol), followed by addition of methylene chloride (8 mL), N- bromosuccinimide (585 mg, 3.29 mmol, 1 eq.) at RT. The mixture was stirred at RT for 8 hrs.

Solvent was evaporated under reduced pressure and product was recrystallized with hot ethanol

(15 mL). Solid product was filtered. Yield- 688 mg, 91%. Product was confirmed with 1H NMR.

1 H NMR (400 MHz, CDCl3) δ 10.25 (s, 1H), 7.56 (d, 1H, J = 8.4 Hz), 6.91 (d, 1H, J = 8.4 Hz),

13 6.11 (s, 1H), 4.00 (s, 3H) ppm. C NMR (125 MHz, CDCl3) δ 191.02, 151.85, 143.38, 127.44,

122.91, 113.03, 109.44, 56.75 ppm. Spectral data matched with those in literature.166

2-bromo-3,4-dimethoxybenzaldehyde (2-6)

To a stirred solution of 2-bromo 3-hydroxy 4-methoxybenzaldehyde (200 mg, 0.87 mmol) in acetone (5 mL) in a scintillation vial was added potassium carbonate (240 mg, 1.74 mmol, 2.0 eq.) followed by addition of methyl iodide (492 mg, 3.46 mmol, 4.0 eq.) at RT. The mixture was heated at 60 °C for 16 hrs. The mixture was cooled to RT and solvent was evaporated under reduced pressure. The crude product was dissolved in ethyl acetate (40 mL), washed with water, brine, and dried over sodium sulfate. The crude product was purified with silica gel flash chromatography

(30%:70% EtOAc:Hexanes). Product was confirmed with 1H NMR. Yield: 178 mg, 84% 1H NMR

(400 MHz, CDCl3) δ 10.24 (s, 1H), 7.73 (d, 1H, J = 8.8 Hz), 6.95 (d, 1H, J = 8.4 Hz), 3.95 (s,

13 3H), 3.87 (s, 3H) ppm. C NMR (125 MHz, CDCl3) δ 191.12, 158.81, 146.54, 127.54, 126.65,

123.29, 111.08, 60.82, 56.46 ppm. Spectral data matched with those in literature.167

Methyl (Z)-2-azido-3-(2-bromo-3,4-dimethoxyphenyl)acrylate (2-7)

To a solution of 2-bromo 3,4-dimethoxy benzaldehyde (100 mg, 0.41 mmol), and methyl 2- azidoacetate (188 mg, 1.63 mmol, 4 eq.) in methanol (0.5 mL) was added sodium Methoxide (1M in methanol, 1 mL, 2.5 eq.) at -15 °C. The mixture was stirred at -15 °C for 2 hours and RT for 18 hrs. TLC confirmed the consumption of benzaldehyde and formation of the product. The mixture was washed with water (20 mL) and extracted with diethyl ether (3x20 mL). The combined organic layers were washed with brine and dried over sodium sulfate. Solvent was evaporated and crude was purified with silica gel (10:90 EtOAc:Hex). Yield-106 mg, 76% Yield 1H NMR (400 MHz,

CDCl3) δ 7.96 (d, 1H, J = 8.8 Hz), 7.28 (s, 1H), 6.90 (d, 1H, J = 8.8 Hz), 3.93 (s, 3H), 3.92 (s,

13 3H), 3.85 (s, 3H) ppm. C NMR (125 MHz, CDCl3) δ 164.13, 154.29, 146.79, 127.16, 126.27,

125.73, 123.98, 121.70, 119.89, 60.59, 52.73, 50.45 ppm. Spectral data matched with those in literature.70

Methyl 4-bromo-5,6-dimethoxy-1H-indole-2-carboxylate (2-8)

5-bromo 3,4-dimethoxy7-ene azidoacetate (116 mg, 0.34 mmol) was dissolved in toluene (3 mL).

The vial was capped with teflon heat resistant cap and tape, and refluxed for 24 hours. Solvent was evaporated under reduced pressure yielding the desired product. Yield-98 mg, 93% 1H NMR (400

MHz, CDCl3) δ 8.89 (N-H), 7.19 (s, 1H), 6.82 (s, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 3.87 (s, 3H)

13 ppm. C NMR (125 MHz, CDCl3) δ 162.22, 153.65, 143.35, 133.45, 126.48, 122.52, 110.44,

109.65, 93.72, 61.15, 56.38, 52.13 ppm. Spectral data matched with those in literature.70

2.4.4 Synthesis of 7’-bromo DMIC (2-12) 3-bromo-4-hydroxy-5-methoxybenzaldehyde (2-9)

In a scintillation vial equipped with a stir bar to a solution of 4-hydroxy 3-methoxybenzaldehyde/ vanillin (500 mg, 3.29 mmol) in acetic acid (11 mL), was added bromine (578 mg, 3.61 mmol, 1.1 eq.) dropwise at RT. The mixture was stirred at RT for 5 hrs. The mixture was poured into ice- water resulting in product precipitation. Solid product was filtered and dried under vacuum. Yield-

1 677 mg, 89%. H NMR (400 MHz, CDCl3) δ 9.79 (s, 1H), 7.64 (d, 1H, J = 2.0Hz), 7.36 (d, 1H,

13 J = 1.6 Hz), 6.49 (s, 1H), 3.99 (s, 3H) ppm. C NMR (125 MHz, CDCl3) δ 189.79, 149.03,

147.83, 130.24, 130.21, 108.34, 108.18, 56.77 ppm. Spectral data matched with those in literature.168 63

3-bromo-4,5-dimethoxybenzaldehyde (2-10)

To a stirred solution of 3-bromo-4-hydroxy-5-methoxybenzaldehyde (523 mg, 2.26 mmol) in acetone (15 mL) in a scintillation vial was added potassium carbonate (626 mg, 4.53 mmol, 2.0 eq.) followed by addition of methyl iodide (1.287 g, 9.06 mmol, 4.0 eq.) at RT. The reaction mixture was heated at 60 °C for 16 hrs. The mixture was cooled to RT and solvent was evaporated under reduced pressure. The crude product was dissolved in ethyl acetate (120 mL) and washed with water, brine and dried over sodium sulfate. The crude product was purified with silica gel flash chromatography (30%:70% EtOAc:Hexanes)and confirmed with 1H NMR. Yield:439 mg,

1 79% H NMR (400 MHz, CDCl3) δ 9.85 (s, 1H), 7.65 (d, 1H, J = 1.6 Hz), 7.39 (d, 1H, J = 1.6

13 Hz), 3.95 (s, 3H), 3.94 (s, 3H) ppm. C NMR (125 MHz, CDCl3) δ 190.03, 154.35, 151.97,

133.20, 128.95, 118.09, 110.22, 60.99, 56.40 ppm. Spectral data matched with those in literature.169

Methyl (Z)-2-azido-3-(3-bromo-4,5-dimethoxyphenyl)acrylate (2-11)

To a solution of 3-bromo-4,5-dimethoxybenzaldehyde (150 mg, 0.61 mmol), and methyl 2- azidoacetate (282 mg, 2.45 mmol, 4 eq.) in methanol (0.5 mL) was added sodium methoxide (1M

64 in methanol, 1.5 mL, 2.5 eq.) at -15 °C. The mixture was stirred at -15 °C for 2 hours and RT for

18 hrs. TLC confirmed the consumption of benzaldehyde and formation of the product. The mixture was washed with water (25 mL) and extracted with diethyl ether (3x25 mL). The combined organic layers were washed with brine and dried over sodium sulfate. Solvent was evaporated and crude was purified with silica gel (10:90 EtOAc:Hex). Yield- 154 mg, 74% Yield 1H NMR (400

MHz, CDCl3) δ 7.58 (d, 1H, J = 2.0 Hz), 7.39 (d, 1H, J = 1.6 Hz), 6.76 (s, 1H), 3.91 (s, 3H), 3.90

13 (s, 3H), 3.89 (s, 3H) ppm. C NMR (125 MHz, CDCl3) δ 163.93, 153.40, 147.53, 127.65, 125.67,

123.81, 117.68, 113.71, 60.88, 56.33, 53.15 ppm. Spectral data matched with those in literature.70

Methyl 7-bromo-5,6-dimethoxy-1H-indole-2-carboxylate (2-12)

Methyl(Z)-2-azido-3-(3-bromo-4,5-dimethoxyphenyl)acrylate(116 mg, 0.34 mmol) was dissolved in toluene (3 mL). The vial was capped with teflon heat resistant cap and tape, and refluxed for 24 hours. Solvent was evaporated under reduced pressure. The crude product was purified with silica gel flash chromatography (30:70 EtOAc:Hex) to get the desired product. Desired product: Methyl

7-bromo-5,6-dimethoxy-1H-indole-2-carboxylate.Yield- 63 mg, 60%1H NMR (500 MHz,

CDCl3) δ 8.82 (N-H), 7.18 (d, 1H, J=2.5 Hz), 7.06 (s, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 3.91 (s, 3H)

13 ppm. C NMR (125 MHz, CDCl3) δ 162.05, 145.92, 139.32, 131.17, 128.17, 125.87, 120.83,

111.93, 108.46, 61.47, 61.21, 52.26 ppm. Side-product: Methyl 5-bromo-6,7-dimethoxy-1H-

1 indole-2-carboxylate. Yield- 31 mg, 30% H NMR (500 MHz, CDCl3) δ 9.01 (N-H), 7.61 (s, 1H),

7.09 (d, 1H, J=2.5 Hz), 3.94 (s, 3H), 3.92 (s, 3H), 3.92 (s, 3H) ppm. Spectral data matched with those in literature.70

2.4.5 Synthesis of di and tri-brominated DMIC (2-14 to 2-17) Synthesis of 3’4’-dibromo DMICA (2-14)

To a scintillation vial equipped with a stir bar was added methyl 4-bromo-5,6-dimethoxy-1H- indole-2-carboxylate (40 mg, 0.12 mmol), followed by addition of acetonitrile (1.2 mL), N- bromosuccinimide (66 mg, 0.12 mmol, 1 eq.) at RT. The mixture was stirred at 65 °C for 5 hrs.

Solvent was evaporated and crude was purified by silica gel flash chromatography. Yield-41 mg,

1 88%. H NMR (500 MHz, CDCl3) δ 8.98 (N-H), 6.78 (s, 1H), 3.97 (s, 3H), 3.91 (s, 3H), 3.86 (s,

13 3H) ppm. C NMR (125 MHz, CDCl3) δ 161.23, 154.05, 144.16, 133.33, 124.21, 118.10,

111.06, 98.31, 93.49, 60.90, 56.33, 52.31 ppm. HRMS (TOF MS ES+) m/z calculated for

+ C12H12NO4Br2 ([M+H] ) 391.9126 Da, found 391.9133 Da.

Synthesis of 3’-7’-dibromo DMICA (2-15)

To a scintillation vial equipped with a stir bar was added methyl 5,6-dimethoxy-1H-indole-2- carboxylate (39 mg, 0.16 mmol), followed by addition of acetonitrile (1.5 mL), N- bromosuccinimide (116 mg, 0.64 mmol, 4 eq.) at RT. The mixture was stirred at 65 °C for 3 hrs.

Solvent was evaporated and crude was purified by silica gel flash chromatography. Yield-50 mg,

1 81%. H NMR (500 MHz, CDCl3) δ 8.91 (N-H), 7.00 (s, 1H), 3.99 (s, 3H), 3.96 (s, 3H), 3.92 (s,

13 3H) ppm. C NMR (125 MHz, CDCl3) δ 160.91, 150.64, 147.84, 129.49, 124.44, 123.83,

101.37, 100.28, 100.28, 98.55, 661.39, 56.57, 52.38 ppm. HRMS (TOF MS ES+) m/z

+ calculated for C12H12NO4Br2 ([M+H] ) 391.9132 Da, found 391.9133 Da.

Synthesis of 3’-4’-7’-tribromo DMICA (2-16)

To a scintillation vial equipped with a stir bar was added methyl 5,6-dimethoxy-1H-indole-2- carboxylate (100 mg, 0.42 mmol), followed by addition of acetonitrile (4 mL), N- bromosuccinimide (303 mg, 1.70 mmol, 4 eq.) at RT. The mixture was stirred at RT for 20 hrs.

Solvent was evaporated under reduced pressure. Crude was purified with silica gel flash chromatography (30:70-EtOAc:Hex). Yield-158 mg, 80%. Product was confirmed with 1H

1 NMR. H NMR (500 MHz, CDCl3) δ 9.05 (N-H), 4.01 (s, 3H), 3.98 (s, 3H), 3.90 (s, 3H) ppm.

13 C NMR (125 MHz, CDCl3) δ 160.54, 150.87, 147.79, 131.86, 125.93, 120.43, 110.27, 98.99,

98.70, 61.60, 61.15, 52.44 ppm. HRMS (TOF MS ES+) m/z calculated for C12H12NO4Br2

([M+H]+) 469.9295 Da, found 469.9272 Da.

Synthesis of 4’-7’-dibromo dimethoxyindole carboxylate (2-17)

In a scintillation vial methyl 3,4,7-tribromo-5,6-dimethoxy-1H-indole-2-carboxylate (46 mg,

0.098 mmol) was dissolved in acetic acid (1 mL) followed by addition of lithium bromide (18 mg, 0.21 mmol, 2.1 eq.), m-dimethoxybenzene (29 mg, 0.21 mmol, 2.1 eq.), and sulfuric acid

(20.5 mg, 0.21mmol, 2.1 eq.). The mixture was heated at 65 °C for 1.5 hrs. After cooling, the solution mixture was added to ice water and resulting precipitate were filtered out. The crude product was purified with silica gel flash chromatography (30:70-EtOAc:Hex). Yield-26 mg,

1 68% H NMR (500 MHz, CDCl3) δ 8.96 (N-H), 7.27 (d, 1H, J=2.0 Hz), 3.97 (s, 6H), 3.92 (s,

13 3H) ppm. C NMR (125 MHz, CDCl3) δ 161.80, 151.23, 143.67, 134.00, 131.51, 127.17,

123.15, 122.56, 60.53, 56.65, 51.28 ppm. HRMS (TOF MS ES+) m/z calculated for

+ C12H12NO4Br2 ([M+H] ) 391.9133 Da, found 391.9123 Da.

2.4.6 Synthesis of DHICA (2-19) and its mono-brominated derivatives (2-21, 2-23, 2-25) 5,6-dimethoxy-1H-indole-2-carboxylic acid (2-18)

To a solution of 5,6-dimethoxyindole carboxylate (20 mg, 0.085 mmol) was added sodium hydroxide (5 eq.) and water:ethanol (1:1, 10 mL). The reaction mixture was stirred at RT for 8 hrs. Solvent was evaporated and pH was adjusted to ~2-3 with 1 N HCl. The crude product was extracted with ethyl acetate (3 x 10 mL). Organic layer was dried with sodium sulfate and

1 solvent was evaporated. Yield: 18 mg, 95% H NMR (500 MHz, CD3CN) δ 9.70(s, N-H), 7.08

13 (s, 1H), 7.05(s, 1H), 6.95 (s, 1H), 3.85 (s, 3H), 3.81 (s, 3H) ppm. C NMR (125 MHz, CD3OD)

δ 164.92, 151.16, 147.16, 134.25, 127.56, 121.82, 109.59, 104.07, 95.41, 56.71, 56.71 ppm.

+ HRMS (TOF MS ES+) m/z calculated for C12H12NO4Br2 ([M+H] ) 222.0764 Da, found

222.0766 Da.

5,6-dihydroxy-1H-indole-2-carboxylic acid (DHICA) (2-19)

To the solution of 5,6-dimethoxyindole carboxylic acid (22 mg, 0.099 mmol) in DCM (0.2 mL)

o was added BBr3 (0.29 mL, 0.29 mmol, 3 eq) dropwise at -78 C. The mixture was stirred for 18 hours at RT. The mixture was quenched with water and precipitate were filtered. The crude

1 product was purified with prep-HPLC. Yield-12 mg, 62% H NMR (500 MHz, CD3CN) δ 9.77

13 (s, N-H), 7.02 (s, 1H), 6.97 (s, 1H), 6.95 (s, 1H) ppm. C NMR (125 MHz, CD3CN) δ 163.44,

146.55, 142.16, 133.72, 126.46, 121.50, 109.04, 105.92, 97.51 ppm. Spectral data matched with those in literature.70

4-bromo-5,6-dimethoxy-1H-indole-2-carboxylic acid (2-20)

To a solution of 4’-bromo 5,6-dimethoxyindole carboxylate (25 mg, 0.079 mmol) was added

Lithium Hydroxide (9.5 mg, 0.39 mmol, 5 eq.) and water:ethanol (1:1, 8 mL). The reaction mixture was stirred at 50 oC for 8 hours. Solvent was evaporated and pH was adjusted to ~2-3 with 1N HCl. Product was extracted with ethyl acetate (3 x 10 mL). The combined organic layer was dried with sodium sulfate and solvent was evaporated. Yield-21 mg, 90% 1H NMR (500

13 MHz, CD3CN) δ 9.95 (s, N-H), 7.05 (s, 1H), 7.00 (s, 1H), 3.89 (s, 3H), 3.79 (s, 3H) ppm. C

NMR (125 MHz, CD3CN) δ 162.26, 154.27, 143.90, 134.78, 127.81, 122.72, 109.90, 109.24,

+ 95.20, 61.16, 56.79 ppm. HRMS (TOF MS ES+) m/z calculated for C12H12NO4Br2 ([M+H] )

299.9867 Da, found 299.9871 Da.

4-bromo-5,6-dihydroxy-1H-indole-2-carboxylic acid (2-21)

To the solution of 4’-bromo-5,6-dimethoxyindole carboxylic acid (14 mg, 0.046 mmol) in

o DCM (0.2 mL) was added BBr3 (0.14 mL, 0.14 mmol, 3 eq) dropwise at -78 C. The mixture was stirred for 20 hours at RT. The mixture was quenched with water followed by filtration of the precipitate. The product crude was purified with prep-HPLC. Yield-10 mg, 80% 1H NMR

13 (500 MHz, CD3CN) δ 9.74 (s, N-H), 6.98 (s, 1H), 6.94 (s, 1H) ppm. C NMR (125 MHz,

CD3CN) δ 161.92, 146.17, 139.75, 132.50, 126.77, 126.70, 122.41, 108.20, 96.93 ppm. HRMS

- (TOF MS ES-) m/z calculated for C12H12NO4Br2 ([M-H] ) 269.9397 Da, found 269.9402 Da.

3-bromo-5,6-dimethoxy-1H-indole-2-carboxylic acid (2-22)

To a solution of 3’-bromo 5,6-dimethoxyindole carboxylate (25 mg, 0.079 mmol) was added

Lithium Hydroxide (9.5 mg, 0.39 mmol, 5 eq.) and water:ethanol (1:1, 8 mL). The reaction mixture was stirred at 50 oC for 5 hours. Solvent was evaporated and pH was adjusted to ~2-3 with 1N HCl. The crude product was extracted with ethyl acetate (3 x 10 mL). The combined organic layer was dried with sodium sulfate and solvent was evaporated. Yield-22 mg, 93% 1H

13 NMR (500 MHz, CD3CN) δ 9.94 (s, 1H), 6.95 (s, 2H), 3.86 (s, 6H) ppm. C NMR (125 MHz,

CD3CN) δ 161.52, 152.11, 148.13, 131.83, 123.14, 121.63, 101.28, 97.80, 95.20, 56.52, 56.48

+ ppm. HRMS (TOF MS ES+) m/z calculated for C12H12NO4Br2 ([M+H] ) 299.9866 Da, found

299.9871 Da.

3-bromo-5,6-dihydroxy-1H-indole-2-carboxylic acid (2-23)

To the solution of 3’-bromo-5,6-dimethoxyindole carboxylic acid (18 mg, 0.06 mmol) in DCM

o (0.25 mL) was added BBr3 (0.18 mL, 0.18 mmol, 3 eq) dropwise at -78 C. The mixture was stirred for 20 hours at RT. The mixture was quenched with water and precipitate were filtered.

The crude product was purified with prep-HPLC. Yield-11 mg, 68% 1H NMR (500 MHz,

13 CD3CN) δ 10.13 (s, N-H), 7.01 (s, 1H), 6.96 (s, 1H) ppm. C NMR (125 MHz, CD3CN) δ

163.27, 146.35, 141.96, 133.47, 132.39, 126.31, 121.27, 108.75, 105.66 ppm. HRMS (TOF MS

- ES-) m/z calculated for C12H12NO4Br2 ([M-H] ) 269.9401 Da, found 269.9399 Da.

7-bromo-5,6-dimethoxy-1H-indole-2-carboxylic acid (2-24)

To a solution of 7’-bromo 5,6-dimethoxyindole carboxylate (25 mg, 0.079 mmol) was added

Lithium Hydroxide (9.5 mg, 0.39 mmol, 5 eq.) and water:ethanol (1:1, 8 mL). The reaction mixture was stirred at 50 oC for 6 hours. Solvent was evaporated and pH was adjusted to ~2-3

71 with 1N HCl. The crude product was extracted with ethyl acetate (3 x 10 mL). The combined organic layer was dried with sodium sulfate and solvent was evaporated. Yield-22 mg, 90% 1H

NMR (500 MHz, CD3CN) δ 9.91(s, N-H), 6.99 (s, 1H), 6.96 (s, 1H), 3.85 (s, 3H), 3.82 (s, 3H)

13 ppm. C NMR (125 MHz, CD3CN) δ 160.25, 153.27, 142.80, 133.75, 127.5, 121.62, 109.50,

108.84, 94.70, 60.86, 56.29 ppm. HRMS (TOF MS ES+) m/z calculated for C12H12NO4Br2

([M+H]+) 299.9867 Da, found 299.9871 Da.

7-bromo-5,6-dihydroxy-1H-indole-2-carboxylic acid (2-25)

To the solution of 7’-bromo-5,6-dimethoxyindole carboxylic acid (20 mg, 0.066 mmol) in

o DCM (0.3 mL) was added BBr3 (0.2 mL, 0.20 mmol, 3 eq) dropwise at -78 C. The mixture was stirred for 20 hours at RT. The mixture was quenched with water and product precipitate were filtered. The crude product was purified with prep-HPLC. Yield-13 mg, 74% 1H NMR (500

13 MHz, CD3CN) δ 9.70 (s, N-H), 6.94 (s, 1H), 6.92 (s, 1H) ppm. C NMR (125 MHz, CD3CN) δ

160.55, 145.20, 138.25, 131.86, 126.18, 125.50, 121.47, 108.00, 97.56 ppm. HRMS (TOF MS

- ES-) m/z calculated for C12H12NO4Br2 ([M-H] ) 269.9398 Da, found 269.9401 Da.

3 SYNTHESIS OF PHEOMELANOID MONOMERS- BENZOTHIAZOLES AND BENZOTHIAZINES 3.1 Introduction Pheomelanin is red-yellow pigment, commonly found in hair and skin. It is derived from DOPA or dopamine in presence of cysteine, and as a result, it possesses nitrogen as well as sulfur in its polymeric structure.10,17,20,55,170 Monomeric eumelanin units (DHI and DHICA) (Chpater-2) have been widely studied due to scope and applications of eumelanin and polydopamine. Synthesis and study of pheomelanin has gained significance due to its role in skin cancer and melanoma.171–174 Pheomelanin is prone to photogdegradation in presence of oxygen and is potentially phototoxic. Intermediates of pheomelaninogenesis from cyteinyldopa to pheomelanin need to be further studied in order to understand their biological function and their phototoxic role in diseases.38

Figure 3-1 Pheomelanin monomeric units.

Pheomelanin found in skin and hair contains DOPA sidechain, whereas pheomelanin found in brain as part of neuromelanin structure contains dopamine sidechain. Pheomelanin consists of benzothiazine and its metabolite benzothiazole as shown in Figure 3-1. Synthesis of benzothiazine with DOPA sidechain was done by Donato and Napolitano from 5,S- cysteinyldopa by potassium ferricyanide catalyzed cyclization.38 A unified strategy to synthesize benzothiazole and benzothiazine with potential access to DOPA and dopamine sidechain still needs to be explored. In this chapter, synthetic equivalent of natural analogues of pheomelanin monomeric units benzothiazole and benzothiazine were synthesized with two different 73 sidechains-ethyl ester (CO2Et) and methylene nitrile (CH2CN). Ethyl ester sidechain can potentially be converted to DOPA sidechain by reduction of ester followed by condensation- hydroamination.175 Methylene nitrile sidechain can be converted to dopamine sidechain by hydrogenation with borane-tetrahydrofuran or transition metal catalyzed hydrogenation. This strategy is advantageous as both types of monomeric units of phenomelanin, benzothiazole and benzothiazine can be accessed from a simple starting material like ortho-methoxyaniline.

Figure 3-2 Unified strategy to access monomeric pheomelanin subunits.

Benzothiazole were synthesized by thiocyanation of o-methoxyaniline compounds which results in aminobenzothiazole (X=NH2). Aminobenzothiazole was modified via Sandmeyer reaction to access benzothiazole (X=H) and bromobenzothiazole (X=Br). Benzothiazine were synthesized by halogenation followed by Ullman type thiolation at ortho position to amine, followed by alkylate condensation using different alkylating reagents such as dichloroethane, bromoacetaldehyde and ethyl bromopyruvate.

3.2 Results and Discussion:

Monomeric pheomelanoids such as benzothiazole (BTZL) and benzothiazine (BTZ) derivatives were synthesized using commercially available starting material like 3-methoxyphenyl acetonitrile and 3-methoxy 4-amino benzoic acid. The key intermediate was ortho-methoxyaniline

(compounds 3-6 and 3-8), which were used to synthesize benzothiazole in one-step. In addition, ortho-methoxyaniline was also used for synthesis of ortho-aminothiol compounds via halogenation followed by Ullman coupling.

3.2.1 ortho-methoxyaniline synthesis

One-pot conversion methods to synthesize anilines from phenols via smiles rearrangement are widely known.176–178 These methods utilize harsher conditions such as use of sodium hydride base and dimethylacetamide solvent, which produces exothermic reaction. As shown by Xie et al. anilines can be synthesized from phenols in one-step using smiles rearrangement using milder conditions.179 Homovanillonitrile was synthesized using vanillin by modifying the sidechain using TosMIC in 66% yield after purification.

Scheme 3-1 Synthesis of ortho-methoxyaniline (CH2CN sidechain)

Smiles reaction was attempted using two different smiles reagents, 2-chloropropionamide and bromoacetamide at a few different temperatures (Table 3-1) and reactions were monitored by

TLC. As shown in Scheme 3-2, the mechanism of the reaction involves formation of 3-3 as the

75 nucleophilic oxygen attacks the halogenated carbon of the smiles reagent resulting in nucleophilic substitution.

Scheme 3-2 Smiles rearrangement of homovanillonitrile.

Nitrogen of the terminal amide is activated by deprotonation by base and it attacks the aromatic carbon. The rearrangement proceeds through an unstable cyclic intermediate, and as a result of this ring-opening, 3-4 is formed. Upon hydrolysis, 3-4 would be converted to the desired aniline product. Table 3-1 shows the results of the smiles rearrangement experiments. 3-3 was observed on TLC and NMR initially when the reaction was being run at higher temperatures (148 or 165

°C) for 5 hours with both the smiles reagents. When the temperature and time were optimized to

180 °C for 18 hours, cyclized 3-4 was observed in NMR along with 3-3.

Table 3-1 Smiles rearrangement-reaction conditions and results

Smiles reagent Base, Conditions Recovered solvent, (Temp., Time) product catalyst o Bromoacetamide K2CO3 90 C- 2 hrs 3-3 (Yield- 61%) DMF 148 oC- 5 hrs KI o K2CO3 100 C- 2 hrs 3-3 (Yield- 51%) DMF 165 oC- 5 hrs KI o 2-chloropropionamide K2CO3 100 C- 2 hrs 3-3 (Yield-34%) DMF 165 oC- 5 hrs & Starting KI Material o K2CO3 100 C- 2 hrs 3-4 (Yield-39%) DMF 180 oC- 18 hrs 3-3 (Yield-12%) KI

Due to poor conversion after multiple synthesis attempts and difficulty in purification due to isolation of multiple intermediates, alternate route, which also involved a two-step synthesis of desired ortho-methoxyaniline compound starting from a simple starting material like 3- methoxyphenyl acetonitrile, was used as shown in Scheme 3-1. Nitration was done using tetrabutylammonium nitrate which gave the desired 2(3-methoxy-4-nitrophenyl)acetonitrile product (3-5) in 47% yield. Reaction also produced 2,6 dinitro compound as side products. As described in previous literature, desired product was purified using silica gel flash chromatography.180,181 This nitro compound undergoes palladium on carbon mediated hydrogenation in presence of hydrogen gas to yield the desired o-methoxyaniline product (3-6).

Ortho-methoxyaniline variant of CO2Et sidechain (3-8) was synthesized by ethylating 4-amino-3- methoxybenzoic acid in ethanol by using sulfuric acid as a catalyst.

3.2.2 Benzothiazole(BTZL) synthesis

Once ortho-methoxyaniline compounds (3-6 and 3-8) were accessed, the next step was to synthesize benzothiazole (synthetic analogues of natural phenomelanin subunit BTZL), which include aminobenzothiazole, benzothiazole, and bromobenzothiazole.

Scheme 3-3 Synthesis of benzothiazoles

Scheme 3-3 was followed in order to access these pheomelanoids. Thiocyanation-cyclization is a commonly used protocol to synthesize aminobenzothiazole in one-step.7-10 Thiocyanation occurs at the ortho position to amine, and in presence of bromine in acetic acid nucleophilic nitrogen of amine cyclizes to form aminobenzothiazole, which is a stable compound. This reaction worked well for ethyl ester sidechain (3-9, Yield-92%), and methylene nitrile sidechain (3-12, Yield-95%) and did not require extensive purification steps. Aminobenzothiazole undergoes deamination in presence of isoamyl nitrite in THF resulting in benzothiazole derivative. Deamination were done 78 successfully to synthesize 3-10 (86%) and 3-13 (89%) from 3-9 and 3-12 respectively.

Benzothiazole (3-10 and 3-13) can be brominated using carbon tetrabromide with potassium tert- butoxide to synthesize bromobenzothiazole resulting in 3-11 (83%) and 3-14 (78%). Alternatively, bromobenzothiazole (3-11 and 3-14) were synthesized directly from aminobenzothiazole via copper catalyzed sandemeyer reaction in 75% and 67% yield respectively. Deamination to synthesize benzothiazole and deamination-bromination to synthesize bromobenzothiazole in one- step from aminobenzothiazole follow Sandmeyer mechanism as discussed in Figure 3-3.

Figure 3-3 Sandmeyer reaction mechanism Sodium nitrite, tert-butyl ammonium nitrite and isoamyl nitrite are used for diazotization of aromatic amines as they generate nitrosonium, which goes under nucleophilic attack, and generate diazonium salts.182 In Sandmeyer mechanism, nucleophile can be altered in order to substitute the aromatic amine with different nucleophilic functional groups. In the case above, when bromination is done on diazonium salt, nitrogen leaves and bromobenzothiazole is recovered as the product

79 whereas if hydrogen adds to the electrophilic carbon of the thiazole, benzothiazole is generated

(Figure 3-3).

3.2.3 Synthesis of key intermediate: ortho-aminothiophenol

The key intermediate to access 1,4-benzothiazine subunits of pheomelanin was ortho- aminothiophenol which could later be alkylated to make benzothiazines. Aminobenzothiazole (3-

7, 3-9 and 3-12) could be synthesized from ortho-methoxyaniline compound in one-step.

Aminobenzothiazole could be reduced in alkaline condition at high temperature in order to get ortho-aminothiophenol. Aminobenzothiazole compound with methyl ester sidechain (3-7) was used as a model to assess the reaction conditions. Upon reduction with sodium or potassium hydroxide, the starting material reduced to the dimer of the desired ortho-aminothiophenol

(Scheme 3-4). In addition to challenges in purification, demethylation occurred at the sidechain as acid product was observed in 1H NMR. Several reaction conditions were attempted by varying base concentration, temperature, and time, in order to optimize reaction conditions. However, the same product was observed in each case. The dimer was reduced to the thiol monomer by breaking the disulfide bond using lithium aluminum hydride, which gave the ortho-aminothiophenol compound with sidechain acid reduced to primary alcohol. Reduction of ethyl ester (3-9) and methylene nitrile (3-12) benzothiazole under similar condition did not yield in formation of desired product due to degradation and purification challenges.

Scheme 3-4 Reduction of aminobenzothiazole.

Due to undesired modification of sidechain while using harsh reducing conditions with aminobenzothiazole and challenges in purification, an alternate pathway was explored in order to get the ortho-aminothiophenol. This pathway involved protection of amine followed by thiocyanation at the ortho position, and reduction and deprotection to get the nucleophilic functionalities as shown in Scheme 3-5. Three different protecting groups were used in these experiments- Boc (di-tert-butyl dicarbonate), Azide (N3), and trityl (triphenylmethyl).

Scheme 3-5 Protection-thiocyanation-reduction scheme for ortho-aminothiophenol synthesis. (Pg-Protecting group)

Table 3-2 Thiocyanation of protected ortho-methoxyaniline.

Aromatic amine was protected with the protecting groups mentioned above and the formation of these products was confirmed with NMR and Infrared spectroscopy. Thiocyanation conditions were similar to benzothiazole synthesis conditions, except in this case ammonium thiocyanate was used instead of potassium thiocyanate. Reactions were ran under ice-bath to control thiocyanation and to prevent cyclization. Table 3-2 shows the results of thiocyanation reactions. With no protecting group present, under these reaction conditions, benzothiazole formed. With Boc- protected amine, thiocyanation was incomplete and major product recovered was the deprotected o-methoxyaniline compound. With azide protected amine, thiocyanation did not work as the starting material was recovered. With trityl protected amine, thiocyanation resulted in deprotection and thiocyanation at the desired ortho position of amine. However, cyclization occurred resulting in benzothiazole formation, as it was recovered as the major product.

Since the pathway discussed above to protect, thiocyanate, and reduce the aniline failed to give the expected outcomes, thiolation via Ullman type coupling was pursued.

Scheme 3-6 Halogenation-Ullman type thiolation. ortho-methoxyaniline compound with ethyl ester sidechain (3-8) was iodinated using iodine monochloride at the ortho position to amine selectively. Following iodination, desired thiol functionality was installed using copper (II) catalyzed single step aryl thiol synthesis via C-S cross coupling using 1,2-ethanedithiol as explored by Liu et al.183 The mechanism of this reaction involves two steps. First step is the catalysis step where halogenated compound undergoes C-S coupling with 1,2-ethanedithiol via copper catalyzed Ullman type mechanism.183

Figure 3-4 Mechanism of thiolation via Ullman type C-S coupling.

As shown in Figure 3-4, thiol nucleophile coordinates to the metal, followed by oxidative addition of aryl halide to the metal complex. Aryl is attached to sulfur of 1,2-ethanedithiol as it reductively eliminates following C-S coupling, as the intermediate shown in Figure 3-4 is generated. The second step of this mechanism is intramolecular sulfide cleavage, as the terminal nucleophilic sulfur of 1,2-dithioethane moiety attacks the neighboring carbon of the aryl attached sulfur forming tricyclic thioether. This thioether is eliminated, and aromatic thiol is generated upon acid workup.

Ullman type thiol installation was done on iodinated and brominated ortho-methoxy aniline compounds. Iodination was successfully done to synthesize 3-21 (78%), 3-22 (75%), and 3-23

(60%). Bromination was done with NBS at ortho-position of amine to synthesize 3-24 (84%), 3-

25 (79%), and 3-26 (81%). Thiolation reactions were carried out on methyl ester (CO2Me) 3-21

84 and 3-24 to assess conditions. However, Ethyl-ester sidechain was later used to optimize the reaction condition due to deesterification observed in compounds with methyl-ester sidechain.

Triphenylphosphine and tributylphosphine were used to reduce the dimer to get the o-aminothiol compound. However, these protocols required an extra purification step and as a result, TCEP was used to reduce the dimer. Ullman type thiolation was done successfully to synthesize 3-27 in overall yield of 54% from ortho-methoxyaniline compound (3-8), and 3-29 was synthesized with overall 47% yield from. 3-6.

3.2.4 Alkylate condensation: The key intermediate ortho-aminothiophenol was synthesized using Ullman type coupling, which was followed by alkylate condensation of different alkylating agents to synthesize monomeric benzothiazines. Synthesis scheme of 1,4-benzothiazine ethyl ester derivatives is shown in Scheme

3-7. Dihydrobenzothiazine (3-30, Yield-71%) was synthesized by condensing dichloroethane on o-aminothiophenol compound (3-27) in DMF. The aliphatic protons of benzothiazine (CH2-CH2) and their splitting were characteristic of the desired dihydrobenzothiazine product. Benzothiazine

(3-31, Yield-59%) was synthesized by condensing bromoacetaldehyde on ortho-aminothiophenol compound (3-27). In the first step, thiophenolate is generated by reaction with sodium hydride.

After addition of bromoacetaldehyde, amino aldehyde is condensed in situ to yield the desired cyclic imine.184

Scheme 3-7 Synthesis of benzothiazines by alkylate condensation.

When this reaction was performed with ortho-aminothiophenol compounds with methylene nitrile sidechain (3-29), isomer of benzothiazine (3-33) was observed in 46% yield. Following similar reaction mechanism, benzothiazine carboxylate (3-32, Yield-66%) was synthesized by condensing ethylbromopyruvate on ortho-aminothiophenol (3-27). Crude was purified with prep-HPLC and products were characterized using NMR. Presence of aliphatic CH2 and proton of ethyl ester sidechain with relevant chemical shift and integration confirmed the formation of benzothiazine carboxylate products. Condensation reactions of 3-29 with dibromoethane and ethyl bromopyruvate were attempted using similar conditions, but product could not be confirmed with

1H or 13C NMR. These reactions could be optimized and scaled to yield these benzothiazine products in a future work. Thus, pheomelanin subunits-benzothiazines (BTZ) and benzothiazloles

(BTZL) were synthesized. Benzothiazoles were synthesized using thiocyanation-cyclization and

86 sandmeyer reaction. Benzothiazines were synthesized using Ullman type thiolation and alkylate condensation reactions. These synthetic pheomelanin subunits could be deprotected to better represent their natural analogues to extend this work.

3.3 Conclusion and future direction Synthetic analogues of benzothiazole and benzothiazine (pheomelanin subunits) derivatives were synthesized from ortho-methoxyaniline derivatives with two different sidechains (CO2Et and

CH2CN) for the first time. Synthesized analogues included aminobenzothiazole compounds with

CO2Et and CH2CN sidechain in 92% and 95% yield, benzothiazole compounds with CO2Et and

CH2CN sidechain in 86% and 89% yield. Bromobenzothiazole compounds with CO2Et and

CH2CN sidechain were synthesized via bromination of benzothiazole with 83% and 78% yield respectively and via Sandmeyer reaction of aminobenzothiazole compounds with 75% and 67% yield, respectively. To synthesize benzothiazine compounds, which are synthetic analogues of

1,4-benzothiazine subunit of pheomelanin, ortho-aminothiol compounds with CO2Et and CH2CN sidechain were synthesized as key intermediates via Ullman type thiolation of ortho-brominated or ortho-iodinated aniline compounds in 69% and 58% yield. Synthetic analogues of 1,4- benzothiazine- dihydrobenzothiazine (71% yield) was synthesized via alkylative condensation of dichloroethane, benzothiazine with CO2Et and CH2CN sidechain were synthesized via alkylate condensation of bromo-acetaldehyde in 59% yield 41% respectively; and benzothiazine dicarboxylate (66% yield) via alkylate condensation of ethyl bromopyruvate.

Ullman type thiolation reaction conditions could be optimized further in future to increase the yield and efficiency. In addition, it may include modifying sidechains of pheomelanin to access

DOPA sidechain to access natural analogues of 1,4-benzothiazine and benzothiazole which are key intermediates of pheomelanogenesis. Degradation experiments followed by HPLC analysis

87 via reductive hydrolysis or oxidative degradation using HI or H2O2 could be performed on synthesized natural analogues to analyze degradation products of melanogenesis.

3.4 Synthesis 3.4.1 General synthesis procedure All reagents were purchased from Sigma-Aldrich and Fisher Scientific and used directly without further purification. Solvents were dried with activated 4 Å molecular sieves or anhydrous solvents were dispensed from Solvent system. Reactions were monitored by TLC or analytical HPLC. All products were purified with silica-gel flash chromatography or preparative HPLC. HPLC was performed on a Gilson analytical to semi-preparative system using Kinetex 5 µm EVO C18 100Å

4.6 x 150 mm for analytical and Kinetex 5 µm EVO C18 100Å 21.1 x 150 mm for preparative purpose. Products were characterized by 1H and 13C NMR and mass spectrometry. NMR characterization results and J values were compared with published results. 1H and 13C NMR spectra were recorded on Agilent/Varian 400 MHz or Bruker Neo 500 MHz and 101 MHz NMR spectrometer, respectively. 1H NMR multiplicities are defined as ‘s’-singlet, ‘d’-doublet, ‘t’- triplet, ‘dd’-doublet of doublets, and ‘m’-multiplet. Mass spectra were recorded using a Synapt G2

Q-TOF ESI mass spectrometer at the Mass Spectrometry Lab at the University of Illinois Urbana

Champagne. Abbreviations: TLC-Thin layer chromatography, HPLC-High Performance Liquid

Chromatography, NMR-Nuclear Magnetic Resonance, Q-TOF-Quadrupole time of flight, ESI-

Electrospray ionization

3.4.2 Synthesis of ortho-methoxy aniline compound 2-(4-hydroxy-3-methoxyphenyl) acetonitrile (3-1)

To the mixture of potassium tert-butoxide (2.24 g, 10 mmol) in THF (15 mL), solution of toluenesulfonylmethyl isocyanide-TosMIC (1.95 g, 10 mmol) in THF (5 mL) was added at -78

°C. Mixture was stirred for 15 minutes followed by addition of vanillin (761 mg, 5 mmol) in

THF (5 mL) dropwise at -78 °C. The mixture was stirred at -78 °C for 2 hours and warmed to rt.

Methanol (5 mL) was added and the mixture was heated at 66 °C for 1.5 hours. Solvent was evaporated under reduced pressure. Water (20 mL) was added to the crude and the crude product was extracted with ethyl acetate (3x20 mL). The combined organic layer was dried with sodium sulfate and evaporated under reduced pressure. The crude product was purified with silica gel

1 flash chromatography (40:60 EtoAc: Hex). 567 mg, 66%. H NMR (400 MHz, CDCl3) δ 6.84

(d, 1H, J=2.0 Hz), 6.82 (d, 1H, J=8.4 Hz), 6.79 (dd, 1H, J=2.0 Hz, 8.4 Hz), 3.85 (s, 3H), 3.62 (s,

13 2H). C NMR (101 MHz, CDCl3) δ 146.63, 146.26, 123.03, 119.76, 118.45, 114.51, 111.29,

56.26, 23.18 ppm. Spectral data matched with those in literature. 185

2-(4-amino-3-methoxyphenyl) acetonitrile synthesis via Smiles rearrangement

General procedure: Cyanovanillin (55 mg, 0.32 mmol) was dissolved in DMF (1.5 mL). Smiles reagent (1.2 eq.) was added to the solution followed by addition of potassium carbonate (2.5 eq.) and potassium iodide (0.2 eq.). The mixture was stirred at variable temperature and time (refer to the Table 3-1-results and discussion). Solvent was evaporated under reduced pressure and crude was purified with silica gel flash chromatography (70:30 EtOAc:Hex).

2-(3-methoxy-4-nitrophenyl) acetonitrile (3-5)

To a solution of tetra-n-butyl ammonium nitrate (1.97 g, 6.45 mmol) and 18-crown-6 (27 mg,

0.102 mmol) in dry DCM (40 mL) cooled in ice-bath was added trifluoroacetic anhydride (2.2 mL) dropwise at 0°C. The solution was stirred in ice bath for 30 minutes and slowly syringed to a solution of 3-methoxyphenyl acetonitrile (1 g, 6.79 mmol) in DCM (70 mL). The mixture was stirred at rt overnight. The crude product solution was washed with sodium bicarbonate (100 mL) and extracted with DCM (3 x 100 mL). The combined organic extracts were dried over sodium sulfate and solvent was evaporated under reduced pressure. Crude was purified with silica-gel flash

1 chromatography (30:70 ethyl acetate:hexanes). 613 mg, 47%. H NMR (500 MHz, CDCl3) δ 7.86

(d, 1H,8 Hz), 7.07 (s,1H), 6.99 (d, 1H, J=8.5 Hz), 3.99 (s, 3H), 3.83 (s, 2H) ppm. 13C NMR (125

MHz, CDCl3) δ 153.62, 136.72, 132.23, 126.74, 120.80, 119.86, 113.13, 56.86, 23.99 ppm.

Spectral data matched with those in literature.180

2-(3-methoxy-4-aminophenyl) acetonitrile (3-6)

To a 20 mL scintillation vial with a septum were added 2-(3-methoxy-4-nitrophenyl) acetonitrile

(150 mg, 0.78 mmol) and palladium on carbon (75 mg). The vial was evacuated and purged with hydrogen, followed by addition of methanol (3.5 mL). Reaction was stirred under hydrogen for 12

90 hours. The catalyst was filtered and solvent was evaporated under reduced pressure to recover 124

1 mg (98%) 2-(3-methoxy-4-aminophenyl) acetonitrile. 122 mg, 98%. H NMR (500 MHz, CDCl3)

δ 6.65 (s,1H), 6.64 (d, 1H,8 Hz), 6.60 (d, 1H, J=7.5 Hz), 3.79 (s, 3H), 3.57 (s, 2H) ppm. 13C NMR

(125 MHz, CDCl3) δ 147.71, 136.13, 121.46, 120.75, 115.07, 110.29, 110.15, 55.72, 23.39 ppm.

Spectral data matched with those in literature.186

3.4.3 Benzothiazole synthesis Methyl 2-amino-4-methoxybenzo[d]thiazole-6-carboxylate (3-7)

In a 7 mL scintillation vial, potassium thiocyanate (322 mg, 3.31 mmol) and acetic acid (0.5 mL) were added, followed by addition of methyl 4-amino-3-methooxybenzoate (150 mg, 0.83 mmol) in acetic acid (1 mL) dropwise at 0 °C. To this mixture was added bromine (146 mg, 0.91 mmol) in acetic acid (0.5 mL) dropwise at 0 °C. The reaction mixture was stirred at rt for 12 h. The crude product mixture was diluted with water and potassium carbonate was added to adjust the pH ~8.

Solid precipitate were filtered and dried under vacuum overnight. 177 mg, 89%. 1H NMR (400

MHz, d6-DMSO) δ 7.94 (d, 1H, J=1.2 Hz), 7.84 (s, N-H, 2H), 7.34 (d, 1H, J=1.6 Hz), 3.87

(s, 3H), 3.83 (s, 3H). 13C NMR (101 MHz, d6-DMSO) δ 169.32, 166.91, 149.68, 146.85,

132.25, 123.03, 116.30, 108.83, 56.39, 52.69 ppm. Spectral data matched with those in literature.187

Ethyl-4-amino3-methoxybenzoate (3-8)

To a 7 mL scintillation vial with a stir bar was added, 4-amino 3-methoxybenzoic acid (200 mg,

1.20 mmol) followed by addition of ethanol (2.4 mL) and sulfuric acid (0.10 mL). The reaction mixture was stirred at 81 °C for 10 h. After cooling to room temperature, the solvent was evaporated under reduced pressure. Saturated sodium bicarbonate (20 mL) was added to the crude and product was extracted with ethyl acetate (2*20 mL). The organic layer was washed with brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure. 220

1 mg, 94%. H NMR (500 MHz, CDCl3) δ 7.56 (dd, 1H, J=1.6, 6.4 Hz), 7.46 (d, 1H, J=1.2

Hz), 6.66 (d, 1H, J=6.4 Hz), 4.33 (q, 2H, J=6 Hz), 3.90 (s, 3H), 1.37 (t, 3H, J=6 Hz) ppm.

13 C NMR (125 MHz, CDCl3) δ 166.96, 146.20, 141.10, 124.05, 119.90, 113.18, 111.20,

60.48, 55.63, 14.51 ppm. Spectral data matched with those in literature.188

Ethyl 2-amino 4-methoxybenzothiazole 6-carboxylate (3-9)

In a 7 mL scintillation vial, potassium thiocyanate (364 mg, 3.75 mmol) and acetic acid (0.6 mL) were added, followed by addition of ethyl 4-amino-3-methooxybenzoate (183 mg, 0.94 mmol) in acetic acid (1 mL) dropwise at 0 °C. To this mixture was added bromine (165 mg, 1.03 mmol) in acetic acid (0.5 mL) dropwise at 0 °C. The reaction mixture was stirred at rt for 12 h. The crude

92 product mixture was diluted with water and potassium carbonate was added to adjust the pH ~8.

Solid precipitate were filtered and dried under vacuum overnight. 220 mg, 92%. 1H NMR (500

MHz, CDCl3) δ 7.97 (d, 1H, J=1.0 Hz), 7.51 (d, 1H, J=1.5 Hz), 5.34 (s, N-H, 2H), 4.40

13 (q, 2H, J=7.5 Hz), 4.03 (s, 3H), 1.41 (t, 3H, J=7.0 Hz). C NMR (125 MHz, CDCl3) δ

165.94, 146.23, 139.17, 126.88, 119.73, 115.73, 109.73, 106.63, 60.81, 56.06, 14.42 ppm.

Spectral data matched with those in literature.189

Ethyl 4-methoxybenzothiazole 6-carboxylate (3-10)

In a 7 mL scintillation vial, ethyl 2-amino 4-methoxybenzoate 6-carboxylate (25 mg, 0.098 mmol) and THF (0.5 mL) were added followed by slow addition of isoamyl nitrite (25 mg, 0.21 mmol).

The mixture was stirred at 66 °C for 1 hour. The mixture was cooled and poured into ice-water (8 mL). The aqueous mixture was extracted with ethyl acetate (3 x 8 mL). The organic extracted were combined and washed with brine, dried over sodium sulfate, and concentrated under reduced

1 pressure. 20 mg, 86%. H NMR (500 MHz, CDCl3) δ 9.05 (s, 1H), 8.29 (d, 1H, J=1.0 Hz), 7.60

(d, 1H, J=1.5 Hz), 4.44 (q, 2H, J=7.5 Hz), 4.12 (s, 3H), 1.43 (t, 3H, J=7.0 Hz). 13C NMR (125

MHz, CDCl3) δ 165.85, 152.43, 145.25, 140.56, 138.59, 129.05, 114.97, 108.01, 61.61,

56.34, 14.36 ppm. Spectral data matched with those in literature.189

Ethyl 2-bromo 4-methoxybenzothiazole 6-carboxylate (3-11)

To a solution of ethyl4-methoxybenzothiazole 6-carboxylate (20 mg, 0.084 mmol) in DMF (0.2 mL) were added carbon tetrabromide (30.5 mg, 0.092 mmol) and potassium tert-butoxide (38 mg,

0.336 mmol). The mixture was stirred at rt for 1.5 hours. The reaction mixture was poured into water (5 mL), extracted with DCM (3 x 5 mL), and dried over sodium sulfate. Solvent was evaporated under reduced pressure to get crude product. The crude product was purified with silica-gel flash chromatography (40:60 ethyl acetate:hexanes). 22 mg, 83%

Sandmeyer reaction

In a 7 mL scintillation vial, to a stirred solution of 2-amino 4-methoxybenzothiazole 6-carboxylate

(20 mg, 0.079 mmol) and copper (II) bromide (30 mg, 0.134 mmol) in acetonitrile (0.5 mL) was added isoamyl nitrite (11 mg, 0.095 mmol) dropwise at 0 °C. The mixture was stirred at rt for 3 hours, after which water (4 mL) was added. The precipitate were filtered and dried under vacuum.

1 19 mg, 75%. H NMR (500 MHz, CDCl3) δ 8.13 (d, 1H, J=1.2 Hz), 7.58 (d, 1H, J=1.2 Hz), 4.43

13 (q, 2H, J=5.6 Hz), 4.09 (s, 3H), 1.43 (t, 3H, J=5.6 Hz). C NMR (125 MHz, CDCl3) δ 165.85,

152.43, 145.25, 140.55, 138.59, 129.05, 114.96, 108.00, 61.61, 56.34, 14.36 ppm. HRMS (TOF

MS ES+) m/z calculated ([M+H]+) 315.9643 Da, found 315.9642 Da.

2-(2-amino-4-methoxybenzothiazol-6-yl) acetonitrile (3-12)

In a 7 mL scintillation vial, potassium thiocyanate (134 mg, 1.38 mmol) and acetic acid (0.2 mL) were added, followed by addition of 2-(3-methoxy-4-aminophenyl) acetonitrile (56 mg, 0.35 mmol) in acetic acid (0.3 mL) dropwise at 0 °C. To this mixture was added bromine (62 mg, 0.38 mmol) in acetic acid (0.5 mL) dropwise at 0 °C. The reaction mixture was stirred at rt for 12 hours.

The crude product mixture was diluted with water and potassium carbonate was added to adjust the pH ~8. Solid precipitate were filtered and dried under vacuum overnight. 73 mg, 95%. 1H

NMR (500 MHz, CDCl3) δ 7.01 (d, 1H,1.5 Hz), 6.58 (d, 1H, J=1.5 Hz), 3.76 (s, 2H), 3.61 (s, 3H).

13 C NMR (125 MHz, CDCl3) δ 166.16, 150.14, 141.96, 132.69, 124.74, 119.96, 113.29, 108.89,

56.34, 22.86 ppm. HRMS (TOF MS ES+) m/z calculated ([M+H]+) 220.0545 Da found 220.0542

Da.

2-(4-methoxybenzothiazol-6-yl) acetonitrile (3-13)

In a 7 mL scintillation vial, 2-(2-amino-4-methoxybenzothiazol-6-yl) acetonitrile (40 mg, 0.18 mmol) and THF (0.9 mL) were added followed by slow addition of isoamyl nitrite (47 mg, 0.40

95 mmol). The mixture was stirred at 66 °C for 1 hour. Mixture was cooled and poured into ice-water

(15 mL). The aqueous mixture was extracted with ethyl acetate (3 x 15 mL). The organic extracted were combined and washed with brine, dried over sodium sulfate, and concentrated under reduced

1 pressure. 33 mg , 89%. H NMR (500 MHz, CDCl3) δ 8.93 (s, 1H), 7.53 (d, 1H, J=1.5 Hz), 6.85

13 (d, 1H, J=1.0 Hz), 4.08 (s, 3H), 3.90 (s, 2H). C NMR (125 MHz, CDCl3) δ 154.24, 153.27,

143.52, 136.33, 128.84, 117.69, 113.20, 106.74, 56.37, 24.20 ppm. HRMS (TOF MS ES+) m/z calculated ([M+H]+) 205.0436 Da found 205.0433 Da.

2-(2-bromo-4-methoxybenzothiazol-6-yl) acetonitrile (3-14)

To a solution of 2-(4-methoxybenzothiazol-6-yl) acetonitrile (17 mg, 0.084 mmol) in DMF (0.2 mL) were added carbon tetrabromide (30.5 mg, 0.092 mmol) and potassium tert-butoxide (38 mg,

0.336 mmol). Mixture was stirred at rt for 1.5 hours. The reaction mixture was poured into water

(5 mL), extracted with DCM (3 x 5 mL), and dried over sodium sulfate. Solvent was evaporated under reduced pressure to get crude product. The crude product was purified with silica-gel flash chromatography (40:60 ethyl acetate: hexanes). 19 mg, 78%

In a 7 mL scintillation vial, to a stirred solution of 2-(2-amino-4-methoxybenzothiazol-6-yl) acetonitrile (46 mg, 0.208 mmol) and copper (II) bromide (79 mg, 0.35 mmol) in acetonitrile (1.5

96 mL) was added isoamyl nitrite (29 mg, 0.25 mmol) dropwise at 0 °C. The mixture was stirred at rt for 3 hours, after which water (4 mL) was added. The precipitate were filtered and dried under

1 vacuum. 40 mg, 67%. H NMR (500 MHz, CDCl3) δ 7.38 (d, 1H, J=1.5 Hz), 6.83 (d, 1H, J=1.0

13 Hz), 4.06 (s, 3H), 3.87 (s, 2H) ppm. C NMR (125 MHz, CDCl3) δ 153.13, 142.36, 139.64,

138.14, 129.12, 112.20, 109.10, 107.39, 56.43, 24.17 ppm. HRMS (TOF MS ES+) m/z calculated

([M+H]+) ([M+H]+) 282.9542 Da found 282.9541 Da.

3.4.4 Synthesis of ortho-aminothiophenol intermediate 4-amino-3-mercapto-5-methoxybenzoic acid (3-17)

Methyl 2-amino-4-methoxybenzo[d]thiazole-6-carboxylate (120 mg, 0.50 mmol) was added to a solution of sodium hydroxide (or potassium hydroxide) (10 mmol, 20 eq.) in water (1.5 mL). The mixture was heated and stirred at 110-140 °C for 2-4 days (Multiple trials). The mixture was poured into ice-water (12 mL), acidified with concentrated HCl to pH 4-5 and extracted with ethyl acetate. The organic layer was washed with water and dried over sodium sulfate. Solvent was evaporated under reduced pressure. 32 mg, 30%. 1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H),

7.28 (d, 1H, J=1.6 Hz), 7.24 (d, 1H, J=1.6 Hz), 5.86 (s, 2H, N-H), 3.84 (s, 3H) ppm. 13C NMR

(101 MHz, DMSO-d6) δ 167.40, 146.08, 144.50, 130.89, 117.41, 115.08, 112.33, 56.40 ppm.

To a solution of 5,5'-disulfanediylbis(4-amino-3-methoxybenzoic acid) (28 mg, 0.07 mmol) in water:acetonitrile (1:1, 5 mL) was added TCEP (tris(2-carboxyethyl)phosphine) (36 mg, 0.126 mmol). Mixture was stirred at room temperature for 3 hours. Solvent was evaporated under

1 reduced pressure to get the desired thiol. 26 mg, 88%. H NMR (400 MHz, CDCl3) δ 7.88 (d, 1H,

J=1.6 Hz), 7.41 (d, 1H, J=1.6 Hz), 4.75 (s, 2H, N-H), 3.92 (s, 3H) ppm. 13C NMR (101 MHz,

DMSO-d6) δ 167.40, 146.08, 144.50, 130.89, 117.41, 115.08, 112.33, 56.40 ppm. Spectral data matched with those in literature.190

Methyl 4-amino-3-methoxy-5-thiocyanatobenzoate

To a mixture of methyl 4-amino-3-methoxy benzoate (75 mg, 0.41 mmol) in methanol (2 mL) were added ammonium thiocyanate (76 mg, 0.99 mmol). The mixture was cooled to 5 °C and bromine (99 mg, 0.62 mmol) in methanol (1 mL) wad added dropwise. The mixture was stirred at

5 °C for 3 hours and at room temperature for 12 hours. Solid was filtered and washed with water.

Aminobenzothiazole product was observed.77 mg, 79%. 1H NMR (400 MHz, d6-DMSO) δ

7.94 (d, 1H, J=1.2 Hz), 7.84 (s, N-H, 2H), 7.34 (d, 1H, J=1.6 Hz), 3.87 (s, 3H), 3.83 (s,

3H). 13C NMR (101 MHz, d6-DMSO) δ 169.32, 166.91, 149.68, 146.85, 132.25, 123.03,

116.30, 108.83, 56.39, 52.69 ppm. Spectral data matched with those in literature. 187

Methyl 4-((tert-butoxycarbonyl) amino)-3-methoxybenzoate (3-18)

In a 7 mL scintillation vial, to a solution of methyl 4-amino 3-methoxy benzoate (196 mg, 0.82 mmol) in THF (2 mL) was added Boc-anhydride (289 mg, 0.98 mmol, 1.2 eq.). The reaction mixture was heated and stirred at 66 °C for 24 hours. Reaction was monitored by TLC. The reaction mixture was cooled, and solvent was evaporated under reduced pressure. Water (10 mL) was added to the crude and product was extracted with diethyl ether (3x10 mL). The combined organic layer was washed with brine and dried over sodium sulfate. Solvent was evaporated under reduced pressure and the crude product was purified using silica gel flash chromatography (20:80

1 EtOAc:Hex) to get the desired product. 171 mg, 74%. H NMR (400 MHz, CDCl3) δ 8.17 (d, 1H,

J= 8.8 Hz), 7.68 (dd, 1H, J=2.0, 8.8 Hz), 7.51 (d, 1H, J=2.0 Hz), 7.28 (s, 1H, N-H), 3.93 (s, 3H),

13 3.89 (s, 3H), 1.53 (s, 9H) ppm. C NMR (125 MHz, CDCl3) δ 167.33, 166.90, 152.31, 146.83,

132.67, 123.47, 116.69, 110.61, 80.96, 55.85, 51.97, 28.28 ppm. Spectral data matched with those in literature.191

Methyl 4-azido-3-methoxybenzoate (3-19)

Methyl 4-amino 3-methoxy benzoate (100 mg, 0.55 mmol) and 2N HCl (1.65 mL) were added to a vial and the solution was cooled to 0 °C. Sodium nitrite (57 mg, 0.83mmol, 1.5 eq.) in water (1.3 mL) was added dropwise to the above solution at 0 °C and the mixture was stirred for 30 minutes.

Sodium azide (142 mg, 2.2 mmol, 4 eq.) in water (2.25 mL) was added to the mixture dropwise and the vial was stirred for 4 hours at room temperature. The reaction mixture was extracted with ethyl acetate (3x8 mL). The organic layers were combined and washed with water. Product solution was dried over sodium sulfate and solvent was evaporated under reduced pressure to get

1 the desired product. 99 mg, 87%. H NMR (400 MHz, CDCl3) δ 7.63 (dd, 1H, J= 2.0, 8.4 Hz),

7.55 (d, 1H, J=2.0 Hz), 7.02 (d, 1H, J=8.4 Hz), 3.93 (s, 3H), 3.91 (s, 3H) ppm. 13C NMR (101

MHz, CDCl3) δ 166.57, 151.82, 133.36, 127.48, 123.27, 120.08, 112.96, 56.32, 52.47 ppm.

Spectral data matched with those in literature.192

Methyl 3-methoxy-4-(tritylamino) benzoate (3-20)

Methyl 4-amino 3-methoxy benzoate (150 mg, 0.83 mmol) was dissolved in dichloromethane (3 mL) in a vial. Triethylamine (168 mg, 1.66 mmol, 2.0 eq.) and trityl chloride (254 mg, 0.91 mmol,

1.1 eq.) were added to the reaction mixture and the vial was stirred at room temperature for 16

100 hours. The reaction mixture was diluted with dichloromethane (25 mL) and washed with water

(3x25 mL). The organic layer was dried over sodium sulfate and solvent was evaporated under

1 reduced pressure to get the desired product. 281 mg, 80%. H NMR (400 MHz, CDCl3) δ 7.34-

7.26 (m, 15H), 7.04 (dd, 1H, J=2.0, 8.4 Hz), 6.16 (s, 1H), 5.92 (d, 1H, 8.4 Hz), 5.76 (s, 1H), 3.91(s,

13 3H), 3.71 (s, 3H). C NMR (101 MHz, CDCl3) δ 167.47, 146.25, 144.90, 140.42, 129.06, 128.08,

127.95, 127.02, 123.02, 117.59, 113.53, 109.54, 71.10, 55.78, 51.56 ppm. Spectral data matched with those in literature.192

Methyl 4-amino-3-iodo-5-methoxybenzoate (3-21)

To a solution of 4-amino 3-methoxy benzene compound (1.65 mmol) and sodium bicarbonate

(279 mg, 3.32 mmol) in DCM (4 mL) was added iodine monochloride (297 mg, 1.83 mmol)

DCM (2.5 mL). Reaction mixture was stirred at room temperature for 24 hours. The mixture was washed with saturated sodium bicarbonate and extracted with DCM (4x15 mL). The combined organic fractions were dried over sodium sulfate and solvent was evaporated under reduced pressure. The crude product was purified with silica gel flash chromatography (30:70

1 EtOAc:Hex). 395 mg, 78%. H NMR (400 MHz, CDCl3) δ 7.99 (d, 1H, J=1.6 Hz), 7.38 (d, 1H,

13 J=1.6 Hz), 4.59 (s, 2H, N-H), 3.88 (s, 3H), 3.86 (s, 3H) ppm. C NMR (101 MHz, CDCl3) δ

166.10, 145.10, 141.82, 133.13, 120.34, 110.55, 80.56, 55.99, 51.95 ppm. Spectral data matched with those in literature.193

101

Ethyl 4-amino-3-iodo-5-methoxybenzoate (3-22)

To a solution of 4-amino 3-methoxy benzene compound (0.80 mmol) and sodium bicarbonate

(134 mg, 1.6 mmol) in DCM (2.5 mL) was added iodine monochloride (143 mg, 0.88 mmol)

DCM (1.5 mL). The reaction mixture was stirred at room temperature for 24 hours. Mixture was washed with saturated sodium bicarbonate and extracted with DCM (4x10 mL). The combined organic fractions were dried over sodium sulfate and solvent was evaporated under reduced pressure. The crude product was purified with silica gel flash chromatography (30:70

1 EtOAc:Hex). 193 mg, 75%. H NMR (400 MHz, CDCl3) δ 7.99 (d, 1H, J=1.6 Hz), 7.40 (d, 1H,

J=1.6 Hz), 4.33 (q, 2H, J=7.2 Hz), 3.90 (s, 3H), 1.37 (t, 3H, J=7.2 Hz) ppm. 13C NMR (101

MHz, CDCl3) δ 165.65, 145.13, 141.72, 133.01, 120.76, 110.58, 80.58, 60.78, 55.99, 14.44 ppm. HRMS (TOF MS ES+) m/z calculated ([M+H]+) 321.9940 Da, found 321.9943 Da.

2-(4-amino-3-iodo-5-methoxyphenyl)acetonitrile (3-23)

To a solution of 4-amino 3-mthoxy benzene compound (0.5 mmol) and sodium bicarbonate (84 mg, 1 mmol) in DCM (1 mL) was added iodine monochloride (89 mg, 0.55 mmol) DCM (1 mL).

Reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was washed with saturated sodium bicarbonate and extracted with DCM (4x5 mL). The combined organic

102 fractions were dried over sodium sulfate and solvent was evaporated under reduced pressure.

Crude was purified with silica gel flash chromatography (30:70 EtOAc:Hex). 86 mg, 60%. 1H

NMR (400 MHz, CDCl3) δ 7.11 (d, 1H, J=2.0 Hz), 6.62 (d, 1H, J=2.0 Hz), 4.22 (s, 2H, N-H),

13 3.80 (s, 3H), 3.55 (s, 2H) ppm. C NMR (101 MHz, CDCl3) δ 146.50, 137.45, 129.60, 120.23,

117.97, 109.59, 82.42, 55.94, 22.64 ppm. HRMS (TOF MS ES+) m/z calculated ([M+H]+)

288.9838 Da, found 288.9835 Da.

Methyl 4-amino-3-bromo-5-methoxybenzoate (3-24)

To a solution of 4-amino 3-mthoxy benzene compound (1.50 mmol) in DCM (5 mL) was added

N-bromosuccinimide (1.50 mmol) at 0 °C. The mixture was stirred at room temperature for 10 hours. The crude was washed with water (40 mL) and extracted with DCM (3x50 mL). The combined organic layers were dried over sodium sulfate and solvent was evaporated under reduced pressure. The crude product was purified with silica gel flash chromatography (30:70

1 EtOAc:Hex). 328 mg, 84%. H NMR (400 MHz, CDCl3) δ 7.81 (d, 1H, J=2.0 Hz), 7.38 (d, 1H,

13 J=2.0 Hz), 4.65 (s, 2H, N-H), 3.91 (s, 3H), 3.87 (s, 3H) ppm. C NMR (101 MHz, CDCl3) δ

161.98, 146.24, 139.26, 127.00, 110.56, 109.78, 106.65, 56.06, 51.96. Spectral data matched with those in literature.194

103

2-(4-amino-3-bromo-5-methoxyphenyl)acetonitrile (3-25)

To a solution of 4-amino 3-mthoxy benzene compound (0.6 mmol) in DCM (2 mL) was added N- bromosuccinimide (0.6mmol) at 0 °C. Mixture was stirred at room temperature for 12 hours. The crude was washed with water (15 mL) and extracted with DCM (3x15 mL). The combined organic layer was dried over sodium sulfate and solvent was evaporated under reduced pressure. The crude product was purified with silica gel flash chromatography (30:70 EtOAc:Hex).130 mg, 79%. 1H

NMR (400 MHz, CDCl3) δ 6.99 (d, 1H, J=2.0 Hz), 6.67 (d, 1H, J=2.0 Hz), 3.88 (s, 3H), 3.64 (s,

13 2H), 3.49 (s, 1H, S-H) ppm. C NMR (125 MHz, CDCl3) δ 147.74, 134.81, 123.80, 119.39,

118.11, 108.80, 108.26, 56.14, 23.04 ppm. Spectral data matched with those in literature.195

2-(4-amino-3-bromo-5-methoxyphenyl)acetonitrile (3-26)

To a solution of 4-amino 3-mthoxy benzene compound (0.35 mmol) in DCM (1.5 mL) was added N-bromosuccinimide (0.35 mmol) at 0 °C. The mixture was stirred at room temperature for 10 hours. The crude product was washed with water (10 mL) and extracted with DCM (3x10 mL). The combined organic layers were dried over sodium sulfate and solvent was evaporated under reduced pressure. The crude product was purified with silica gel flash chromatography

1 (30:70 EtOAc:Hex). 68 mg, 81%. H NMR (400 MHz, CDCl3) δ 6.99 (1H, J=2.0 Hz), 6.66

13 (1H, J=2.0 Hz), 4.25 (2H, N-H), 3.87 (3H), 3.63 (2H) ppm. C NMR (101 MHz, CDCl3) δ

104

147.74, 134.81, 123.80, 119.39, 119.12, 108.80, 108.26, 56.14, 23.04 ppm. HRMS (TOF MS

ES+) m/z calculated ([M+H]+) 240.9977 Da, found 240.9976 Da.

Methyl 4-amino-3-mercapto-5-methoxybenzoate

To a 7 mL vial evacuated and purged with N2, methyl-4-amino-3-halo-5-methoxy benzoate (70 mg, 0.27 mmol) was added followed by addition of copper sulfate (2.2 mg, 5 mol%), potassium hydroxide (76 mg, 1.35 mmol) in DMSO (0.6 mL), water (0.06 mL) were added. 1,2- ethanedithiol (51 mg, 0.54 mmol) was added dropwise under N2 to the above mixture. Mixture was stirred at 90 °C for 36 hours. The reaction mixture was quenched with 5% HCl and extracted with ethyl acetate. The organic layer was washed with water, brine and dried over sodium sulfate. Solvent was evaporated under reduced pressure and crude was purified with silica gel

1 flash chromatography (40:60 EtOAc:Hex). 32 mg, 59%. H NMR (400 MHz, CDCl3) δ 7.88 (d,

1H, J=1.6 Hz), 7.41 (d, 1H, J=1.6 Hz), 4.75 (s, 2H, N-H), 3.92 (s, 3H) ppm. 13C NMR (101

MHz, DMSO-d6) δ 167.40, 146.08, 144.50, 130.89, 117.41, 115.08, 112.33, 56.40 ppm. Spectral data matched with those in literature.190

105

Diethyl 5,5'-disulfanediylbis(4-amino-3-methoxybenzoate) (3-28)

To a 7 mL vial evacuated and purged with N2, ethyl-4-amino-3-halo-5-methoxy benzoate (223 mg, 0.69 mmol) was added followed by addition of copper sulfate (11 mg, 10 mol%), cesium carbonate (1.1 g, 3.45 mmol) in DMSO (3 mL) were added. 1,2-ethanedithiol (98 mg, 1.04 mmol) was added dropwise under N2 to the above mixture. The mixture was stirred at 110 °C for 20 hours.

The reaction mixture was quenched with 5% HCl and extracted with ethyl acetate. The organic layer was washed with water, brine and dried over sodium sulfate. Solvent was evaporated under reduced pressure and the crude product was purified with silica gel flash chromatography (40:60

1 EtOAc:Hex). 116 mg, 74%. H NMR (400 MHz, CDCl3) δ 7.48 (d, 2H, J=1.6 Hz), 7.42 (d, 2H,

J=1.6 Hz), 4.97 (s, 4H, N-H), 4.24 (q, 4H, J=7.2 Hz), 3.90 (s, 6H), 1.30 (t, 6H, J=7.2 Hz). 13C

NMR (125 MHz, CDCl3) δ 166.17, 146.01, 143.48, 131.30, 118.38, 112.11, 107.55, 55.97, 54.48,

25.04 ppm.

106

Ethyl 4-amino-3-mercapto-5-methoxybenzoate (3-27)

To a solution of diethyl 5,5’-disulfanediylbis(4-amino-3-methoxybenzoate) (30 mg, 0.06 mmol) in water:acetonitrile (1:1, 5 mL) was added TCEP (tris(2-carboxyethyl)phosphine) (36 mg, 0.126 mmol). The mixture was stirred at room temperature for 3 hours. Solvent was evaporated under

1 reduced pressure to get the desired thiol. 28 mg, 93%. H NMR (400 MHz, CDCl3) δ 7.78 (d,

1H, J=2.0 Hz), 7.39 (d, 1H, J=2.0 Hz), 4.33 (q, 2H, J=7.2 Hz), 3.90 (s, 3H), 2.96 (s, 1H, S-H),

13 1.37 (t, 3H, J=7.2 Hz) ppm. C NMR (125 MHz, CDCl3) δ 166.17, 146.01, 143.48, 131.30,

118.38, 112.11, 107.55, 60.74, 55.97, 14.30 ppm. HRMS (TOF MS ES+) m/z calculated

([M+H]+) 228.1007 Da, found 228.1005 Da.

2-(4-amino-3-mercapto-5-methoxyphenyl)acetonitrile (3-29)

To a 7 mL vial evacuated and purged with N2, 2-(4-amino-3-halo-5-methoxyphenyl)acetonitrile

(50 mg, 0.21 mmol) was added followed by addition of copper sulfate (4 mg, 10 mol%), cesium carbonate (367 mg, 1.15 mmol) in DMSO (1 mL) were added. 1,2-ethanedithiol (39 mg, 0.42 mmol) was added dropwise under N2 to the above mixture. The mixture was stirred at 110 °C for

107

20 hours. The reaction mixture was quenched with 5% HCl and extracted with ethyl acetate. The organic layer was washed with water, brine and dried over sodium sulfate. Solvent was evaporated under reduced pressure and the crude product was purified with silica gel flash

1 chromatography (40:60 EtOAc:Hex). 24 mg, 58%. H NMR (400 MHz, CDCl3) δ 6.94 (d, 1H,

J=2.0 Hz), 6.62 (d, 1H, J=1.6 Hz), 4.26 (s, 2H, N-H), 3.98 (s, 2H), 3.86 (s, 3H), 1.58 (s, 1H, S-

13 H). C NMR (101 MHz, C3D6O) δ 146.50, 137.45, 129.60, 120.23, 117.97, 109.59, 82.42,

55.94, 22.64 ppm. HRMS (TOF MS ES+) m/z calculated ([M+H]+) 195.0592 Da, found

195.0586 Da.

Ethyl 5-methoxy-3,4-dihydro-2H-benzo[b][1,4]thiazine-7-carboxylate (3-30)

To Ethyl 4-amino-3-mercapto-5-methoxybenzoate (9 mg, 0.04 mmol) and potassium carbonate

(0.02 mmol) in DMF (0.4 mL) was added 1,2-dichloroethane or 1,2-dibromoethane (0.05 mmol).

Mixture was stirred at 60 °C for 10 hours. DMF was evaporated under reduced pressure. The crude was washed with brine and extracted with ethyl acetate (3x 4 mL). The combined organic layer was dried over sodium sulfate and solvent was evaporated under reduced pressure. The crude product was purified with silica gel flash chromatography or HPLC. 7 mg, 71%. 1H NMR

(400 MHz, CDCl3) δ 7.44 (d, 1H, J=2.0 Hz), 7.21 (d, 1H, J=1.6 Hz), 4.98 (s, 1H, N-H), 4.31 (q,

13 2H), 3.88 (s, 3H), 3.75 (m, 2H), 3.02 (m, 2H), 1.36 (t, 3H). C NMR (125 MHz, CDCl3) δ

163.19, 139.16, 130.93, 129.26, 116.91, 111.32, 109.32, 60.59, 55.68, 42.78, 36.90, 14.44 ppm.

HRMS (TOF MS ES+) m/z calculated for ([M+H]+) 254.1006 Da, found 254.1007 Da.

108

Ethyl 5-methoxy-2H-benzo[b][1,4]thiazine-7-carboxylate (3-31)

To a solution of sodium hydride (9 mg, 0.04 mmol) in anhydrous THF (0.2 mL) was added 2-(4-

o amino-3-mercapto-5-methoxyphenyl) (0.04 mmol) THF (0.2 mL) was added under N2 at 0 C.

Reaction mixture was stirred at RT for 2 hours followed by addition of bromoacetaldehyde (0.05 mmol) in THF (0.1 mL) and molecular sieves. The mixture was stirred at RT for 16 hours. The crude product was filtered through celite and purified with silica gel flash chromatography.6 mg,

1 59%. H NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 8.30 (d, 1H, J=1.2 Hz), 7.61 (d, 1H, J=1.2

13 Hz), 4.44-4.42 (m, 4H), 4.12 (s, 3H), 1.42 (t, 3H). C NMR (125 MHz, CDCl3) δ 167.95,

143.92, 131.17, 126.02, 116.58, 111.20, 110.85 109.60, 58.47, 56.34, 24.54 ppm. HRMS (TOF

MS ES+) m/z calculated for ([M+H]+) 252.0652, found 252.0654.

2-(5-methoxy-4H-benzo[b][1,4] thiazine-7-yl)acetonitrile

To a solution of sodium hydride (0.035 mmol) in anhydrous THF (0.2 mL) was added 2-(4-

o amino-3-mercapto-5-methoxyphenyl) (0.04 mmol) THF (0.2 mL) was added under N2 at 0 C.

Reaction mixture was stirred at RT for 2 hours followed by addition of bromoacetaldehyde (0.05 mmol) in THF (0.1 mL) and molecular sieves. The mixture was stirred at RT for 16 hours. The

109 crude product was filtered through celite and purified with silica gel flash chromatography. 3 mg,

1 41%. H NMR (400 MHz, CDCl3) δ 7.70 (d, 1H, J=3.2 Hz), 7.21 (d, 1H, J= 3.6 Hz), 7.01 (d,

13 1H, J=1.6 Hz), 7.68 (d, 1H, J=1.6 Hz), 4.21 (s, 2H), 3.83 (s, 3H). C NMR (125 MHz, CDCl3)

δ 143.50, 137.45, 135.91, 129.60, 128.70, 117.97, 113.25, 112.98, 106.59, 55.94, 24.64 ppm.

HRMS (TOF MS ES+) m/z calculated for ([M+H]+) 219.0516, found 219.0512.

Diethyl 5-methoxy-2H-benzo[b][1,4]thiazine-3,7-dicarboxylate (3-32)

To a solution of sodium hydride (8 mg, .035 mmol) in anhydrous THF (0.2 mL) was added 2-(4-

o amino-3-mercapto-5-methoxyphenyl) (0.035 mmol) THF (0.2 mL) was added under N2 at 0 C.

Reaction mixture was stirred at RT for 2 hours followed by addition of ethyl bromopyruvate

(0.05 mmol) in THF (0.1 mL) and molecular sieves. The mixture was stirred at RT for 16 hours.

The crude product was filtered through celite and purified with silica gel flash chromatography.7

1 mg, 66%. H NMR (400 MHz, CDCl3) δ 8.26 (d, 1H, J=1.2 Hz), 7.60 (d, 1H, J=1.2 Hz), 4.54

(q, 2H), 4.44 (q, 2H), 4.11 (s, 3H), 3.90 (s, 2H), 1.46 (t, 3H), 1.43 (t, 3H). 13C NMR (125 MHz,

CDCl3) δ 166.66, 143.75, 141.40, 139.41, 131.10, 125.70, 117.14, 112.35, 109.45, 60.82, 56.15,

55.91, 31.17, 14.66, 14.50 ppm. HRMS (TOF MS ES+) m/z calculated for ([M+H]+) 324.0890, found 324.0852.

110

4 SYNTHESIS OF REGIOSELECTIVELY FUNCTIONALIZED EUMELANOIDS MIXED MELANOIDS AND THEIR PHOTOPHYSICAL STUDIES 4.1 Introduction

Mixed melanin such as neuromelanin are melanin biopolymer which contain conjugated pheomelanin and eumelanin units. Human neuromelanin is extracted from one of the following organelles: substantia nigra, putamen, premotor cortex, and cerebellum. The process involves multiple steps of gradient centrifugation.53 Access to these natural melanin have been limited to extraction and biosynthesis.53,121 Oligomerization of pheomelanin subunits in order to access intermediates to pheomelanogenesis has been done by D’Ischia.87,196 Similarly, oligomerization of eumelanin subunits, DHI and DHICA has been developed in order to access polydopamine based synthetic eumelanin for applications in polymer stabilization126,127, surface coating and bio-adhesion62,128, biosensing, nano particle and nanocapsule preparation for drug delivery, light harvesting and cell interfacing.62,66,125,129–135 Eumelanin and pheomelanin have unique photoprotective and material properties. Conjugation of eumelanin and pheomelanin subunits would form a synthetic subunit of mixed melanin. Oligomerization of this new type of melanin could have interesting photophysical and materials properties.

Suzuki coupling has been used to couple brominated DHICA compounds to borylated aromatic species.73 However, brominated indoles have not been functionalized before in order to conjugate these species with halogenated aromatics via Suzuki coupling. One of the research goals of this project was to functionalize regioselectivity brominated eumelanin subunits- DMIC.

This would be followed by coupling of these functionalized DHICA subunits with brominated pheomelanin subunits (benzothiazole) to access subunits of mixed melanin.

111

Figure 4-1 Target synthetic mixed melanoid synthesized from synthetic monomeric subunits of pheomelanin and eumelanin.

Functionalizing monomeric eumelanin units would be advantageous to couple them selectively with pheomelanin monomeric units such as benzothiazole and benzothiazine. In this work, monomeric eumelanin units of mono brominated DMIC were functionalized and coupled with benzothiazole. Benzothiazole with CO2Et sidechain was used as a model substrate to assess the coupling reactions which would yield a mixed melanoid. Once Suzuki coupling was shown to be successful to form the new C-C bond, benzothiazole with CH2CN sidechain was used, which would allow to access natural analogues of dopamine derived mixed melanin (potential monomeric subunit of neuromelanin) in future.

4.2 Results and Discussion:

Scheme 4-1 describes the two reaction pathways attempted to functionalize eumelanoid and conjugate it to benzothiazole. Initially aldehyde was installed at the 4’-position via lithiation of the 4’-bromo eumelanoid (3-8) using organolithium reagent followed by KI catalyzed coupling of aldehyde with benzothiazole (ethyl ester sidechain) in aqueous solution in presence of tert- butylhydroperoxide.

112

Scheme 4-1 Reaction pathways for mixed melanoid (4-4) synthesis.

After several unsuccessful attempts of KI catalyzed coupling, Miyuara borylation of brominated

DMIC compounds followed by Pd catalyzed Suzuki coupling was successfully used to access two different types of mixed melanin subunits.

4.2.1 Eumelanoid functionalization Initial pathway to functionalize bromo-DMIC derivatives included installing aldehyde at the brominated position via lithiation. 4’-bromo DMIC was used as a model substrate. Using organolithium reagent n-BuLi followed by reaction with DMF (dimethylformamide) installed aldehyde at the 4’-position via electrophilic addition. The second step of this pathway was KI catalyzed coupling of aldehyde of 4’-eumelanoid and benzothiazole (ethyl ester sidechain in this

11 - case) in aqueous solution in presence of tert-butylhydroperoxide. I or I2 would form tert- butylperoxyl radical which would form the product via annulation. The mechanism of this reaction

113 was studied by controlled experiments by Gao et al and it has been shown in Figure 4-2 for 4’-

DMIC aldehyde and benzothiazole.11 Benzothiazole would react with water in presence of tert- butyl hydroperoxide to give the dihydrobenzothiazole.

Figure 4-2 Mechanism of KI catalysed coupling of benzothiazole with aromatic aldehyde.11

Nitrogen of the benzothiazole reacts with benzaldehyde forming aminal (Figure 4-2) after nucleophilic addition and protonation. In the dehydration step, loss of water results in formation of Schiff’s base. Tert-butylperoxyl radicals promote formation of intermediate as they are being reduced. This unstable and reactive intermediate undergoes annulation resulting in formation of intermediate which after deprotonation results in desired product (Figure 4-2) with extended

114 conjugation. Lithiation step was done with 4’ and 7’ bromo DMIC (2-8 and 2-12) successfully, however the purification of the product was difficult and the yields of both these products were low (30%-55%). In addition, the 7’-aldehyde DMIC product (4-3) was observed to have butylester at indole ester position as compared to methylester sidechain in the starting material. The KI catalyzed coupling step was unsuccessful as significant amount of unreacted starting material was recovered after up to 24 to 48 hours into the reaction. Using a different organlithium reagent may have helped with increasing the yield of the first step. However, due to low yields in the lithiation step, failure to consume starting material in the coupling step and limited substrate scope (limited to BTZL) of KI-catalyzed coupling, a different reaction pathway was used.

In this alternate pathway-Suzuki-Miyaura coupling was used to synthesize mixed melanoids.

Bromo DMIC derivatives (2-8 ans 2-12) were borylated via Miyaura borylation followed by Pd catalyzed Suzuki coupling of the functionalized eumelanoid with bromobenzothiazole. 4’-bromo eumelanoid (2-8) was used as model substrate to assess and optimize the reaction conditions.

Initially, borylation via lithiation was attempted but due to poor conversion of the starting material different Pd catalysts were utilized. Pd catalysts such as PdCl2(dppf), PdCl2(PPh3)2, Pd2(dba)3, and

Pd(PPh3)4 were assessed to optimize borylation. Reactions were monitored with analytical-HPLC to observe consumption of starting material (2-8), formation of product (4-5), and formation of protodeboronated product (2-3) at different time points. Pd(PPh3)4 along with reaction conditions listed below were observed to be the optimized conditions to maximize conversion of 2-8 to 4-5 and minimize the formation of 2-3. Reaction was monitored with analytical HPLC and mole fraction data were plotted for comparison. Tetrakistriphenyl palladium- Pd(PPh3)4 in dioxane with potassium acetate base and bispinacolatodiboron as borylating agent yielded the best results. The optimum conversion was observed at 36 hours (Figure 4-3).

115

4'-bromo eumelanoid- Borylation 1 0.9 0.8 0.7 0.6 (2-8) 0.5 (4-5) 0.4 (2-3) 0.3 Molefraction(X) 0.2 0.1 0 0 5 10 15 20 25 30 35 40 45 Time(hrs)

Figure 4-3 Mole fractions of 4’-bromo DMIC (2-8) borylation reaction. (Calculated from HPLC data)

As observed in Figure 4-3, in HPLC chromatograms, in addition to the starting material and product traces, a trace of Methyl 5,6-dimethoxyindole carboxylate (2-3) was observed. This could be due to protodeboronation of the starting material. However, this degradation was negligible for

4’-bromo DMIC, it was a significant issue in 3’-bromo DMIC borylation.

116

Figure 4-4 Borylation and hydrolysis of pinacol ester monitored by HPLC. (C-18 stationary phase, Mobile phase gradient 50%:50% water:can to 100% AcN in 10 minutes)

Crude of borylation reaction mixture was purified using C18 prep-HPLC. It was observed that after purification on C-18 with water:acetonitrile as mobile phase, the 4’-pinacol ester product(4-

5) had partially hydrolyzed into the 4’-boronic acid product(4-6). Figure 4-4 details HPLC chromatograms of reaction mixture at the beginning containing starting material (2-8) at 3.83 minutes, crude at 36 hours shows pinacol ester product (4-5) at 4.28 minutes, and a mixed trace after purification which shows 4’-boronic acid product (4-6) at 1.81 minutes with 4’-pinacol ester product (4-5) at 4.28 minutes. For all of the HPLC traces in Figure 4-4, HPLC conditions including the C-18 column, temperature (30 ℃), and solvent gradient (50%:50% water AcN to 100% AcN in 10 minutes) were kept constant. Most of the prep-HPLC fractions contained 4’-boronic acid as the major product and non-hydrolyzed pincol ester as the minor product. Since boronic acid functionalized aromatics are known to be more reactive in Suzuki coupling, 4’-pinacol ester

117 product (4-5) was hydrolyzed to 4’-bronic acid (4-6) in 50:50 water:AcN at RT in 24 hours. 4-6 was used for Suzuki coupling reactions.

Figure 4-5 Borylation of 7’-bromo DMIC (2-11).

Similar to 4’-bromo DMIC (2-8) borylation to syntheize 4’-boronic acid product (4-6), borylation of 7’-bromo DMIC (2-11) was performed using Pd catalyst to install pinacol ester (4-8). 7’-pinacol ester product (4-8) as shown in Figure 4-5, was hydrolyzed using 1:1 water:acetonitrile in 24 hours to yield 7’-boronic acid product (4-9) in 77% yield over 2 steps. These reactions were monitored with analytical HPLC (Figure D-1, Appendix D) and showed similar trend as 2-8 borylation, with starting material (2-11) retention at 4.25 minutes, 7’-pinacol ester product (4-8) retention at 5.01 minutes, and 7’-boronic acid product (4-9) retention at 1.90 minutes. 7’-boronic acid product (4-

9) was used in Suzuki coupling.

Borylation of 3’-bromo DMIC (2-4) was attempted using several reaction conditions as significant protodeborylation was observed in these reactions. Reactions were monitored with C-18 analytical

HPLC. An example HPLC chromatogram (Figure D-2, Appendix D), which shows HPLC traces for 3’-bromo DMIC starting material (2-4) at 2.94 minutes, protodeboronated product (2-3) at 2.24 minutes, and desired 3’-borylated product (4-7) at 3.33 minutes. For each reaction, small fractions were taken out every 4 hours and HPLC conditions including the C-18 column, temperature (30

℃), and solvent gradient (50%:50% water AcN to 100% AcN in 10 minutes) were kept constant.

Mole fractions of the optimization experiments with variable changes have been shown in Table

118

4-1. Temperature optimization was done with PdCl2(dppf) catalyst and dioxane as solvent. All three reactions were running at the same indole concentration (0.28 M) and for the same duration.

Figure 4-6 3’-borylation reaction monitored by analytical HPLC.

Table 4-1 3’-borylation reaction optimization and relevant mole fractions (from HPLC data).

No Temp Catalyst/ Solvent Time ᵡ (2-3) ᵡ (4-7) ᵡ (2-4) Ligand (hrs)

1 RT PdCl2(dppf) Dioxane 24 0.048 0.012 0.94

2 50 °C PdCl2(dppf) Dioxane 24 0.53 0.04 0.43

3 50 °C Pd(PPh3)4 Dioxane 24 0.35 0.076 0.57

4 100 °C PdCl2(dppf) Dioxane 24 0.93 0.029 0.041

5 100 °C Pd2(dba)3 Dioxane 24 0.28 0.03 0.69 X-phos

6 100 °C Pd(PPh3)4 Dioxane 24 0.76 0.03 0.21

7 65 °C Pd(PPh3)4 THF 24 0.11 0.09 0.80

o 8 65 C Pd(PPh3)4 THF 36 0.30 0.12 0.58

119

As observed by the trend in mole fractions of each compound, the product formation increases as the temperature is increased initially as the SM conversion is observed to increase, however it lowers at high temperature (100 °C). Most importantly, the amount of protodeboronated product increases as the temperature is increased. This indicates that the rate of borylation is much slower than protodeboronation under the reaction conditions. Consistent catalytical loading (5 mol%) was done with other Pd catalysts used [Pd(PPh3)4 and Pd2(dba)3 with X-phos] while keeping the reaction conditions same. Both of these catalysts gave similar yield of the product, however,

Pd(PPh3)4 resulted in higher conversion of SM compared to slower reaction of Pd2(dba)3. Despite generation of higher debrominated product, following higher conversion of SM and better borylation yields in previous indole borylation, Pd(PPh3)4 was utilized as the catalyst and conditions were further optimized. Solvent was changed to THF in order to increase solubility and reflux temperature (65 °C) was used for these optimizations. Entry 8 in Table 4-1 indicates the optimized conversion of SM to desired borylated product and recovered yield was ~10%. A parallel reaction was ran for 60 hours and monitored with HPLC, but it indicated degradation of product to deboronated compound. Borylation of 3’-bromo DMIC (2-4) needs to be further optimized in future possibly with different bases or catalysts, and the mechanism of protodeboronation could be studied.

Figure 4-7 Borylation of bromobenzothiazole (3-14).

120

In order to circumvent the protodeboronation problem of 3’-borylation, and to access 3’- conjugated mixed melanoid, borylation of bromobenzothiazole was attempted. Borylation of bromo-BTZL (3-14) was attempted using Pd(PPh3)4 and PdCl2(dppf) as catalyst with similar reaction conditions to bromo-DMIC borylation. Reactions were monitored with HPLC, and it was observed that pinacol ester of the BTZL is generated as a minor product (22%) with the major product (63%) being the protodeboronation product of BTZL (3-13). Pinacol ester could be converted to boronic acid BTZL (4-13) in 77% yield. Borylation reaction of bromo-BTZL (3-14) needs to be optimized in a future study to access 3’-DMIC conjugated mixed melanoid.

4.2.2 Mixed melanoid synthesis via Suzuki Coupling:

Figure 4-8 Mechanism of Suzuki coupling.

Mixed melanoid synthesis was carried out with 4’-and 7’-boronic acid compounds as they were conjgated with pheomelanoids using Suzuki coupling. Bromobenzothiazole (3-11) was used as a model monomeric pheomelanin substrate due to ease of synthesis and scalability. Mechanism of

Pd catalyzed Suzuki coupling is shown in Figure 4-8. Bromobenzothiazole (3-11) would

121 oxidatively add to Pd(0) as it oxidizes to Pd(II) during oxidative addition. In transmetallation step, in presence of base-potassium phosphate (tribasic) 4’-boronic acid DMIC (4-6) adds to the metal complex as the boronic acid halogenates resulting in both the aryl compounds complexed to the

Pd(II) metal. In the reductive elimination step, the new C-C bond is formed between the aryl compounds (4-6 and 3-11) as the mixed melanoid compound (4-10) is generated resulting in restoration of Pd oxidation state Pd(0).

Figure 4-9 Suzuki coupling to synthesize mixed melanoids.

Suzuki coupling was done successfully on 4’-boronic acid DMIC, where it was coupled with bromobenzothiazole (R=CO2Et or CH2CN) in presence of Xphos Pd G2 catalyst with yields of

96% (R=CO2Et) and 88% (R=CH2CN). Products were characterized using combination of NMR experiments including 1D NMR experiments such as 1H, 13C and 2D NMR experiments such as

COSY, HSQC, and HMBC. 2D HMBC was key in characterizing and confirming the conjugation of the melanoids. A specific section of HMBC spectra of ethyl 2-(5,6-dimethoxy-2-

(methoxycarbonyl)-1H-indol-4-yl)-4-methoxybenzo[d]thiazole-6-carboxylate is shown in Figure

4-10.

122

Figure 4-10 2D HMBC (Indole proton-focused) of mixed melanoid [ethyl 2-(5,6-dimethoxy-2- (methoxycarbonyl)-1H-indol-4-yl)-4-methoxybenzo[d]thiazole-6-carboxylate] (4-4).

Correlation of indole proton(1H shift-7.61 ppm-highlighted red) to the neighboring carbons(13C) is observed. The correlation and as a result the signal becomes weaker as the bond distance between the proton and the carbon increases. HMBC does not show one bond correlation, however

2 3 2 two ( JCH) to three bond correlation ( JCH) are commonly observed. Two bond correlation ( JCH)

(highlighted green) were observed between the indole proton and the neighboring aromatic

3 carbons. Three bond correlation ( JCH) (highlighted blue) were observed between indole proton and two aromatic carbons as well as the carbonyl carbon of the methyl ester. Four bond

4 correlations( JCH) are rare in HMBC, but it was observed (highlighted red) for the indole proton correlating to the benzothiazole carbon of the conjugated benzothiazole. This analysis along with the NMR analysis mentioned above confirmed the formation of mixed melanoids. 7’-boronic acid

123

DMIC (4-9) was coupled with bromo-BTZL (3-14) via Suzuki coupling resulting in 7’-conugated mixed melanoid (4-11) in 70% yield. Once the mixed melanoids (4-4, 4-10 and 4-11) were accessed, initial photophysical properties of these compounds were studied using spectramax i3 spectrophotometer.

4.2.3 UV-Vis and Fluroscence properties of mixed melanoids Overlayed UV-vis spectra of 5,6-DMIC (2-3), benzothiazole (3-13), and their mixed melanoid

(4-10) has been shown in Figure 4-11. Broad absorption spectra are characteristic of melanin derivatives. Absorption at 260 nm and 280 nm were observed as λmax for BTZL (3-13) and 5,6-

DMIC (2-3) respectively. Mixed melanoid (4-10) is a conjugate of 3-13 and 2-3, and it shows absorbance 280 nm however, the λmax was observed at low energy UV wavelength 360 nm resulting from extended aromaticity of the conjugated system.

Figure 4-11 UV-Vis absorbance of 5,6-DMIC (2-3), BTZL (3-13), and their mixed melanoid (4- 10)- 50 µM in ethanol.

124

Similar UV-Vis absorption spectra were observed for mixed melanoids 4-4 and 4-11. For compound 4-4 (Figure E-1, Appendix E) absorption peaks were observed at 280 nm and λmax at

350 nm and another absorption peak (shoulder) at 390 nm. For compound 4-11 (Figure E-3,

Appendix-E), which contains the same sidechain (CH2CN) in the pheomelanoid unit as 4-10,

UV-Vis absorption spectrum showed peaks at 280 nm. λmax was observed at low energy UV wavelength 350 nm and an absorption band (shoulder) at 380 nm similar to 4-10.

Figure 4-12 Fluorescence spectra of 4-10.

Preliminary fluorescence properties of mixed melanoids (4-4, 4-10 and 4-11) were studied. All three of these mixed melanoid products visibly luminesce under 365 nm light source. As shown in excitation-emission spectra of compound 4-10 in Figure 4-12, excitation at 280 nm and 360 nm results in emission at 425 nm. Similarly, as described in Figure E-2 (Appendix E), compound

4-4 exhibits luminescence (emission) at 455 nm upon excitation at 280 nm and 354 nm; and

125 compound 4-11 (Figure E-4, Appendix E) exhibits luminescence (emission) at 450 nm upon excitation at 280 nm and 350 nm. These luminescence results of a synthetic mixed melanin dimer are comparable to those of synthetic eumelanin solution where excitation at 360-380 nm showed emission at 460-480 nm.160 However, synthetic eumelanin solution exhibited excitation wavelength dependent emission as it breaks Kasha’s rule, and emission spectra does not mirror the respective absorption spectra violating the mirror image rule.160,197 As shown in Figure 4-12, luminescence spectra of 4-10 follows Kasha’s rule where emission is independent of excitation wavelength as it stays constant (420 nm) for two different excitation wavelengths (280 nm and

197 360 nm) resulting from photon transfer from lowest excitation state S1 to ground state S0. In addition, it follows the mirror image rule for organic chromophores as the emission spectra (ex-

280 nm and 360 nm) mirror the absorbance wavelengths. This preliminary data indicates that these mixed melanoid compounds possess interesting photophysical properties which can be studied further for materials applications, and these compounds can be studied as organic chromophores.

4.3 Conclusion and future direction: Eumelanin subunit (5,6-DMIC) functionalization and conjugation with pheomelanin subunit

(benzothiazole) was attempted using KI catalyzed coupling and Suzuki-Miyaura coupling.

Miyaura borylation was successful in functionalizing 4’-bromo DMIC as it was borylated to form 4’-boronic acid DMIC in 61% yield over 2 steps. 7’-bromo DMIC was borylated to form

7’-boronic acid DMIC with 77% yield over 2 steps. Functionalized Eumelanin DMIC subunit 4-

6 was coupled with bromobenzothiazole via Pd catalyzed Suzuki coupling to synthesize mixed melanin subunit with CO2Et and CH2CN sidechain with 96% and 88% yields respectively. 7’- boronic acid DMIC was coupled with in similar manner to synthesize 7’-functionalized mixed melanoid in 70% yield. Structures of these compounds were confirmed with 1D and 2D NMR as 126 well as HRMS. Preliminary photophysical data was collected to study UV absorbance and luminescence behavior. Mixed melanin subunits exhibit broad absorption bands in UV-Vis absorption spectra with λmax ~360 nm, and luminescence at 425 nm resulting from extended conjugation of the heteroaromatic system.

This work can be extended further to resolve the limitations regarding the substrate scope by functionalizing 3’-bromo DMIC and bromobenzothiazole by borylation for it to be used in

Suzuki coupling. In addition, synthesis and functionalization of 5,6-dimethoxyindole (5,6-DMI) would give access to the 5,6-dihydroxyindole (DHI) mixed melanins. These would give access to a variety of dimeric, trimeric, and tetrameric mixed melanin subunits. In addition, deprotection via sidechain hydrogenation of pheomelanin subunit, saponification of methyl ester, and demethylation of methoxy groups of mixed melanoids would result in formation of natural analogues of mixed melanin subunits which could be key intermediates in melanogenesis of neuromelanin. To further explore the applications of these N-heterocycles for as organic chromophores, the next step should be a detailed study of their photophysical properties.

4.4 Synthesis 4.4.1 General Synthetic procedure: All reagents were purchased from Sigma-Aldrich and Fisher Scientific and used directly without further purification. Solvents were dried with activated 4 Ao molecular sieves or anhydrous solvents were dispensed from Solvent system. Reactions were monitored by TLC or analytical

HPLC. All products were purified with silica-gel flash chromatography or preparative HPLC.

HPLC was performed on a Gilson analytical to semi-preparative system using Kinetex 5 µm EVO

C18 100Å 4.6 x 150 mm for analytical and Kinetex 5 µm EVO C18 100Å 21.1 x 150 mm for preparative purpose. Products were characterized by 1H and 13C NMR and mass spectrometry.

NMR characterization results and J values were compared with published results. 1H and 13C NMR 127 spectra were recorded on Agilent/Varian 400 MHz or Bruker Neo 500 MHz and 101 MHz NMR spectrometer, respectively. Mass spectra were recorded using a Synapt G2 Q-TOF ESI mass spectrometer at the Mass Spectrometry Lab at the University of Illinois Urbana Champagne.

Abbreviations: TLC-Thin layer chromatography, HPLC-High Performance Liquid

Chromatography, NMR-Nuclear Magnetic Resonance, Q-TOF-Quadrupole time of flight, ESI-

Electrospray ionization

4.4.2 Conjugation via oxidation: Methyl 4-formyl-5,6-dimethoxy-1H-indole-2-carboxylate

To a stirred solution of methyl 4-bromo-5,6-dimethoxy-1H-indole-2-carboxylate (19 mg, 0.06 mmol) in dry THF (0.6 mL) at -78 °C under N2 was added 2.5 mM n-BuLi (0.30 mmol, 5 eq.) dropwise. The reaction mixture was stirred at room temperature for 2 hours and dimethylformamide (0.30 mmol) was added at 0 °C. The reaction mixture was stirred at room temperature for 5 hours. Ice pieces were added to the reaction mixture followed by 1N HCl (~1 mL) and extracted with ethyl acetate (3x10 mL). The combined organic layer was dried over sodium sulfate and solvent was evaporated under reduced pressure. The crude product was purified with silica gel flash chromatography (30:70 EtOAc:Hex). 9 mg, 55%. 1H NMR (400

MHz, CDCl3) δ 10.64 (s, 1H), 8.13 (s, 1H, N-H), 7.22 (d, 1H, 2.0 Hz), 7.15 (s, 1H), 3.97 (s, 3H),

3.93 (s, 3H), 3.91 (s, 3H) ppm. Spectral data matches with those in literature.198

128

Methyl 7-formyl-5,6-dimethoxy-1H-indole-2-carboxylate

To a stirred solution of To a stirred solution of To a stirred solution of methyl 7-bromo-5,6- dimethoxy-1H-indole-2-carboxylate (15 mg, 0.049 mmol) in dry THF (0.4 mL) at -78 °C under

N2 was added 2.5 mM n-BuLi (0.24 mmol, 5 eq.) dropwise. The reaction mixture was stirred at room temperature for 2 hours and dimethylformamide (17.5 mg, 0.24 mmol) was added at 0 °C.

Mixture was stirred at room temperature for 5 hours. Ice pieces were added to the reaction mixture followed by 1N HCl (~1 mL) and extracted with ethyl acetate (3x5 mL). The combined organic layer was dried over sodium sulfate and solvent was evaporated under reduced pressure.

The crude product was purified with silica gel flash chromatography (20:80 EtOAc:Hex). Yield-

1 6 mg, 38%. H NMR (500 MHz, CDCl3) δ 10.54 (s, 1H), 10.02 (s, 1H, N-H), 7.40 (s, 1H), 6.18

(d, 1H, 2.0 Hz), 4.02 (s, 3H), 3.93 (s, 3H), 1.31-1.18 (m, 9H) ppm.

Conjugation (Failed)

To a 7 mL scintillation vial were added methyl 4-formyl-5,6-dimethoxy-1H-indole-2-carboxylate

(4 mg, 0.015 mmol), ethyl 4-methoxybenzo[d]thiazole-6-carboxylate (2.5 mg, 0.01 mmol), tert-

129 butyl hydroperoxide (70% aq., 0.03 mmol), potassium iodide (0.001 mmol, 10 mol%) and water

(0.3 mL). Mixture was heated at 100 °C for 24 hours. Starting material was not consumed as monitored by TLC and crude NMR. Acetonitrile (0.2 mL) was added and mixture was stirred at

85 °C for 18 hours.

Methyl 5,6-dimethoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-2- carboxylate

To a mixture of methyl 4-bromo-5,6-dimethoxy-1H-indole-2-carboxylate (70 mg, 0.224 mmol), tetrakis(triphenylphosphine)palladium(0) (13 mg, 0.0112 mmol, 0.05 eq.), potassium acetate (66 mg, 0.672 mmol, 3 eq.), and bis(pinacolato)diboron (74 mg, 0.291 mmol, 1.3 eq.) evacuated and

o purged with N2 was added dry-dioxane (0.4 mL) under N2. The mixture was stirred at 100 C for

36 hours. Reaction was monitored by HPLC. The catalyst was filtered, and the crude was used for hydrolysis.

(5,6-dimethoxy-2-(methoxycarbonyl)-1H-indol-4-yl)boronic acid

130

The crude mixture from the borylation reaction was stirred in water:acetonitrile (1:1, 5 mL) for

24 hours. Solvent was evaporated and the crude product was purified with prep-reverse phase

HPLC. After purification prep fractions containing product were analyzed using NMR and

1 analytical HPLC. 38 mg, 61%. H NMR (500 MHz, C3D6O) δ 10.66 (s, 1H), 7.71 (s, 1H), 7.51

13 (s, 2H), 7.21 (s, 2H), 3.92 (s, 3H), 3.89 (s, 3H), 3.86 (s, 3H) ppm. C NMR (125 MHz, C3D6O)

δ 162.79, 152.67, 135.71, 127.20, 125.37, 112.66, 108.97, 103.73, 98.47, 61.97, 56.05, 51.72 ppm. HRMS (TOF MS ES+) m/z calculated ([M+H]+) 280.0992 Da, found 280.0986 Da.

Methyl 5,6-dimethoxy-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-2- carboxylate

(Reaction conditions are listed in Table 4-1)

General procedure: To a mixture of methyl 3-bromo-5,6-dimethoxy-1H-indole-2-carboxylate (20 mg, 0.064 mmol), Pd(0) catalyst (13 mg, 0.0112 mmol, 0.05 eq.), base (1.7- 3 eq.), and bis(pinacolato)diboron (19.5 mg, 0.077 mmol, 1.2 eq.) evacuated and purged with N2 was added dry-dioxane or THF (0.3 mL) under N2. Mixture was stirred at (a temp. between room temperature-100 oC) for 24-36 hours. Reaction was monitored by HPLC.

131

Methyl 5,6-dimethoxy-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-2- carboxylate

To a mixture of methyl 7-bromo-5,6-dimethoxy-1H-indole-2-carboxylate (12 mg, 0.038 mmol), tetrakis(triphenylphosphine)palladium (0) (2.2 mg, 0.0019 mmol, 0.05 eq.), potassium acetate

(11 mg, 0.114 mmol, 3 eq.), and bis(pinacolato)diboron (13 mg, 0.05 mmol, 1.3 eq.) evacuated

o and purged with N2 was added dry-dioxane (0.2 mL) under N2. The mixture was stirred at 100 C for 36 hours. Reaction was monitored by HPLC. The catalyst was filtered, and the crude was used for hydrolysis.

(5,6-dimethoxy-2-(methoxycarbonyl)-1H-indol-7-yl)boronic acid

The crude mixture from the borylation reaction was stirred in water:acetonitrile (1:1, 5 mL) for

24 hours. Solvent was evaporated and the crude product was purified with prep-reverse phase

HPLC. After purification prep fractions containing product were analyzed using NMR and

1 analytical HPLC. 8 mg, 77%. H NMR (500 MHz, C3D6O) δ 10.09 (s, 1H), 7.79 (s, 1H), 7.40

13 (s, 1H), 7.09 (s, 3H), 3.98 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H) ppm. C NMR (125 MHz, C3D6O)

δ 162.43, 156.46, 149.16, 132.60, 129.42, 127.79, 124.04, 108.72, 108.17, 62.01, 56.38, 52.00 ppm. HRMS (TOF MS ES+) m/z calculated ([M+H]+) 280.0992 Da, found 280.0993 Da. 132

Ethyl 2-(5,6-dimethoxy-2-(methoxycarbonyl)-1H-indol-4-yl)-4-methoxybenzo[d]thiazole-6- carboxylate (4-4)

To a 7 mL vial were added XPhosPdG2(4 mol%), (5,6-dimethoxy-2-(methoxycarbonyl)-1H- indol-4-yl) boronic acid (0.021 mmol), ethyl 2-bromo-4-methoxybenzo[d]thiazole-6-carboxylate

(0.0216 mmol, 1.03 eq.). The vial was evacuated and purged with N2 followed by addition of

1,4-dioxane (0.35 mL) and 0.5 M potassium phosphate tribasic (3 eq.). The reaction mixture was heated at 75 ℃ for 5 hrs. The reaction mixture was quenched with water (4 mL) and extracted with ethyl acetate (5x4 mL), dried with sodium sulfate and solvent was evaporated under reduced pressure. The crude product was purified with silica gel flash chromatography (50:50

1 EtOAc:Hex). 9 mg, 96%. H NMR (500 MHz, CDCl3) δ 9.10 (s, 1H, N-H), 8.43 (d, 1H, J=2.0

Hz), 8.29 (d, 1H, J=1.5 Hz), 7.59 (d, 1H, J=1.5 Hz), 7.04 (s, 1H), 4.43 (q, 2H), 4.17 (s, 3H),

13 3.99 (s, 3H), 3.98 (s, 3H), 3.95 (s, 3H), 1.43 (t, 3H) ppm. C NMR (125 MHz, CDCl3) δ

166.52, 164.23, 162.45, 153.23, 152.37, 145.89, 145.52, 137.08, 133.95, 128.10, 127.28, 119.63,

119.17, 115.72, 112.14, 107.34, 97.32, 61.28, 60.96, 56.41, 56.22, 51.92, 14.42 ppm. HRMS

(TOF MS ES+) m/z calculated ([M+H]+) 471.1226 Da, found 471.1226 Da.

133

Methyl 4-(6-(cyanomethyl)-4-methoxybenzo[d]thiazol-2-yl)-5,6-dimethoxy-1H-indole-2- carboxylate (4-11)

To a 7 mL vial were added XPhosPdG2(4 mol%), (5,6-dimethoxy-2-(methoxycarbonyl)-1H- indol-4-yl) boronic acid (0.021 mmol), 2-(2-bromo-4-methoxybenzo[d]thiazol-6-yl)acetonitrile

(0.0216 mmol, 1.03 eq.). The vial was evacuated and purged with N2 followed by addition of

1,4-dioxane (0.35 mL) and 0.5 M potassium phosphate tribasic (3 eq.). The reaction mixture was heated at 75 oC for 6 hrs. The reaction mixture was quenched with water (4 mL) and extracted with ethyl acetate (5x4 mL), dried with sodium sulfate and solvent was evaporated under reduced pressure. The crude product was purified with silica gel flash chromatography (50:50

1 EtOAc:Hex). 8 mg, 88%. H NMR (500 MHz, CDCl3) δ 8.85 (s, 1H, N-H), 8.41 (d, 1H, J=2.0

Hz), 7.52 (d, 1H, J=1.5 Hz), 7.02 (s, 1H), 6.85 (d, 1H, J=1.5 Hz), 4.16 (s, 3H), 4.00 (s, 3H), 3.99

13 (s, 3H), 3.96 (s, 3H), 3.91 (s, 2H) ppm. C NMR (125 MHz, CDCl3) δ 152.44, 142.72, 128.99,

128.97, 128.77, 128.40, 127.86, 127.19, 126.02, 119.73, 112.53, 112.10, 106.82, 96.90, 61.00,

56.47, 56.20, 51.90, 24.11 ppm. HRMS (TOF MS ES+) m/z calculated ([M+H]+) 438.1124 Da, found 438.1112 Da.

134

Methyl 7-(6-(cyanomethyl)-4-methoxybenzo[d]thiazol-2-yl)-5,6-dimethoxy-1H-indole-2- carboxylate (4-11)

To a 7 mL vial were added XPhosPdG2(4 mol%), (5,6-dimethoxy-2-(methoxycarbonyl)-1H- indol-7-yl) boronic acid (0.015 mmol), 2-(2-bromo-4-methoxybenzo[d]thiazol-6-yl) acetonitrile

(0.0155 mmol, 1.03 eq.). The vial was evacuated and purged with N2 followed by addition of

1,4-dioxane (0.35 mL) and 0.5 M potassium phosphate tribasic (3 eq.). The reaction mixture was heated at 75 ℃ for 6 hrs. The reaction mixture was quenched with water (4 mL) and extracted with ethyl acetate (5x4 mL), dried with sodium sulfate and solvent was evaporated under reduced pressure. The crude product was purified with silica gel flash chromatography (50:50

1 EtOAc:Hex). 5 mg, 70%. H NMR (500 MHz, CDCl3) δ 8.83 (s, 1H, N-H), 8.43 (d, 1H, J=2.0

Hz), 7.49 (d, 1H, J=1.5 Hz), 7.06 (s, 1H), 6.90 (d, 1H, J=1.5 Hz), 4.11 (s, 3H), 3.99 (s, 3H), 3.98

13 (s, 3H), 3.94 (s, 3H), 3.92 (s, 2H) ppm. C NMR (125 MHz, CDCl3) δ 153.16, 143.50, 129.79,

129.42, 128.27, 128.05, 126.89, 127.19, 126.51, 118.93, 112.95, 112.50, 107.20, 96.90, 61.49,

56.92, 56.65, 52.15, 24.60 ppm. HRMS (TOF MS ES+) m/z calculated ([M+H]+) 438.1124 Da, found 438.1112 Da.

135

5 SYNTHESIS, STRUCTURE AND PHOTOPHYSICAL PROPERTIES OF METAL DICYANIDE DONORS COORDINATED TO AZA[5]HELIZENE VIOLOGEN ACCEPTORS 5.1 Introduction This chapter describes our collaborative work with HHP group (UMaine) on synthesis and study of 2D network of gold-heli viologen as published in Dalton Transactions.199 Viologens are redox active, strong electron accepting diquaternary salts of 4, 4’-bipyridine with the ability to form ion- pair charge transfer complexes (IPCT) with donors such as dicyanoaurate. Charge transfer properties of IPCT can be applied in photochromic and electrochromic devices.200–203 The photoreduction properties of viologens have been known for several years with applications as herbicides, photocleaving agents, and electronic devices. However, the photophysical and photochemical properties of viologens are mostly unexplored.141 Phenylviologens such as 1,1’- bis-(2,4-dinitrophenyl)-4,4’-bipyridinium (DNP2+) have shown desirable optical and electronic properties due to extended π conjugation.150,204 The π–π stacking interactions of phenyl viologens are being explored in chemistry and biology due to its effect on the photophysical properties of 2D and 3D networks.205 In conjunction, dicyanoaurate(I) complexes are also of interest as they form one of the most stable two coordinate complexes, with reports of high stability constants as well as potential applications in semiconductor materials and photonic devices.147,148 Dicyanoaurate(I) has also unique geometric and charge-transfer properties as the d10 Au(I) metal center favors

− − aurophilic interactions and aggregation of [Au(CN)2 ]n , which allow tuning of the optical properties.149,150 Due to their strong electron accepting potential, viologens are luminescence quenchers of triplet metal-to-ligand charge transfer (MLCT) luminescence in complexes containing Au(I).

136

Figure 5-1 Previously studied DNP viologen and target Gold-heli viologen (5-13).

In previous studies by HHP group at University of Maine, low energy phosphorescence was observed from a 3D network consisting of DNP2+ coupled with dicyanoaurate(I) dimers (Figure

5-1).150 These results were surprising given that DNP2+ is non-luminescent in its free state. The observed luminescence in this complex revealed unique photophysical properties that were attributed to an IPCT transition arising from the heavy atom effect due to gold-viologen orbital overlap.150,206 It would be interesting to study the photochemical interaction of other methylated phenyl viologens which possess extended π conjugation and are luminescent in the free state with dicyanoaurate(I).

5,10-dimethyl-5,10-diaza[5] helicene bis-tetrafluoroborate viologen was synthesized a few years ago and the synthetic route has recently been optimized.141,206 In addition, it is one of the few viologens known which has chirality due to the helical aromatic heterocycle.141,207 It was shown that this compound has energy holding abilities and is a good electron transfer contender due to its

Ve being greater than Vh (electronic coupling for electron and hole respectively).208 The conductivity spectra and refractive index of this viologen highlighted its ability of high intra/inter molecular charge transfer within the crystal and ability to yield significant outputs with low input.

137

In this work, we extend the previous work of HHP group on methyl viologen (MV2+) systems by synthesizing 5,10-dimethyl-5,10-diaza[5]helicene (heli-viologen2+) and coupling it with two

− molecules of [Au(CN)2] to form complex 8 as shown in Figure 5-1. Synthesis and characterization of the N,N-dimethylaza[5]helicene dicyanoaurate (Au and Ag salts) complex was done using nuclear magnetic resonance (NMR). The crystal structure of Au heli-viologen was determined by single crystal X-ray diffraction. Optical properties of this complex were studied using temperature variable photoluminescence, diffuse reflectance spectroscopy (DRS), and Stern–Volmer analysis.

Density Functional Theory (DFT) calculations were performed to assist with interpretation of the experimental data.

5.2 Results and Discussion 5.2.1 Synthesis

Scheme 5-1 Retrosynthetic scheme for viologen synthesis.

In order to access the Gold and silver heli-viologens, the key intermediate was quinolyl-E-stilbene, which can be cyclized to form the aza-helicene structure. As shown in Scheme 5-1, aza-helicene can be methylated to get MV2+ species of the desired viologen, which can be coupled with gold or silver anionic species to get the desired metal-heli viologens. The key intermediate, quinolyl-E- stilbene was initially synthesized from stepwise wittig route, and the method was improved by utilizing Pd catalyzed Stille coupling or Hiyama-Heck coupling.

138

Scheme 5-2 Step-wise Wittig route to access quinolyl-E-stilbene (5-5).

As shown in Scheme 5-2, stilbene intermediate (5-5) was synthesized in four steps from quinoline-

3-carbaldehyde (5-1) via Wittig reaction.13 5-1 was reduced with sodium borohydride to yield primary alcohol of quinoline followed by chlorination using thionyl chloride to access 5-3. In order to perform Wittig reaction, Wittig salt (5-4) was synthesized from 5-3. Wittig reaction of 5-4 with aldehyde 5-1 resulted in formation of the new C-C bond resulting in quinolyl-E-stilbene (5-5).

Overall yield of this four-step route was ~20% due to poor yields at Wittig salt synthesis and Wittig reaction steps. One of the major reasons for product loss was due to difficulty in silica gel purification of product 5-5 due to presence of triphenylphosphine oxide. To improve the yield and efficiency of stilbene (5-5) synthesis, Pd catalyzed Stille coupling was explored. To perform Stille coupling, bis-tributylstannyl ethylene linker (5-8) was required, which was synthesized from tributylstannyl hydride (5-6) and tributylstannyl acetylene (5-7) in good yield (90%).

139

Table 5-1 Catalyst optimization for Stille coupling.

Pd(0) Catalyst Ligand Yield

Tetrakistriphenylphosphine palladium - 20% Pd(PPh ) 3 4 Triphenylphosphine PPh3 25%

Tris(dibenzylideneacetone)dipalladium Triphenylphosphine PPh3 27% Pd (dba) 2 3

Tris(o-tolyl)phosphine P(o-tolyl) 49% 3

2-dicyclohexylphosphino-2’,4’,6’- 57% triisopropylbiphenyl X-phos

140

Reaction condition optimizations were done in order to improve the yield of 5-5. Table 5-1 shows the reaction conditions and catalyst optimization reactions for Stille coupling. Reaction conditions shown above with Pd2(dba)3 as Pd(0) catalyst and X-phos as the ligand were the optimized condition resulting in 57% of the desired product. Later, Pd(II) catalyzed Hiyama–Heck cross- coupling was used with triethoxyvinylsilane to get better scalability and higher yield (91%) as shown by Clennan and coworkers in 2016.141

Figure 5-2 Photocyclization mechanism of 5-5.

Following the synthesis of Stilbene intermediate (5-5), photocyclization was done to synthesize,

5,10-Diaza[5]helicene (5-9). As described by Woodward-Hoffman rules of cyclization, 4n+2 cyclization of 5-5 is conrotatory and as a result is photochemically allowed.209 Figure 5-2 described the mechanism of photocyclization to synthesize 5-9. Several photocyclization conditions were explored based on UV-Vis absorption spectrum (Figure 5-3).

141

Figure 5-3 UV-Vis absorption spectrum of 5-5

Table 5-2 Light source optimization for photocyclization of 5-5

Light Source Intensity Time %Conversion %Yield(major

(lumens) (Hours) product)

Incandescent Lamp 600 48-52 90% 65%

Halogen Flood lamp 5000 55-60 86% 63%

Photo reactor (350 nm) 24000 4-6 100% 73%

Table 5-2 shows different light sources used to optimize photocyclization reaction yields.

Photocyclization generates desired product 5-9 and a side product 5-10. As shown in Table 5-2, incandescent lamp and halogen flood lamp gave good conversion and yield of the desired product, however the reaction times were longer, and reproducibility was inconsistent due to low amount of UV emitted by these light sources. Photocyclization in a photoreactor (350 nm),

142 which was equipped with a magnetic stir plate, and cooling fan to minimize the heating resulted in 100% conversion and 71% yield of the desired product in 4 to 6 hours.

Scheme 5-3 Gold-heli viologen (5-13) synthesis.

Subsequently, Methylation of 5-9 with trimethyloxonium tetrafluoroborate under nitrogen produced the BF4 salt of heli[5]viologen (5-11) which was purified with preparative HPLC. In order to couple the heli viologen with gold or silver dicyanide, the reaction needed to be done in aqueous solution. Due to the limited solubility of 5-11 in aqueous solvents, the tetrafluoroborate counterion was exchanged for chloride counterions using DOWEX ion-exchange resin. The chloride heli-viologen (5-12) showed high solubility in aqueous solvents and was further reacted with potassium dicyanoaurate to produce gold-coupled viologen, 5-13 in powder form. The powder was dried and used for luminescence experiments. Three crystallization methods were attempted in order to get single crystals for X-ray crystallography: Slow evaporation, liquid-liquid diffusion, and vapor diffusion method. Among which vapor diffusion using the solvent combinations below gave small single crystals for X-ray crystallography.

143

Figure 5-4 Single crystal synthesis via vapor-diffusion method.

The crystallization conditions via a vapor diffusion method for Au heli-viologen (5-13) were optimized and methanol: n-hexane and ethanol: cyclohexane were used as solvent : anti-solvent to get single crystals for X-ray diffraction analysis.210

5.2.2 Single crystal X-ray diffraction The solid state structure of Au heli-viologen crystallized in the centrosymmetric monoclinic space

- group C2/c and contained one viologen dication and two [Au(CN)2] anions on general positions as the elements of the asymmetric unit (Figure 5-5). When looking into the bc-plane of the unit

- cell, one of the [Au(CN)2] anions lies perpendicular to this plane (and parallel to the a-axis) at a distance of 3.3773(6) Å between the Au(I) atom of the dicyanoaurate anion and the centroid of the aromatic ring sitting at the turn of the right-handed helical viologen. Typically, dicyanoaurate anions are linear with N–Au–N angles approaching 180°; however within the structure of Au heli-

- viologen, the [Au(CN)2] anion lying above the turn of the helicate deviates from linearity, showing an N–Au–N angle of 174.0(3)°.211 Such a deviation from linearity can be rationalized upon the application of symmetry to the asymmetric unit. After applying crystallographic symmetry, this dicyanoaurate anion dimerizes with itself and supports the formation of a bond between the monovalent gold atoms at a distance of 3.3098(11) Å, a value on par with those published in a previous report.212

144

Figure 5-5 View along the a-axis of Au heli-viologen crystal. The dotted lines show dimer - interactions among adjacent [Au(CN)2] anions.

- As a direct consequence of the binding of the neighboring, symmetry-equivalent [Au(CN)2] anions to one another, their dimerization imparts some structural rigidity as evidenced by the smaller size of the thermal parameters of the dimer’s constituent gold, carbon and nitrogen atoms.

Projections of the asymmetric unit along the b-axis (Figure 5-5) demonstrated strong one-

- dimensional overlap between the dimerized [Au(CN)2] anions and the nearly-planar region of the viologen dications parallel to that axis. Such an orientation of these moieties relative to one another within the solid-state structure may provide evidence for the onset of interesting spectroscopic properties. Despite the potential for the dicyanoaurate anion located above the turn of the helical viologen in concert with its propensity to dimerize upon the application of crystallographic

- symmetry to generate interesting spectroscopic features, the second [Au(CN)2] anion in the asymmetric unit does not dimerize upon the application of crystallographic symmetry. Unlike the anion in the asymmetric unit which does dimerize, the second anion has much more linear geometry as evidenced by its N–Au–N angle of 176.5(5)°. Thermal parameters for the second dicyanoaurate anion also demonstrate more thermal motion as evidenced by the larger anisotropic

145 displacement ellipsoids for its constituent gold, carbon and nitrogen atoms. Between the anion and the helical viologen, the closest contact occurred between the centroid of the phenyl at the turn in the helix and N4 from the terminal nitrile at a distance of 3.351(19) Å. This distance suggests the onset of a potential dipole–dipole interaction given the positioning of the electron-rich nitrile relative to the electron deficient phenyl ring of the dication.31 Also, given the less intimate molecular orbital overlap between this latter dicyanoaurate and the planar surface of the viologen,

- suggests that unlike the former [Au(CN)2] , the latter is more spectroscopically innocent. As a consequence, Au heli-viologen can be described as a 2D network consisting of a series of

2- 2+ noninteracting stacked [Au(CN)2]2 / heli-viologen slabs.

- Analysis of the bond distances within the different [Au(CN)2] anions as a function of their being free or dimerized in the solid state also revealed subtle differences. Within the dicyanoaurate anion that dimerizes, the bond distances between the nitriles and the Au(I) were measured at distances of 2.007(16) and 2.011(15) Å while those in the free anion were measured at 1.98(2) and 1.94(2)

Å respectively. The nitrile distances from the different anions also revealed differences as those from the dimerized anion were measured at 1.09(2) and 1.132(19) Å while their analogues from

- the free [Au(CN)2] revealed distances of 1.12(3) and 1.17(3) Å between the constituent carbon and nitrogen atoms. Such a difference can be hypothesized on the basis of dimerization between the neighboring dicaynoaurate anions upon the application of symmetry. Electron density is donated from the nitrile into the corresponding Au–C bond, resulting in a shortening of the former and a lengthening of the latter. This build-up of electron density on the monovalent Au atom, in concert with the nearby positioning of a symmetry-related anion, supports bond formation between the gold atoms. Relevant bond distances within the free dicyanoaurate anion are similar to those

146 reported in the literature as no intramolecular donation of electron density to support Au–Au bond formation occurs.211

5.2.3 Infrared spectroscopy

Figure 5-6 Infrared spectrum of Au heli viologen (5-13).

Au heli-viologen (5-13) is a coordination polymer, where a dominant vibrational feature is the

-1 -1 cyanide stretch. The νs(CN) of the gold(I) dicyanide anions are observed at 2140 cm , 2152 cm ,

2160 cm-1, and 2165 cm-1, indicating that the CN ligands are slightly inequivalent throughout the crystal motif. This split is in agreement with the structural data which shows the presence of

- 2- monomer [Au(CN)2] and dimer [Au(CN)2]2 units. For oligomer units of metal cyanides, the

νs(CN) vibrational mode moves to lower energies as the oligomer size increases due to the increased electron density of the CN π* LUMO.213 In keeping with this, we have assigned the

147

-1 -1 -1 bands at 2140 cm and 2152 cm to the νs(CN) vibration modes of the dimers and the 2160 cm and 2165 cm-1 bands to the vibrational modes of the monomer subunits.

5.2.4 Photophysical studies To investigate the absorption of Au heli-viologen, we have performed diffuse reflectance measurements of solid microcrystalline samples of both Au heli-viologen (5-13) and MV2+ at 298

K (Figure 5-7). Solid samples of gold coordinated MV2+ are yellow in color and absorb strongly in the UV.

heli[5]viologen

Au heli-viologen Normalized Normalized Absorbance

225 325 425 525 625 725 Wavelength (nm)

Figure 5-7 Solid state DRS absorption spectrum of solid samples of Au heli-viologen and MV2+ at 298 K converted via Kubelka-Munk.

As seen in the spectra, the absorption falls off sharply around 475 nm with an optical absorption edge of 2.39 eV. This edge is slightly more variable for the gold free heli-viologen2+ which slowly tappers off through the visible range and is identical to previous reports of MV2+ which are

141 - assigned to a π → π* state. Introduction of [Au(CN)2] results in few changes in the DRS spectra in comparison to heli-viologen2+.

148

Figure 5-8 Luminescence spectra of Au heli-viologen between 10 K and 298 K. Excitation (dashed) and emission (solid).

The appearance of higher energy bands at 268 nm not observed in the gold free heli-viologen2+ are

- 2- assigned to a metal to ligand charge transfer (MLCT) localized to the [Au(CN)2] /[Au(CN)2]2 subunits consistent with reports elsewhere.214 The majority of the band remains a π → π* state of the heli-viologen2+.

As an aromatic cation, heli-viologen2+ is well equipped to accept electrons from the electron rich gold(I) dicyanide anions as reported for other viologen like molecules. To probe this temperature variable photoluminescence measurements of microcrystalline samples of Au heli-viologen between 298 K and 10 K were performed. The spectra are shown in Figure 5-8 and are overlaid to demonstrate the change in peak shape and relative intensity with respect to temperature. We observe that the excitation spectrum of Au heli-viologen (5-13) consists of two bands: one high energy band at 375 nm, and one low energy band at 470 nm. A shift in the relative intensity towards the high energy band is observed upon temperature change. No significant shift in the position of

149 either excitation band is observed at lower temperatures. At room temperature, compound 5-13 in microcrystalline form shows one broad triplet emission band at 580 nm (τ298 K = 1.01 μs, τ78 K =

1.12 μs). At lower temperatures the relative emission intensity increases and a second, lower- energy band appears at 608 nm. This band first appears at 129 K and does not shift during cooling.

Time-dependent measurements show much shorter lifetime of this band (τ78 K = 130 ns) in comparison to that of the band at 580 nm.

2+ Heli-viologen

Figure 5-9 Energy diagram of emission pathways for Au heli-viologen. At higher temperatures 2+ -2 the heli-viologen emission by T1→S0 is favored along with quenching of [Au(CN)2] via the 2+ excited S1 to heli-viologen S1. At low temperatures (<129 K) electron transfer is absent resulting -2 in [Au(CN)2] S1→S0 emission. S0, ground state; S1, first excited state; T1, first triplet state; NR, nonradiative decay; ET, electron transfer

Based on previous reports of heli-viologen2+ the data reported here, the 580 nm band is assigned to a triplet π → π* localized to heli-viologen2+. The emission band observed at 608 nm is a new feature and was not observed in previous reports of heli-viologen2+. Both the life-time and

150 wavelength of this band are too long for cation S1→S0 emission. Instead, these values better fit

- - 2+ expected values for [Au(CN)2] emission. We have observed similar behavior in Au(CN)2 /DNP

- structures where low temperature emission is dominated by [Au(CN)2] bands but quenched at 298

150 - 2+ K. The absence of [Au(CN)2] emission above 129 K indicates quenching by heli-viologen at high temperatures. It is attributed to an oxidative electron transfer in the excited state at higher

- 2+ temperatures between [Au(CN)2] and heli-viologen (Figure 5-9).

5.2.5 Electron transfer analysis An analysis of this system was performed via Stern–Volmer and a Rehm–Weller to support the proposed electron transfer. First, a Stern–Volmer analysis was performed in order to examine the

2+ - nature of heli-viologen quenching of [Au(CN)2] , specifically to determine if the observed quenching is due to electron transfer or resonance.215 First, the ground state absorption spectra of the components were compared to the combined mixture.

2.5 Potassium dicyanoaurate

2 Heli[5]viologen Au heli-viologen

1.5 Absorbance

0.5

0 200 220 240 260 280 300 320 340 Wavelength (nm)

Figure 5-10 Absorption spectra of potassium dicyanoaurate, heli[5]viologen, and Au heli- viologen in methanol (5 x 10-4 M).

Figure 5-10 shows that the absorption spectrum of a mixture of heli-viologen2+ and potassium dicyanoaurate is the simple sum of the two component absorption spectra. The lack of an additional

151 absorption band in the mixed spectrum indicates that no ground state electronic interaction occurs between the two species. From this observation, we conclude that no static quenching occurs in this system. While our previous experiment indicates no ground-state quenching, the possibility of quenching in the excited-state was further investigated. The effect of heli-viologen2+ as a dynamic

- quencher of [Au(CN)2] photoluminescence was investigated by measuring the photoluminescence of a series of heli-viologen2+/dicyanoaurate solutions. Luminescence intensity associated with

- 2+ [Au(CN)2] was observed to decrease as the concentration of heli-viologen was increased. Since we observe photoluminescence quenching upon addition of heli-viologen2+, this data can be fit to the Stern–Volmer equation to further analyze the photophysical kinetics of the system.

1.8

1.5

0 I/I y = 5167.3x + 0.9869 1.2 R² = 0.9973

0.9 0 0.00004 0.00008 0.00012 [Q] (M) Figure 5-11 Stern-Volmer plot for aqueous Au heli-viologen.

Figure 5-11 shows the Stern–Volmer plot constructed for this experiment. From this plot a KSV

-1 value can be determined (KSV = 5170 M ). The data show that quenching is observed for this system. This indicates that there is a significant interaction between gold dicyanoaurate species and heli-viologen2+ during the photophysical excitation and relaxation process. This observation

152 provides further support for our assignment of the emission band observed at 608 nm as a

- 2+ [Au(CN)2] →heli-viologen electron transfer.

The Rehm–Weller equation35 can be used to support the proposed electron transfer by determining whether the process is thermodynamically favorable.

In the Rehm–Weller equation shown above, ΔGET is the free energy change associated with the

2+ electron transfer process, Ered and Eox are the reduction potential of the viologen heli-viologen

2- acceptor and oxidation potential of the [Au(CN)2]2 donor respectively. Es represents the energy of the singlet state energy at 78 K (average of the lowest energy excitation band and highest energy

2 206,216 emission band), and eo /εa is taken as the attraction that the ion pair experiences. We have calculated the Eox (1.74 eV) via DFT by taking the difference in energy between an optimized

2- - structure of [Au(CN)2]2 and [Au(CN)2] . For the Au heli-viologen, ΔGET is therefore calculated as: ((1.74 + 0.22)-2.32-0.15) = -0.51 eV, indicating the electron transfer is spontaneous. This calculated free energy change supports an electron transfer process as a feasible explanation for the observed quenching system.

5.2.6 Ag-Heli Viologen (5-14) Chloride-heli viologen (5-12) was coupled with potassium dicyanoargentate in order to synthesize silver-heli[5] viologen (5-14). Several attempts were made to synthesize and recover 5-14, which were semi-successful as the powder did not precipitate out of solution as easily. When precipitation was induced by solvent evaporation, inorganic salts precipitated out along with the desired metal organic complex and isolation of 5-14 could not be confirmed. As shown in Figure 5-12, potassium

-1 dicyanoargentate showed that νs(CN) vibration mode is observed at 2136 cm . Synthesized and

-1 precipitated potential Ag-heli viologen sample showed νs(CN) vibrational mode at 2162 cm

153 similar to Au-heli viologen. In addition low energy vibrational modes of aza-helicine system are observed below 1600 cm-1.

Figure 5-12 Infrared spectra of KAg(CN)2 (upper) and potential Ag-Heli viologen (lower)

However, this product could not be confirmed with 1H NMR, 13C NMR or MS. Single crystal synthesis was attempted on precipitated powder using vapor-diffusion methods but these attempts were unsuccessful. Synthesis and photophysical properties of Ag-heli viologen need to be studied in a future to compare the 2D structure of Ag-heli viologen to the synthesized Au-heli viologen.

5.3 Conclusion and Future direction Au-heli viologen (5-13) was synthesized from 4-bromoquinoline via Pd catalyzed Stille coupling/ Pd catalyzed Hiyama-Heck coupling, photocyclization, methylation and metal

154 coupling reactions with overall yield of 22%. Single crystals of Au-heli viologen (5-13) were accessed via vapor-diffusion, and characterized by single crystal X-ray diffraction. The 2D

− network was found to consist of [Au(CN)2] dimers and monomers trapped within heli- viologen2+ units. Photophysical studies show that at 298 K this complex emits at 580 nm via a triplet π → π* transition localized to the heli-viologen2+. Cooling to low temperatures results in a new emission band at 608 nm. Lifetime measurements and DFT calculations indicate that this

2− new band is the appearance of [Au(CN)2]2 emission. The lack of this emission at temperatures higher than 129 K indicates that quenching occur from heli-viologen2+. Stern–Volmer and

Rehm–Weller analysis of this transition supports our assignment by revealing that quenching of

2− 2+ [Au(CN)2]2 by heli-viologen involves the transfer of an electron between ion pairs. Synthesis of Ag-heli viologen was attempted but crystallization and structure confirmation were unsuccessful. In a future study 2D/3D networks of Ag-heli viologen involving better electron donors such as dicyanoargentate would provide insight into the electron-transfer phenomenon investigated here.

5.4 Experimental 5.4.1 General Information All reagents were purchased from Sigma-Aldrich and Fisher Scientific and used directly without further purification. All products were purified with silica-gel flash chromatography or preparative HPLC. HPLC was performed on a Gilson analytical to semi-preparative system using Kinetex 5 µm EVO C18 100Å 4.6 x 150 mm for analytical and Kinetex 5 µm EVO C18 100Å 21.1 x 150 mm for preparative purpose. Products were characterized by 1H and 13C NMR and mass spectrometry. NMR characterization results and J values were compared with published results. 1H and 13C NMR spectra were recorded on Agilent/Varian 400 MHz and 101 MHz NMR spectrometer respectively. Mass spectra

155 were recorded using a Synapt G2 Q-TOF ESI mass spectrometer at the Mass Spectrometry lab at the University of Illinois Urbana Champagne.

5.4.2 Synthesis of (E)-1,2-di(quinoline-3-yl)ethene (5-5) 5.4.2.1 Step-wise Wittig route Quinolin-3-ylmethanol (5-2)

In a 7 mL scintillation vial, 3-quinolinecarboxaldehyde (150 mg, 0.95 mmol) was dissolved in ethanol (2 mL) followed by addition of sodium borohydride (50 mg, 1.32 mmol, 1.4 eq.).

Mixture was stirred at rt for 2 hrs. The mixture was diluted with diethy ether (5 mL) and washed with water (2 x 5 mL). The aqueous phase was extracted with diethyl ether (2 x 5 mL) and combined organic phases were dried with sodium sulfate (Na2SO4) and

1 concentrated. 132 mg, 87% yield. H NMR (400 MHz, CDCl3) δ 8.74 (d, 1H, J=2.0 Hz),

8.06 (s, 1H), 8.00 (d, 1H, J=8.8 Hz), 7.70 (d, 1H, J=8.0 Hz), 7.62 (m,1H), 7.48 (m, 1H),

4.83 (s, 2H) ppm. Spectral data matched with those in literature.207

3-chloromethylquinoline (5-3)

In a 7 mL scintillation vial quinolin-3-ylmethanol (130 mg, 0.82 mmol) in dry methylene chloride (5 mL) under N2 at 0 °C was added thionyl chloride (1.1 mL, 16.3 mmol, ~20 eq.) was added dropwise. The mixture was warmed up to rt and stirred for 1 hr. The mixture was concentrated to dryness under vacuum to give 3-chloromethylquinoline. 140 mg, 96% 156

1 yield. H NMR (400 MHz, CDCl3) δ 9.42 (s, 1H), 9.33 (s, 1H), 8.38 (d, 1H, J=8.4 Hz),

8.33 (d, 1H, J=8.8 Hz), 8.20 (m,1H), 8.01 (m, 1H), 5.12 (s, 2H) ppm. Spectral data matched with those in literature.207

Triphenyl(quinolin-3-ylmethyl)phosphonium chloride (5-4)

In a 20 mL scintillation vial, 3-chloromethylquinoline (135 mg, 0.76 mmol) was dissolved in toluene (7 mL) followed by addition of triphenylphosphine (200 mg, 0.76 mmol).

Reaction mixture was refluxed at 110 °C for 6 hrs. Solid was filtered, washed with toluene

1 and dried to get wittig salt. 187 mg, 56%. H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H, J=2.0

Hz), 8.04 (s, 1H), 7.80 (d, 1H, J=8.4 Hz), 7.69 (m, 8H,), 7.66 (d,1H), 7.55 (m, 7H), 7.51

(m, 1H), 7.37 (m, 1H), 5.60 (d, 2H, JH-P=14.8 Hz ) ppm. Spectral data matched with those in literature.207

Wittig reaction (synthesis of (E)-1,2-di(quinoline-3-yl)ethane) (5-5)

In a 7 mL scintillation vial, to a solution of triphenyl(quinolin-3-ylmethyl)phosphonium chloride (117 mg, 0.25 mmol) and 3-quinolinecarbaldehyde (40 mg, 0.25 mmol) in methanol (4 mL) was added sodium methoxide (0.3 mL, 0.3 mmol, 1M in MeOH) dropwise. Mixture was refluxed at 65 °C for 4 hrs. After cooling the mixture, water (20 mL) was added the mixture was extracted with methylene chloride (4 x 20 mL). Due to

157 significant presence of triphenylphosphine oxide, the product was purified with silica gel flash chromatography (60:40 EtOAc:Hexane). 31 mg, 43%

5.4.2.2 Stille coupling to synthesize 5-5 Bis-1,2-tributylstannyl ethylene synthesis (5-9)

Tributylstannyl acetylene (328 mg, 1 mmol) & tributylstannyl hydride (291 mg, 1 mmol) were added to a vial with AIBN (3.3 mg, 0.02 mmol). The mixture was heated and stirred at 100 oC for 6 hrs. Water was added to the reaction mixture and the product was extracted with four aliquots of methylene chloride and dried over Na2SO4. The solvent was evaporated under reduced pressure yielding 5-9. This product was used in the following

1 step without further purification. 549 mg, 90%. H NMR (400 MHz, CDCl3) δ 6.89 (s,

2H), 1.46-1.64 (m, 12H), 1.25-1.44 (m, 12H), 0.80-0.98 (m, 30H) ppm. Spectral data matched with those in literature.217

(E)-1,2-di(quinoline-3-yl)ethane(5-5)

3-Bromoquinoline (131 mg,0.63 mmol) and bis-1,2-tributylstannyl ethylene (194 mg, 0.32 mmol) were added to a vial purged with N2. Pd2(dba)3 catalyst (5 mol%) & X-phos (0.1 eq.) were added to the reaction vial, followed by addition of dry toluene (4 mL) under N2.

The reaction mixture was heated at 110 oC. The solvent was evaporated under reduced pressure and the crude product was purified with flash chromatography using 70%:30%

158 ethyl acetate:hexane mixture with 1% triethylamine to get (E)-1,2-Di(quinoline-3- yl)ethane. 51 mg, 57%. Same procedure was followed for other catalysts and ligands (Table

5-1) during optimizations with catalyst amounts varying from 5-10 mol% and ligand (0.1-

0.2 eq.).

5.4.2.3 Hiyama-Heck Coupling

A mixture of triethoxyvinylsilane (0.43 g, 2.25 mmol) and sodium hydroxide (0.5 M, 18 mL) was added to a 40 mL pressure vial, followed by addition of 3-bromoquinoline (1)

(0.75 g, 3.60 mmol) & palladium acetate (0.5 mol%). The reaction vial was sealed with a heat resistant teflon resistant screw cap and the mixture was stirred at 140 oC for 6 hrs.

After cooling, the green precipitate was filtered and washed with 10:1 ethyl acetate:chloroform. The solvent was evaporated under vacuum. 460 mg, 91%. 1H NMR

(400 MHz, CDCl3) δ 9.19 (d, 2H, J=2.1 Hz), 8.25 (d, 2H, J=2.1 Hz), 8.11 (d, 2H, J=8.4

Hz), 7.86 (d, 2H, J=8.0 Hz), 7.72 (ddd, 2H, J=8.3,7.0,0.9 Hz), 7.58 (ddd, 2H, J=8.1, 6.7,

0.9 Hz), 7.47 (s, 2H) ppm. 13C NMR (101 MHz, DMSO-d6) δ 175.40,

151.16,149.62,145.85, 137.60, 130.34, 130.07, 128.96, 127.61, 116.79 ppm. HRMS (TOF

+ MS ES+) m/z calculated for C20H15N2 ([M+H] ) 283.1235 Da, found 283.1231Da.

159

5.4.3 Photocyclization to synthesize 5,10-diaza[5]helicene (5-9)

(E)-1,2-di(quinoline-3-yl)ethane (0.6 g, 2.1 mmol) was dissolved in ethyl acetate (500 mL) in a round bottom flask. A condenser was attached to the top and the setup was mounted inside an RPR-100 photoreactor on a stir-plate.

The mixture was stirred and irradiated at 350 nm for 7 hrs. The crude product was purified with silica-gel chromatography using 70%:30% ethyl acetate:hexane. 430 mg, 73%. 1H

NMR (400 MHz, CDCl3) δ 9.46 (s, 2H), 8.65 (dd, 2H, J=8.4, 1.2 Hz), 8.29 (dd, 2H, J=8.3,

1.3 Hz), 8.14 (s, 2H), 7.76 (ddd, 2H, J=8.3, 7.0, 1.3 Hz), 7.44 (ddd, 2H, J=8.4, 7.0, 1.3 Hz)

13 ppm. C NMR (101 MHz, CDCl3) δ 152.53, 145.88, 129.71, 129.48, 129.43, 128.17,

127.32, 127.26, 125.61, 124.38 ppm. HRMS (TOF MS ES+) m/z calculated for C20H13N2

([M+H]+) 281.1079 Da, found 281.1070 Da. Benzo-[b]-1,8-diaza[4]-helicene (6) was

160

1 recovered as a side-product. Yield: 110 mg, 19%. H NMR (400 MHz, CDCl3) δ 11.37 (d,

1H), 9.37 (s, 1H), 8.75 (s, 1H), 8.49 (d, 2H, J=8.7 Hz), 8.30 (d, 2H, J=8.1 Hz), 8.09-7.98

(m, 2H), 7.93-7.83 (m, 4H), 7.67 (dd, 1H, J=7.96, 6.92 Hz) ppm.

5.4.4 Synthesis of dimethylaza[5]helicene Dimethylaza[5]helicene bis-tetrafluoroborate (5-11)

To trimethyl oxonium tetrafluoroborate (Meerwein’s salt, 0.356 g, 2.39 mmol) in a 40 mL scintillation vial was added anhydrous methylene chloride (100 mL) under N2. 5,10- diaza[5]helicene (5-9) (0.223 g, 0.80 mmol) in anhydrous methylene chloride (20 mL) was then added to the vial under N2. The vial was stirred under N2 for 24 hrs. Methylene chloride was removed under vacuum. The dried yellow solid was triturated with warm 90% ethanol.

The crude (5-11) was further purified with preparative HPLC using 5% to 15% acetonitrile

1 in 10 minutes. Yield: 233 mg (60%). H NMR (400 MHz, CD3CN) δ 10.04 (d, 2H, J=2.2

Hz), 8.72 (d, 2H, J=8.5 Hz), 8.67 (d, 2H, J=1.5 Hz), 8.57 (d, 2H, J=8.5 Hz), 8.24 (ddd, 2H,

J=8.7, 7.4, 1.3 Hz), 7.88 (ddd, 2H, J=8.3, 7.3, 1.0 Hz), 4.82 (s, 6H) ppm. 13C NMR (101

MHz, CD3CN) δ 154.82, 136.93, 135.12, 133.74, 131.78, 131.06, 129.86, 129.32, 127.33,

+2 120.34, 47.60 ppm. HRMS (TOF MS ES+) m/z calculated for C20H15N2 ([M] ) 155.0735

Da, found 155.0731 Da.

161

Dimethylaza[5]helicene dichlorate (5-12)

DOWEX 1×8 200-400 mesh ion-exchange resin (Acros) (11.25 g) was loaded onto a column and washed with methanol followed by equilibration with 50% aqueous ethanol.

Compound 5-11 (225 mg, 0.46 mmol) dissolved in acetonitrile (0.5 mL) was loaded into the column. The column was washed with 50% aqueous ethanol. The fractions were concentrated and reloaded on the column and collected two more times. Combined fractions were concentrated under reduced pressure to yield the product (5-12). Yield: 167

1 mg (95%). H NMR (400 MHz, CD3OD) δ 10.54 (d, 2H, J=2.2 Hz), 8.86 (d, 2H, J=8.4

Hz), 8.82 (d, 2H, J=1.2 Hz), 8.75 (d, 2H, J=8.7 Hz), 8.28 (ddd, 2H, J=8.6, 7.3, 1.3 Hz),

13 7.92 (ddd, 2H, J=8.2, 7.3, 1.0 Hz), 4.99 (s, 6H) ppm. C NMR (101 MHz, CD3OD) δ

155.89, 137.21, 135.33, 133.99, 132.12, 130.97, 130.46, 129.73, 127.63, 120.59, 47.12

+2 ppm. HRMS (TOF MS ES+) m/z calculated for C20H15N2 ([M] ) 155.0735 Da, found

155.0735 Da.

162

5.4.5 Synthesis of Au heli-viologen(5-13)

To a solution of N,N-Dimethylaza[5]helicene dichloride (5-12) (40 mg, 0.104 mmol) in water (30 mL), potassium dicyanoaurate (60 mg,0.21 mmol) in water was added while stirring. The precipitate was filtered after 10-12 hours and solvent was slowly evaporated to complete the precipitation of N,N-Dimethylaza[5]helicene dicyanoaurate (5-13). Yellow solid was dried under reduced pressure and used for crystallization. Yield- 49 mg (58%).

Crystallization. Au-heli viologen (Complex 5-13) (10 mg) was dissolved in ethanol (0.2 mL) in a 2 mL scintillation vial. This vial was covered with porous aluminum foil and placed in a 20 mL scintillation vial containing cyclohexane (2 mL). This closed system was kept inside the fume hood for 8 to 9 days. Small crystals formed through slow vapor diffusion. Results were reproduced with methanol as a solvent and n-hexane as anti- solvent.210

1 H NMR (400 MHz, CD3OD) δ 10.49 (d, 2H, J=2.2 Hz), 8.89 (d, 2H, J=8.8 Hz), 8.80 (d,

2H, J=2.1 Hz), 8.75 (d, 2H, J=9.0 Hz), 8.29 (ddd, 2H, J=8.6, 7.5, 1.1 Hz), 7.95 (ddd, 2H,

13 J=8.6, 7.5, 1.1 Hz), 4.97 (s, 6H) ppm. C NMR (101 MHz, D2O) δ 153.67, 135.70, 134.10,

133.08, 130.69, 129.96, 129.93, 128.96, 128.51, 126.43, 119.03 ppm.

5.4.6 Crystallography A small yellow block-like crystal of Au heliviologen having dimensions 0.057 × 0.063 × 0.092 mm3 was secured to a Mitegen cryomount using Paratone oil. Single crystal reflection data were

163 collected on a Rigaku Oxford Diffraction (ROD) Synergy-S X-ray diffractometer equipped with a

HyPix-6000HE hybrid photon counting (HPC) detector. The data were collected at 100 K using

Mo Kα1 radiation from a data collection strategy calculated using CrysAlisPro which was also responsible for unit cell determination, initial indexing, data collection, frame integration, Lorentz- polarization corrections and final cell parameter calculations.218 A numerical absorption correction via face indexing was performed using the SCALE3 ABSPACK algorithm integrated into

CrysAlisPro.17. The crystal structure was solved via intrinsic phasing using ShelXT and refined using olex2. refine within the Olex2 graphical user interface.219–221 The structural model’s space group was unambiguously verified by PLATON.21

Structural refinement of the reflection data for 8 resulted in the identification of electron density peaks associated with a highly disordered interstitial CH2Cl2 molecule that could not be satisfactorily modeled. This minor impurity may have been trapped during screening of solvents for crystallization. The data were treated using the SQUEEZE routine in PLATON, then refined on F2 to acceptable levels.222 The final structural refinement included anisotropic temperature factors on all nonhydrogen atoms and hydrogen atoms were attached via the riding model at calculated positions using appropriate HFIX commands. The crystallographic and refinement data for Au heli-viologen is listed in Table 1. Crystallographic data for this paper has been deposited with the Cambridge Crystallographic Data Centre with the CCDC number 1558366.

5.4.7 Spectroscopy studies Steady-state photoluminescence scans were collected between 298 K and 10 K. Spectra were taken with a Model Quantamaster-1046 photoluminescence spectrophotometer from Photon Technology

International using a 75 W xenon arc lamp combined with two excitation monochromators and one emission monochromator. A photomultiplier tube at 800 V was used as the emission detector.

The solid samples were mounted on a copper plate using non-emitting copper-dust high vacuum

164 grease. All scans were collected under vacuum with a Janis ST-100 optical cryostat. Infrared spectra were collected on solid samples at 298 K using a PerkinElmer FT-IR Spectrum Two equipped with a Universal Attenuated Total Reflectance (UATR) accessory and a LiTaO3 MIR detector. Diffuse reflectance spectra were collected on solid samples at 298 K. The light source was a Mikropack DH-2000 deuterium and halogen light source coupled with an Ocean Optics

USB4000 detector. Scattered light was collected with a fiber optic cable. Spectra was referenced with polytetrafluoroethylene. Data was processed using SpectraSuite 1.4.2_09.

5.4.8 Stern–Volmer quenching experiments For quenching experiments, heli-viologen2+ was used as the quencher. After determining proper

- quencher concentration ranges, the quenching experiment performed using the [Au(CN)2] sample

-4 solutions. A stock solution of 5 x 10 M K[Au(CN)2]2 was first prepared, and then 1 mL stock solution was distributed among all quenching vials. The quencher solution was then added to the quenching vials in increments of 50 μL. The contents of each vial were then transferred to cuvettes and the emission spectrum of each sample was measured separately. Emission scans were conducted on a Jobin Yvon Systems Fluorolog 3 Spectrofluorometer. The excitation monochromator was referenced to a secondary detector, and the emission monochromator was calibrated to a water Raman peak to ensure the accuracy of scans.

165

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APPENDIX A NMR SPECTRA FOR CHAPTER 2

Figure A 1 1H NMR spectrum of 2-1

Figure A 2 13C NMR spectrum of 2-1

186

Figure A 3 1H NMR of 2-2

Figure A 4 13C NMR of 2-2

187

Figure A 5 1H NMR of 2-3

Figure A 6 13C NMR of 2-3 188

Figure A 7 1H NMR of 2-18

Figure A 8 13C NMR of 2-18

189

Figure A 9 1H NMR of 2-19

Figure A 10 13C NMR of 2-19

190

Figure A 11 1H NMR of 2-4

Figure A 12 13C NMR of 2-4

191

Figure A 13 1H NMR of 2-22

Figure A 14 13C NMR of 2-22

192

Figure A 15 1H NMR of 2-23

Figure A 16 13C NMR of 2-23

193

Figure A 17 1H NMR of 2-5

Figure A 18 13C NMR of 2-5

194

Figure A 19 1H NMR of 2-6

Figure A 20 13C NMR of 2-6

195

Figure A 21 1H NMR of 2-7

Figure A 22 13C NMR of 2-7

196

Figure A 23 1H NMR of 2-8

Figure A 24 13C NMR of 2-8

197

Figure A 25 1H NMR of 2-20

Figure A 26 13C NMR of 2-20

198

Figure A 27 1H NMR of 2-21

Figure A 28 13C NMR of 2-21

199

Figure A 29 1H NMR of 2-9

Figure A 30 13C NMR of 2-9

200

Figure A 31 1H NMR of 2-10

Figure A 32 13C NMR of 2-10

201

Figure A 33 1H NMR of 2-11

Figure A 34 13C NMR of 2-11

202

Figure A 35 1H NMR of 2-12

Figure A 36 13C NMR of 2-12

203

Figure A 37 1H NMR of 2-24

Figure A 38 13C NMR of 2-24

204

Figure A 39 1H NMR of 2-25

Figure A 40 13C NMR of 2-25

205

Figure A 41 1H NMR of 2-14

Figure A 42 13C NMR of 2-14

206

Figure A 43 1H NMR of 2-15

Figure A 44 13C NMR of 2-15

207

Figure A 45 1H NMR of 2-17

Figure A 46 13C NMR of 2-17

208

Figure A 47 1H NMR of 2-16

Figure A 48 13C NMR of 2-16

209

APPENDIX B NMR SPECTRA FOR CHAPTER 3

Figure B 1 1H NMR of 3-5

Figure B 2 13C NMR of 3-5

210

Figure B 3 1H NMR of 3-6

Figure B 4 13C NMR of 3-6

211

Figure B 5 1H NMR of 3-7

Figure B 6 13C NMR of 3-7

212

Figure B 7 1H NMR of 3-8

Figure B 8 13C NMR of 3-8

213

Figure B 9 1H NMR of 3-9

Figure B 10 13C NMR of 3-9

214

Figure B 11 1H NMR of 3-10

Figure B 12 13C NMR of 3-10

215

Figure B 13 1H NMR of 3-11

Figure B 14 13C NMR of 3-11

216

Figure B 15 1H NMR of 3-12

Figure B 16 13C NMR of 3-12

217

Figure B 17 1H NMR of 3-13

Figure B 18 13C NMR of 3-13

218

Figure B 19 1H NMR of 3-14

Figure B 20 13C NMR of 3-14

219

1 Figure B 21 H NMR of 3-17

Figure B 22 13C NMR of 3-17

220

Figure B 23 1H NMR of 3-18

Figure B 24 13C NMR of 3-18

221

Figure B 25 1H NMR of 3-19

Figure B 26 13C NMR of 3-19

222

Figure B 27 1H NMR of 3-20

Figure B 28 13C NMR of 3-20

223

Figure B 29 1H NMR of 3-21

Figure B 30 13C NMR of 3-21

224

Figure B 31 1H NMR of 3-22

Figure B 32 13C NMR of 3-22

225

Figure B 33 1H NMR of 3-23

Figure B 34 13C NMR of 3-23

226

Figure B 35 1H NMR of 3-24

Figure B 36 13C NMR of 3-24

227

Figure B 37 1H NMR of 3-26

Figure B 38 13C NMR of 3-26

228

Figure B 39 1H NMR of 3-27

Figure B 40 13C NMR of 3-27

229

Figure B 41 1H NMR of 3-29

Figure B 42 13C NMR of 3-29

230

Figure B 43 1H NMR of 3-30

Figure B 44 13C NMR of 3-30

231

Figure B 45 1H NMR of 3-31

Figure B 46 13C NMR of 3-31

232

Figure B 47 1H NMR of 3-33

Figure B 48 13C NMR of 3-33

233

Figure B 49 1H NMR of 3-32

Figure B 50 13C NMR of 3-32

234

APPENDIX C NMR SPECTRA FOR CHAPTER 4

Figure C 1 1H NMR of 4-1

Figure C 2 1H NMR of 4-3

235

Figure C 3 1H NMR of 4-6

Figure C 4 13C NMR of 4-6

236

Figure C 5 1H NMR of 4-9

Figure C 6 13C NMR of 4-9

237

Figure C 7 1H NMR of 4-4

Figure C 8 13C NMR of 4-4

238

Figure C 9 2D (1H-1H) COSY of 4-4

239

Figure C 10 2D (1H-13C) HSQC of 4-4

Figure C 11 2D (1H-13C) HMBC of 4-4

240

Figure C 12 1H NMR of 4-10

Figure C 13 13C NMR of 4-10

241

Figure C 14 2D (1H-1H) COSY of 4-10

242

Figure C 15 2D (1H-13C) HSQC of 4-10

243

Figure C 16 2D (1H-13C) HMBC of 4-10

244

Figure C 17 1H NMR of 4-11

Figure C 18 13C NMR of 4-11

245

APPENDIX D A. HPLC CHROMATOGRAM OF CHAPTER-4

Figure D 1 HPLC chromatogram of 7’-bromo DMIC (2-12) borylation

246

Figure D 2 HPLC chromatogram of 3’-bromo DMIC (2-4) borylation optimization reactions

247

APPENDIX E B. PHOTOPHYSICAL DATA FOR CHAPTER-4

5,6-DMICA Bz(ethyl ester sidechain)

Dimer-1 Normalized absorbance absorbance Normalized

230 330 430 530 630

Wavelength(nm)

Figure E 1 UV/Vis spectra of 5,6-DMIC (2-3), Benzothiazole (3-10), and mixed melanoid (4-4)

Ex-290 nm

Ex-354 nm

Em-455 nm Normalized intensity Normalized

200 300 400 500 600 700 Wavelength(nm) Figure E 2 Luminescence spectra of mixed melanoid (4-4)

248

Normalized absorbance Normalized

230 330 430 530 630 730 Wavelength (nm)

Figure E 3 UV/Vis spectrum of mixed melanoid (4-11)

Em-450 nm Ex-350 nm

Ex-280 nm Normalized intensity Normalized

230 330 430 530 630 730 Wavelength (nm)

Figure E 4 Luminescence spectra mixed melanoid (4-11)

249

APPENDIX F NMR SPECTRA FOR CHAPTER 5

Figure F 1 1H NMR of 5-2

250

Figure F 2 1H NMR of 5-3

251

Figure F 3 1H NMR of 5-4

252

Figure F 4 1H NMR of 5-9

253

Figure F 5 1H NMR of 5-5

Figure F 6 13C NMR of 5-5

254

Figure F 7 1H NMR of 5-9

Figure F 8 13C NMR of 5-9

255

Figure F 9 1H NMR of 5-11

Figure F 10 13C NMR of 5-11

256

Figure F 11 1H NMR of 5-12

Figure F 12 13C NMR of 5-12

257

Figure F 13 1H NMR of 5-13

Figure F 14 13C NMR of 5-13

258

APPENDIX G C. IR SPECTRA FOR CHAPTER 5

%Trans

3,950 3,450 2,950 2,450 1,950 1,450 950 450 -1 Wavenumber (cm )

Figure G 1 Full IR spectrum of microcrystalline Au heli-viologen (5-13) at 298 K

259

APPENDIX H D. BOND LENGTHS AND BOND ANGLES OF AU-HELI VIOLOGEN

Au(1)-C(1) 2.0107(1) C(9)-C(10) 1.4369(1)

Au(1)-C(2) 2.0068(1) C(10)-C(11) 1.4131(1)

Au(2)-C(4) 1.9837(1) C(10)-C(19) 1.3644(1)

Au(2)-C(3) 1.9433(1) C(13)-C(18) 1.4101(1)

N(1)-C(1) 1.1318(1) C(13)-C(14) 1.4156(1)

N(2)-C(2) 1.0913(1) C(14)-C(15) 1.3473(1)

N(3)-C(3) 1.1708(1) C(15)-C(16) 1.3870(1)

N(4)-C(4) 1.1198(1) C(16)-C(17) 1.3998(1)

N(5)-C(5) 1.4637(1) C(17)-C(18) 1.3923(1)

N(5)-C(6) 1.2860(1) C(18)-C(19) 1.4561(1)

N(5)-C(26) 1.4028(1) C(19)-C(20) 1.4407(1)

N(6)-C(11) 1.3096(1) C(20)-C(21) 1.4663(1)

N(6)-C(12) 1.4724(1) C(21)-C(26) 1.4320(1)

N(6)-C(13) 1.4031(1) C(21)-C(22) 1.4159(1)

C(6)-C(7) 1.4320(1) C(22)-C(23) 1.3267(1)

C(7)-C(8) 1.4136(1) C(23)-C(24) 1.4234(1)

C(7)-C(20) 1.4379(1) C(24)- (C25) 1.3774(1)

C(8)-C(9) 1.3398(1) C(25)-C(26) 1.3754(1)

Table H 1 Select bond lengths of Au heli-viologen

260

C(1)-Au(1)-C(2) 176.62(1) N(6)-C(13)-C(14) 120.77(1)

C(3)-Au(2)-C(4) 177.67(1) C(13)-C(14)-C(15) 120.78(1)

Au(1)-C(1)-N(1) 177.15(1) C(14)-C(15)-C(16) 121.68(1)

Au(1)-C(2)-N(2) 175.42(1) C(15)-C(16)-C(17) 117.87(1)

Au(2)-C(3)-N(3) 175.29(1) C(16)-C(17)-C(18) 122.24(1)

Au(2)-C(4)-N(4) 176.96(1) C(13)-C(18)-C(17) 117.82(1)

C(11)-N(6)-C(13) 119.08(1) C(13)-C(18)-C(19) 118.40(1)

C(12)-N(6)-C(13) 119.16(1) C(17)-C(18)-C(19) 118.40(1)

C(11)-N(6)-C(12) 121.62(1) C(10)-C(19)-C(20) 116.99(1)

C(5)-N(5)-C(26) 118.65(1) C(10)-C(19)-C(18) 116.94(1)

C(6)-N(5)-C(26) 122.98(1) C(18)-C(19)-C(20) 125.97(1)

C(5)-N(5)-C(6) 117.87(1) C(7)-C(20)-C(21) 116.80(1)

N(5)-C(6)-C(7) 123.13(1) C(19)-C(20)-C(21) 126.22(1)

C(6)-C(7)-C(8) 120.84(1) C(7)-C(20)-C(19) 116.64(1)

C(8)-C(7)-C(20) 121.99(1) C(20)-C(21)-C(26) 119.32(1)

C(6)-C(7)-C(20) 117.14(1) C(22)-C(21)-C(26) 116.97(1)

C(7)-C(8)-C(9) 119.55(1) C(20)-C(21)-C(22) 122.64(1)

C(8)-C(9)-C(10) 118.18(1) C(21)-C(22)-C(23) 121.43(1)

C(9)-C(10)-C(19) 124.38(1) C(22)-C(23)-C(24) 120.64(1)

C(11)-C(10)-C(19) 120.09(1) C(23)-C(24)-C(25) 119.77(1)

C(9)-C(10)-C(11) 115.47(1) C(24)-C(25)-C(26) 119.63(1)

N(6)-C(11)-C(10) 122.91(1) N(5)-C(26)-C(21) 117.72(1)

N(6)-C(13)-C(18) 120.16(1) C(21)-C(26)-C(25) 120.78(1)

C(14)-C(13)-C(18) 118.99(1) N(5)-C(26)-C(25) 121.41(1)

Table H 2 Select bond angles of Au heli-viologen

261

APPENDIX I E. COMPUTATIONAL MODELLING All geometry parameters were determined using Gaussian ‘16 software (Gaussian Inc.) with the

University of Maine Advanced Computing Group.23 All ground state and excited state calculations were performed using density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations using the M06 metahybrid functional24,25 with the CEP-

31G(d) basis set employed for all atoms.26,27 XRD structures were used as initial structural input.

From the structural data we have developed a neutral model that is composed of two heli-

2+ 2- viologen units bridged by a dimer [Au(CN)2 and capped with terminal [Au(CN)2] monomer units. This model contains all subunits present within the crystal structure. Isodensity representations of molecular orbitals were generated using GaussView 5.0 software (Gaussian

Inc.). MOs were generated and visualized using the Avogadro 1.2.0 software program.28

DFT calculations were performed to interpret the photophysical changes and emission spectra of

Au heli-viologen upon excitation. A neutral model of gold(I) dicyanide coordinated to heli-

2+ - 2- viologen , shown in Fig. 9, was developed containing both [Au(CN)2] and [Au(CN)2]2 subunits.

Calculated ground state geometry parameters, Table S3 (ESI†), are in good agreement with experimental values with short Au⋯Au distances supportive of aurophilic interactions.

- -2 Figure I 1 DFT M06/CEP-31G(d) calculated ground state of [Au(CN)2] /[Au(CN)2]2 coordinated heli-viologen2+. Close Au⋯Au distance (3.435 Å) shown

262

2- Calculations at the [Au(CN)2]2 center accurately predict a short Au⋯Au distance of 3.435 Å compared to an experimental value of 3.310 Å. The close contact distance between the CN- anion and the N+ cation of the phenyl (3.155 Å) is slightly shorter than the experimental value of 3.216

- Å. We attribute this shortened distance to the lack of surrounding [Au(CN)2] ions normally present. The loss of surrounding anions causes our model to overly rely on the central electron rich

2- [Au(CN)2]2 dimer to balance the cationic viologen ion. TD-DFT calculated UV-vis spectra, Fig.

S14 (ESI†), show agreement of our model with the experimental UV-Vis results with both having strong absorption bands at energies higher than 500 nm. An excited state at 399 nm is predicted with a strong oscillator strength of 0.0132. This excited state energy aligns with our experimental luminescence excitation value of 400 nm. The highest contributing molecular orbital transition calculations performed for this excited state are shown in Figure I 2.

263

Figure I 2 TD-DFT M06/CEP-31G(d) energy diagram calculated for Au heli-viologen at 399 nm excitation for top 3 transitions. Transition percent contributions are noted.

TD-DFT calculations predict that two general types of electron transitions occur upon excitation at 399 nm. The first centers on the electron donating MOs (HOMO-9, and HOMO-11) which are primarily composed of the heli-viologen2+ π* with minor contribution from the neighboring

- 2+ [Au(CN)2] anion. The electron accepting MO (LUMO) is composed of the heli-viologen π* and clearly represents a π → π* transition. In the second case, the electron donating MO (HOMO) is

2- exclusively composed of the dimerized [Au(CN)2]2 subunit with no contribution from the neighboring heli-viologen2+ cations. This molecular orbital calculated transition is clearly an

- electronic transfer and demonstrates the emissive quenching of [Au(CN)2] by the viologen in the solid state observed in the experimental photophysical data.

264

Distance (Å) / Angle (o)

Experimental* Calculated*

Au...Au 3.310 3.327

... NAu(CN)2 Naza[5]helicene 3.216 3.088

Au-C 2.010 2.029

C-Au-C 176.6 166.8

N-C-Au 177.1 169.6

*Average distances

Figure I 3 TD-DFT M06/CEP-31G(d) energy diagram calculated for Au heli-viologen at 399 nm excitation for top 3 transitions. Transition percent contributions are noted

265

APPENDIX J F. UV-VIS, DFT AND MO FOR CHAPTER 5

M06/CEP-31G

Experimental Normalized Normalized Intensity (a.u.)

250 350 450 550 650 750 Wavelength (nm)

Figure J 1 TD-DFT M06/CEP-31G calculated UV-vis of Au heli-viologen compared to experimental UV-Vis

266

Excited State Excited State Excited State

Number Energy f-oscillation

7 428 nm 0.0078

8 425 nm 0.0067

9 423 nm 0.0014

10 421 nm 0.0000

11 421 nm 0.0069

12 420 nm 0.0027

13 406 nm 0.0004

14 404 nm 0.0009

15 401 nm 0.0028

16 399 nm 0.0132

17 392 nm 0.0011

18 390 nm 0.0083

Table J 1 TD-DFT calculated excited states of of Au heli-viologen with corresponding energy and f-oscillation

267

Orbital Transition % Contribution

HOMO-9→LUMO+1 25%

HOMO-11→LUMO 21%

HOMO→LUMO+2 18%

HOMO→LUMO+3 16%

HOMO-9→LUMO 12%

HOMO-11→LUMO+1 8%

Table J 2 Calculated MO transitions of Au heli-viologen for excited state at 399 nm with percent contribution

268

Figure J 2 Isodensity representations of MO transitions of excited state at 399 nm for Au heli- viologen with percent contribution

269

APPENDIX K G. COPYRIGHT PERMISSIONS FROM AUTHORS AND PUBLISHERS

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272

273

274

275

276

277

278

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BIOGRAPHY OF THE AUTHOR

Ealin N. Patel was born in Gujarat, India on November 2, 1990. He graduated high school from Sardar Vallabhbhai Vidyalaya in Vadodara in 2008. He attended University at Buffalo and graduated in 2013 with BA in Chemistry and BS in Medicinal Chemistry. He attended a year of graduate school in SUNY college at Buffalo before transferring to graduate program in Chemistry at the University of Maine in August 2015. Ealin has worked as a Teaching assistant and a

Research Assistant at the University of Maine. After receiving his degree, Ealin will be joining

Janda lab at the Scripps Research Institute in La Jolla, CA as a Postdoctoral Associate in Medicinal

Chemistry. He is a Candidate for the Doctor of Philosophy degree in Chemistry from the

University of Maine in May 2021.

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