Design and Synthesis of Fluorescent Probes A

Home , Rhodamine 6G, Umbelliferone

DESIGN AND SYNTHESIS OF FLUORESCENT PROBES

A dissertation submitted

to Kent State University in partial

fulfillment of the requirements for the

Degree of Doctor of Philosophy

Prabin Rai

August, 2013

Dissertation written by Prabin Rai M.Sc. Tribhuvan University, 1994 Ph.D., Kent State University, USA, 2013

Approved by

, Chair, Doctoral Dissertation Committee Dr. Robert J. Twieg

, Members, Doctoral Dissertation Committee Dr. Paul Sampson

Dr. Scott Bunge

Dr. Brett Ellman

______Dr. Hiroshi Yokoyama

Accepted by

, Chair, Department of Chemistry Dr. Michael Tubergen

, Associate Dean, College of Arts and Sciences Dr. Raymond Craig

TABLE OF CONTENTS

LIST OF FIGURES ...... ix

LIST OF SCHEMES ...... xvii

LIST OF TABLES ...... xxv

LIST OF ABBREVIATIONS ...... xxvii

ACKNOWLEDGMENTS ...... xxxii

CHAPTER 1 Introduction ...... 2

1.1.A brief introduction of Super Resolution Imaging ...... 2

1.2 Single molecule detection and its application in super resolution imaging ...... 4

1.3 Single molecule imaging...... 5

1.4 Super-resolution imaging by single molecule localization ...... 8

1.5 Fluorescent probes for super resolution imaging ...... 10

1.6 Photoswitchable fluorescent probes utilized in super resolution imaging ...... 12

1.7 Problems faced by the existing photoswitchable probes...... 18

1.8 Scope of this thesis...... 19

CHAPTER 2 Design and Synthesis of rhodamine spirolactams ...... 22

2.1 A brief overview of Rhodamine Spirolactams ...... 22

2.2 Rhodamine spirolactam derivatives: ...... 26

2.3 Synthesis of rhodamine derivatives: ...... 28

2.4 Synthesis of tetra-N-ethyl-spiro[benzo[4,5]imidazo[2,1-a]isoindole-11,9'-xanthene]- 3',6'-diamine (P-71) ...... 28

2.5 Literature approaches to 1H-isoindolo<1,2-a>benzimidazole-1-one (2.6) ...... 29

iii

2.6 Attempts on synthesis of rhodamine spirolactams (2.17) based on 7H- Benzimidazo[2,1 a]benz[de ] isoquinolin-7-one ...... 39

2.7 Synthesis of 7H-Benzimidazo[2,1 a]benz[de ] isoquinolin-7-one (2.15) ...... 40

2.8 Attempts on synthesis of rhodamine spirolactams (2.20) based on 12H-phthaloperin- 12-one ...... 41

2.9 Synthesis of 12H-phthaloperin-12-one (2.18) following the literature ...... 41

2.10 Synthesis of Rhodamine salts following literature procedure ...... 43

2.11 Synthesis of Rhodamine spirolactams from N-(9-(2-carboxyphenyl)-6- (dialkylamino)-3H-xanthen-3-ylidene)-N-methylmethanaminium perchlorate ...... 45

2.12 Rhodamine Spirolactam incorporating other dyes ...... 59

2.13 Synthesis of Rhodamine derivatives with different amine donor groups: ...... 63

2.13.1 Synthesis of (1-(9-(2-carboxyphenyl)-6-(pyrrolidin-1-yl)-3H-xanthen-3-ylidene) pyrrolidinium chloride) ...... 63

2.13.2 Synthesis of Rhodamine 101 spirolactams ...... 64

2.13.3 Attempt in synthesizing Rhodamine spirolactam incorporated with 7- azanorbornane as donor group ...... 65

2.13.4 Synthesis of 7-azanorbornane hydrogen chloride by Mitsunobu reaction following literature procedure: ...... 66

CHAPTER 3 Photophysical properties of rhodamine spirolactams ...... 70

3.1 Photophysical properties of rhodamine spirolactams ...... 70

3.2 UV-Vis absorption of the rhodamine lactam in their closed state ...... 70

3.3 Effect of substitution on the absorbance of the closed form ...... 74

3.4 Preliminary study of photoswitching efficiency of rhodamine spirolactams ...... 78

3.5 Rhodamine spirolactams under the influence of pH ...... 81

3.6 Single molecule detection in neutral and acid conditions ...... 93

3.7 Summary ...... 94

CHAPTER 4 Modifications of DCDHF azides and strained alkenes for efficient 1,3 dipolar cycloadditions ...... 97

4.1 Introduction ...... 97

4.2 A brief introduction to azido functionalized chemistry ...... 98

4.3 A different approach of azide chemistry with strained alkenes ...... 102

4.4 Some Preliminary Studies on functionalized strained alkenes...... 107

4.5 A 1,3-dipolar reaction between azido functionality and norbornene and to understand the kinetics of the addition...... 119

4.6 Synthesis and Result ...... 127

4.6.1 Synthesis of norbornene with ester functional group ...... 127

4.6.2 Synthesis of norbornene with benzo-group: ...... 128

4.6.3 Synthesis of norbornene with alkyl group on a bridge head position ...... 128

4.6.4 Synthesis of norbornene with cyclopropane bearing alcohol group ...... 131

4.7 Synthesis of DCDHF dyes with an azido group ...... 132

4.8 Synthesis of DCDHF dyes incorporating CF3 group with azido group on it...... 134

4.9 Synthesis of DCDHF incorporating two F atoms in π-system ...... 138

4.10 Synthesis of DCDHF incorporating CF3 and Ph ring...... 138

4.11 Attempts on the synthesis with two CF3 groups on DCDHF head ...... 147

4.12 Synthesis of the DCDHF unit incorporating a thiazole ring: ...... 148

4.13 Conclusion: ...... 154

CHAPTER 5 Some Cell Imaging technique By Chemical activation ...... 157

5.1 Introduction ...... 157

5.2 A brief overview of Point Accumulation for Imaging in Nanoscale Topography (PAINT)63: ...... 158

5.3 Factors Essential to PAINT fluors ...... 159 v

5.4 Rationale for screening PAINT fluors ...... 162

5.5 Photophysical properties of some PAINT fluors ...... 163

5.6 Spectra in Phosphate Buffer Saline (PBS) (~µM of dye concentration) ...... 165

5.7 Fluorescence of some individual PAINT fluor candidates: ...... 166

5.7.1 Nile red sulfate (P193), NL03008, and Nile Red ...... 166

5.7.2 Fluorescence QY increases with viscosity ...... 170

- 5.7.3 A comparison of fluorescence properties of Nile red-SO3 (P-193), NL03008 and Nile red with respect to viscosity ...... 171

5.8 Preliminary Comparison Results ...... 173

5.9 Results and discussion ...... 174

5.10 Synthesis of hydroxy-Nile red phenol: ...... 175

5.11 Conclusion ...... 177

5.12 Oxy-DCDHFs for potential application in β-Lactamase Active Control Scheme .. 178

5.12.1 Introduction: ...... 178

5.12.2 Result and discussion: ...... 181

5.12.3 Synthesis of oxy-DCDHF ...... 181

5.12.4 Photophysical study of oxy-DCDHFs for potential application in β-Lactamase cleavage...... 183

5.12.5 Conclusion ...... 187

5.13 Tetrazine based fluorophores for potential use in imaging applications...... 188

5.13.1 Introduction: ...... 188

5.14 Combinations of strained alkenes and alkynes derivatives with tetrazine derivatives for fast cycloaddition DA reaction...... 190

5.15 Tetrazine-linked with DCDHF derivatives and Nile red derivatives...... 197

5.16 Synthesis of Tetrazine derivatives ...... 197

5.17 Synthesis of tetrazine-linked-DCDHF derivatives ...... 200

5.18 Synthesis of pyridine containing tetrazines ...... 202

5.19 Synthesis of DCDHF derivatives with carboxylic end ...... 205

5.20 Alternate proposed route for synthesizing 5.54 ...... 207

5.21 A synthetic proposal for tetrazine linked Nile red ...... 208

5.22 Summary ...... 208

CHAPTER 6 Cofacial Structures of 1:1 Complexes of Perfluorophenazine with Polynuclear Aromatic Compounds ...... 211

6.1 Introduction ...... 211

6.2.0 Results and discussion ...... 213

6.2.1 Synthesis of perfluorophenazine ...... 213

6.2.2 X-ray crystallographic discussion of PFPpolynuclear aromatic compounds ...... 214

6.2.3 X-ray crystallography of Perfluorophenazine ...... 217

6.2.4 X-ray crystallography of Perfluorophenazine∙Naphthalene ...... 218

6.2.5 X-ray crystallography of perfluorophenazine and anthracene ...... 223

6.2.6 X-ray crystallography of perfluorophenazine and phenanthrene ...... 227

6.2.7 X-ray crystallography of perfluorophenazine and tetracene ...... 230

6.2.8 X-ray crystallography of PFP∙DTT (dithieno[3,2-b’:2’,3’-d]thiophene) ...... 232

6.2.9 Summary ...... 240

CHAPTER 7. Synthesis of partially fluorinated heteroaromatic compounds for possible use in Organic Semiconductors ...... 242

7.1 Introduction ...... 242

7.2 Option for avoiding ionization potential issue to improve charge mobility ...... 246

7.3 Results and discussion ...... 249

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7.4 Synthesis of partially fluorinated acridine and its derivatives ...... 249

7.5 Synthesis of dibenzo[a,c]phenazine and partially fluorinated dibenzophenazine derivatives ...... 251

7.6 Synthesis of 6-nitroperfluoroaniline ...... 253

7.7 Synthesis of perfluoroaniline-1,2-phenylenediamine following literature procedure ...... 253

7.8 Synthesis of partially fluorinated phenanthrophenazine derivatives ...... 254

7.9 Synthesis of 10,11,12,13-tetrafluorphenathro[4,5-abc]phenazine ...... 254

7.10 X-ray crystallographic discussion of 10,11,12,13-tetrafluorphenathro[4,5- abc]phenazine (12)...... 255

7.11 Crystal parameters of 10,11,12,13-tetrafluorophenathro[4,5-abc]phenazine (7.16) ...... 259

7.12 10,11,12,13-tetrafluorophenanthro[4,5-abc]phenazine-4,5-dione ...... 259

7.13 Concluding remarks ...... 260

CHAPTER 8. Experimental ...... 263

Experimental of Chapter 2 ...... 264

Experimental of Chapter 4 ...... 317

Experimental of Chapter 5 ...... 353

Experimental of Chapter 6 ...... 380

Experimental of Chapter 7 ...... 388

CHAPTER 9. REFERENCES ...... 398

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LIST OF FIGURES

Figure 1.1 Epifluorescence microscopy: a schematic of a representative experimental set up...... 6

Figure 1.2 Imaging single molecules ...... 7

Figure 1.3 A cartoon showing the key idea of super-resolution imaging of a structure by

SMACM ...... 9

Figure 1.4 Structures of some fluorescent dyes utilized in super resolution imaging...... 13

Figure 1.5 The basic principles of FRET operation...... 16

Figure 1.6 Antibody conjugated with Cy3/Cy5 pair which communicates eachother ..... 17

Figure 1.7 Structure of Cy3, Cy5, Alexa-647 with R group as the binding site with antibody 59 ...... 17

Figure 1.8 Schematic representation of trimethoprim-dihydrofolate reductase protein from E. Coli (TMP-eDGHFR labeling system)...... 18

Figure 2.1 Molecular structures of xanthene (1), rhodamine dyes, and (3) rhodamine spirolactams ...... 22

Figure 2.2 (A) simplified Jablonski diagram showing the photophysics of rhodamine spirolactam isomerization and representative bulk spectra...... 23

Figure 2.3 Photoswitching properties of a rhodamine dye developed in Hell’s lab...... 24

Figure 2.4 Reconstructed images of the tubulin network in a PtK2 cell stained with 5-

NHSS69...... 25

Figure 2.5 A Few representative Rhodamine derivatives A, B71 and C72 applied in super resolution imaging by Hell's group...... 26

Figure 2.6 The structural features of the rhodamine spirolactams...... 27

Figure 2.7 Retro-synthesis of rhodamine derivative P-71 ...... 29

Figure 2.8 Structure of 2.7 as determined by X-ray crystallographic study77...... 30

Figure 2.9 Halochromic properties of rhodamine spirolactam P-71...... 34

Figure 2.10 UV-Vis of rhodamine derivative P-71 (the peak at 560 nm is due to the open form P-71) ...... 36

Figure 2.11 Photoswitching of the rhodamine spirolactams favors the ring closed colorless state...... 36

Figure 2.12 Steric hindrance may a reason for the unsuccessful synthesis of hypothetical rhodamine derivatives 2.16 and 2.19 ...... 42

Figure 2.13 A resonating structure of aryl amino version of DCDHF ...... 53

Figure 3.1 Rhodamine lactams with short wavelength absorbance in acetonitrile (278 nm

- 317 nm); ...... 74

Figure 3.2 Rhodamine lactams with medium wavelength absorbance in acetonitrile

(325nm - 360 nm); ...... 75

Figure 3.3 Rhodamine lactams with long wavelength absorbance in acetonitrile (385 -

396 nm); ...... 76

Figure 3.4 Relationship between unsaturation and abs > 400 nm of closed rhodamine lactams...... 77

(Courtesy: Marissa Lee, Stanford University) ...... 77

Figure 3.5 The absorbance of closed form and excitation/emission of the open form of rhodamine spirolactam (P-93)...... 79

Figure 3.6 A representative imaging experiment for the rhodamine lactam (P-82) after activation by 403 nm irradiation (10% PMMA)...... 81

Figure 3.7 Spectra showing reversible stabilization of open isomer at low pH. The solid lines are absorption (left axis) and the dashed lines are fluorescence emission (right axis).

...... 82

Figure 3.8 The absorption/emission properties of a number of rhodamine spirolactams as a function of pH...... 87

Figure 3.9 Spectral changes in rhodamine spirolactam P-146 with acid titration

(acetonitrile/H2O 1:1 mix)...... 89

Figure 3.10 Change in absorbance of rhodamine lactam (P-146) as a function of pH. The isosbestic point is at 337 nm for open and closed isomers...... 92

Figure 3.11 (A) photon counts of rhodamine lactam P-146 in neutral and acidic condition

(B) single molecule detection...... 93

Figure 4.1152 (Top)152Strategy for metabolic labeling of cell-surface glycans with bioorthogonal chemical reporter...... 100

Figure 4.2 A general scheme showing the transformation of the fluorogen to fluorophore by chemical process...... 103

Figure 4.3 DCDHF possessing three critical components; Donor – π system – Acceptor

...... 104

Figure 4.4 A series of strained alkenes(a) norbornene (b) bicyclo[2.2.2]octane (c) bicyclo[2.1.1]hexane (d) Tricyclo[3.3.0.0]oct-3-ene...... 107

Figure: 4.5 A proposed modification in azido-Ph-DCDHF by incorporating electron withdrawing groups in π-system and DCDHF head...... 107

Figure 4.6 A kinetic study of 1,3-dipolar cycloaddition between azido-DCDHF 4.2 and strained alkene 4.22...... 112

Figure 4.7 A kinetic study of 1,3-dipolar cycloaddition between azido-DCDHF 4.2 and strained alkene 4.24...... 114

Figure 4.8 1,3-dipolar cycloaddition reaction between norbornene and azido-Ph-DCDHF-

CF3 monitored by HNMR with consumption of the starting material and formation dihydrotriazole and rearranged product...... 122

Figure 4.9 Reactants (norbornene), intermediate (dihydrotriazole) and final rearranged product (secondary amine) distribution curve with time in T-ATA process ...... 124

Figure 4.10 A comparison of rate of norbornene consumption, dihydrotriazole formation and rearranged product (secondary amine) formation between norbornene vs azido-Ph-

DCDHF-CH3 and norbornene vs azido-Ph-DCDHF-CF3; ...... 125

Figure 4.11 A comparison of rate of norbornene consumption, dihydrotriazole formation and rearranged product (secondary amine) formation between norbornene 37 vs azido-

Ph-DCDHF-CH3 and norbornene vs azido-Ph-DCDHF-CF3; ...... 126

Figure 4.12 Comparing absorption of two compounds (E)-2-(4-(4-diethylamino) styryl)benzylidene)malononitrile (4.125) and (E)-2-((2-(4-diethylamino)styryl) thiazol-5- yl)methylene)malononitrile (4.126) ...... 149

Figure 4.13 Electron density distributions in thiazole unit and matched and mismatched case according the orientation of thiazole unit...... 149

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Figure 4.14 A comparison of absorption for the different orientation of the thiazole unit.

...... 150

Figure 4.15 Proposed structure of azido DCDHF incorporating a thiazole ring...... 150

Figure 4.16 The reactivity of lithiated thiophene with a TMS protected cyanohydrin ... 152

Figure 5.1 Fluorescence image of LUVs63 ...... 161

Figure 5.2 Probes utilized in preliminary screening for PAINT technique...... 163

Figure 5.3 Absorption and emission spectra of PAINT fluor candidates in PBS (~µM of dye) ...... 166

- Figure 5.4 (1) Absorbance and emission of Nile Red-SO3 (P-193) in DMSO and water;

- (2) fluorescence of NileRed-SO3 with increasing viscosity (glycerol/water) ...... 168

Figure 5.5 Fluorescence of NL03008 and Nile Red with increasing viscosity ...... 169

Figure 5.6 A measurement of fluorescence (QY) with respect to viscosity...... 170

- Figure 5.7 A comparison of normalized fluorescence area of the Nile red-SO3 (P-193),

NL03008 and Nile Red with respect to viscosity of the medium...... 172

Figure 5.8 2D image of e.Coli using Nile Red-Peg (Source: Stanford University) 175

Figure 5.9 List of oxy-DCDHF derivatives potential candidates for subdiffraction imaging by enzymatic action...... 181

Figure 5.10 The absorption and emission spectrum of oxy-DCDHFs which are of particular interest given their Stokes shifts ...... 185

Figure 5.11 Absorbance of DCDHF 202 depends on pH...... 187

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Figure 5.12 General Scheme for the process involving a tetrazine-linked fluorophore which undergoes ‘turn on’ after inverse DA reaction with the bioorthogonal dienophiles.

...... 196

Figure 6.1 Molecular packing of perfluorophenazine in its pure state adapting a herringbone motif along the c-axis, green-F, blue-N, grey-C...... 215

Figure 6.2 Capped stick plots of the perfluorophenazine showing herringbone packing along different axes (hydrogen atoms have been omitted for clarity) ...... 217

Figure 6.3 The thermal ellipsoid plot of co-crystal PFPnaphthalene (50% probability)

...... 218

Figure 6.4 PFP molecules in one column and the PFP molecules in the next column intersect by an angle 50.91° ...... 219

Figure 6.5 Capped stick plots of the co-crystal PFP.naphthalene showing herringbone packing along different axes (hydrogen atoms have been omitted for clarity) ...... 220

Figure 6.6 The inter-planar distance between PFP and naphthalene...... 221

Figure 6.7 A Slip distance of a molecule with respect to the similar molecule...... 222

Figure 6.8 PFP∙naphthalene co-crystal showing close contacts ...... 223

Figure 6.9 The thermal ellipsoid plot of co-crystal PFP∙anthracene (50% probability) . 224

Figure 6.10 Capped stick plots of the co-crystal PFP∙anthracene showing almost perfect parallel packing along different axis and face to face stacking to adjacent molecules

(hydrogen atoms have been omitted for clarity) ...... 225

Figure 6.11 PFP∙anthracene co-crystal showing close contacts ...... 226

xiv

Figure 6.12 The thermal ellipsoid plot of co-crystal PFP∙phenanthrene (50% probability)

...... 227

Figure 6.13 Capped stick plots of the co-crystal PFP∙phenanthrene showing almost perfect parallel packing along different axes (hydrogen atoms have been omitted for clarity) ...... 229

Figure 6.14 The thermal ellipsoid plot of co-crystal PFP∙tetracene (50% probability) .. 230

Figure 6.15 Capped stick plots of the co-crystal PFP∙tetracene showing almost perfect parallel packing along different axes and face to face stacking to adjacent molecules

(hydrogen atoms have been omitted for clarity) ...... 231

Figure 6.16 Thermal ellipsoid plot of co-crystal PFP∙DTT (50% probability) ...... 233

Figure 6.17 Capped stick plots of the co-crystal PFP∙DTT showing herringbone pattern packing along different axes (hydrogen atoms have been omitted for clarity) ...... 235

Figure: 6.18 PFP∙DTT co-crystal showing close contacts ...... 236

Figure 6.19 Schematic representation of the intermolecular parameter ...... 237

Figure 7.1. Packing motifs of organic molecule in crystal ...... 244

Figure 7.2 (A) A face to face (cofacial) packing of 1,2,3,4-tetrafluoronaphthalene molecules (B) A cofacial molecular packing of 1,2,3,4-tetrafluoroanthracene ...... 247

Figure 7.3 Molecular packing of 1,2,3,4-tetrafluoro-7-methoxyacridine in a perfect parallel fashion...... 249

Figure 7.4 Capped stick plots of 10,11,12,13-tetrafluorphenanthro[4,5-abc]phenazine showing along different crystallographic axis (H atoms have been omitted for clarity). 256

Figure 7.5 The close contact of atoms between 10,11,12,13-tetrafluorophenathro[4,5- abc]phenazine molecules ...... 258

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LIST OF SCHEMES

Scheme 2.1 Synthesis of 1H-isoindolo<1,2-a>benzimidazole-1-one by direct condensation between phthalic anhydride and phenylene-1,2-diamine ...... 29

Scheme 2.2 Synthesis of 2.9 by stepwise route (a) THF, rt, 4 hrs; (b) stirring THF...... 31

Scheme 2.3 Synthesis of 2.6 from 2.9 by ring closing condensation; (a) NMP, 200°-

210°C, 12hr ...... 31

Scheme 2.4 Synthesis of 2.6 using microwave reactor ...... 32

Scheme 2.5 Synthesis of rhodamine spirolactam P-71 by literature procedure...... 33

Scheme 2.6 Synthesis of P-71 in a microwave reactor ...... 35

Scheme 2.7 A proposed synthetic route of synthesizing derivative of P-71 which may have longer absorption...... 37

Scheme 2.8 Attempted synthesis of rhodamine derivative 2.13 from 2.12 and 2.6 ...... 39

Scheme 2.9 Retrosynthesis of 2.16 via 2.15 with starting material 2.4 and 2.14...... 39

Scheme 2.10 Synthesis of 2.15 by microwave reactor ...... 40

Scheme 2.11 Retrosynthesis of 2.19 via 2.18 with starting material 1,8 naphthalic anhydride and 1,2-phenylenediamine...... 41

Scheme 2.12 Synthesis of 2.19 by microwave reactor ...... 42

Scheme 2.13 Synthesis of N-(9-(2-carboxyphenyl)-6-(dialkylamino)-3H-xanthen-3- ylidene)-N-methylmethanaminium perchlorate ...... 43

Scheme 2.14 An attempt on synthesizing rhodamine salt (2.25) from 1,8-naphthalic anhydride (2.16) (a) 160°C ...... 44

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Scheme 2.15 Attempts in synthesizing rhodamine salt from 2,3-pyrazine carboxylic anhydride by fusion...... 44

Scheme 2.16 A general route for synthesis of Rhodamine spirolactams; aromatic/aliphatic amines (Y); (a) p-TsCl, DMAP, DCM/CH3CN, Y ...... 46

Scheme 2.17 An alternate scheme of synthesizing P-85 ...... 50

Scheme 2.18 Attempt on synthesis of 2.31 ...... 51

Scheme 2.19 An alternate route for synthesizing P-150 ...... 52

Scheme 2.20 Synthesis of P-89 by two different routes ...... 53

Scheme 2.21 Synthesis of iodide salt (P-93) of P-89...... 54

Scheme 2.22 Synthesis of P-101 by Sonogashira reaction...... 55

Scheme 2.23 Synthesis of P-109 by Sonogashira reaction between P-91 and 2.45 ...... 56

Scheme 2.24 Synthesis of P-145 using Buchwald Hartwig chemistry ...... 57

Scheme 2.25 Synthesis of P-137 ...... 58

Scheme 2.26 Synthesis of P-146 with NHS and P-140 with water soluble moiety ...... 59

Scheme 2.27 A proposed rhodamine dye incorporating Nile red in a possible switching mechanism...... 60

Scheme 2.28 A proposed synthesis of P150 and P-460 by direct condensation with P-406

...... 61

Scheme 2.29 Attempt on synthesis of P-152 by Buchwald Hartwig reaction ...... 62

Scheme 2.30 Attempt to synthesize P-157 incorporating Nile red; ...... 63

Scheme 2.31 Synthesis of P-253 ...... 64

Scheme 2.32 Synthesis of P-485 from RhD101 salt ...... 65

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Scheme 2.33 Synthesis of 7-azanorbornane HCl (2.64) by Mitsunobu reaction ...... 66

(a) p-TsCl, NaOH, THF, H2O; (b) PPh3, DIAD, THF; (c) Sodium naphthelenide, THF,

NaHCO3, K2CO3 ...... 66

Scheme 2.34 Synthesis of 2.64 by another route...... 67

Scheme 2.35 Alternate synthetic route for 2.64 ...... 68

Scheme 2.36 Synthesis of 43 in a microwave reactor; (a) Hunig’s base, ZnCl2 anhyd. .. 68

Scheme 4.1 (A) Click chemistry catalyzed by Cu using unstrained alkyne with azide (B)

Bioorthogonal Click chemistry using strained alkyne in the absence of Cu catalyst with azide ...... 99

Scheme 4.2 A plausible mechanism in a T-ATA process between norbornene 1 and azido-Ph-DCDHF4.2 in a dipolar cycloaddition reaction...... 105

Scheme 4.3 Cycloaddition between semifluorinated azido Ph-V-DCDHF 4.9 and norbornene 4.1...... 106

Scheme 4.4 A dipolar 1,3-cycloaddition between 4.12 and 4.2 ...... 108

Scheme 4.5 A dipolar 1,3-cycloaddition between 4.14 and 4.2 ...... 108

Scheme 4.6 A 1,3-dipolar cycloaddition between strained alkene (4.16) and phenylazide

(4.17)...... 109

Scheme 4.7 A cycloaddition between electron rich strained alkene 4.20 and azido

DCDHF 4.2...... 110

Scheme 4.8 A 1,3-dipolar cycloaddition between 4.2 and strained alkene 4.22 ...... 110

Scheme 4.9 A cycloaddition between strained alkene 4.24 and azido-DCDHF 4.2 ...... 113

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Scheme 4.10 A 1,3-dipolar cycloaddition between strained alkene 4.27 and azido-

DCDHF 4.2 ...... 115

Scheme 4.11 A 1,3-dipolar cycloaddition between strained alkene 4.30 and azido-

DCDHF 4.2 ...... 116

Scheme 4.12 A derivative of strained alkene 4.33 in a 1,3-dipolar cycloaddition reaction with azido-Ph-DCDHF 4.2...... 116

Scheme 4.13 A derivative of strained alkene 4.36 in a 1,3-dipolar cycloaddition reaction with azido-Ph-DCDHF 4.2...... 117

Scheme 4.14 A derivative of strained alkene 4.39 in a 1,3-dipolar cycloaddition reaction with azido-Ph-DCDHF 4.2...... 118

Scheme 4.15 A derivative of strained alkene 4.42 in a 1,3-dipolar cycloaddition reaction with azido-Ph-DCDHF 4.2...... 118

Scheme 4.16 A kinetic study of norbornene 4.1 and azido-Ph-DCDHF-CF3 (4.45) ..... 120

Scheme 4.17 Synthesis of strained alkenes 4.12 and 4.16 from dimethyl fumarate 4.49 and dimethyl acetylenedicarboxylate 4.50 respectively...... 128

Scheme 4.18 Synthesis of benzonorbornene 4.24 ...... 128

Scheme 4.19 A proposed synthetic route of 4.58 ...... 130

Scheme 4.20 Synthesis of cyclopropane ring possessing norbornene with alcohol tail for possible bioconjugation ...... 131

Scheme 4.21 Synthesis of 4.2; (a) diallylamine, py, rt, 24h; (b) N,N’dimethylbarbituric acid, Pd(PPh3)4 DCM; (c) NaNO2/HCl, NaN3; (d) NaN3, DMSO...... 132

Scheme 4.22 Synthesis of 4.47 (a) TMSCN, n-BuLi, THF, 0ºC; (b) Mg, THF; (c) diallylamine, pyridine; (d) malononitrile, pyridine, CH3COOH; (e) Pd(PPh3)4, N,N’- dimethylbarbituric acid, DCM; (f) t-BuNO2, TMSN3, CH3CN or NaNO2, NaN3,

CH3COOH ...... 135

Scheme 4.23 Attempt to synthesize 4.71...... 137

Scheme 4.24 Attempt to synthesize 4.77 (a) NaN3, DMSO, rt ...... 137

Scheme 4.25 Synthesis of 4.82 following Scheme 4.22; (a) Mg, THF, rt followed by

4.80; (b) HCl (aqueous ) ...... 138

Scheme 4.26 Synthesis of 4.88, a precursor for DCDHF head possessing CF3 and Ph; 139

Scheme 4.27 Synthesis of precursor of catalyst (NHC); ...... 140

Scheme 4.28 Synthesis of hydroxyketone using intermolecular cross benzoin condensation reaction...... 140

Scheme 4.29 Syntheses of 92 (a) Mg, ether and later DMF and then work up...... 141

Scheme 4.30 An alternate path to synthesize 4.88 and 4.96...... 141

Scheme 4.31 Attempt to displace F in 4.88 by diallylamine to afford 4.1.04; ...... 142

Scheme 4.32 Attempt to synthesize 4.106 from 4.96 ...... 143

Scheme 4.33 Attempt in synthesizing 4.109 from 4.96 ...... 143

Scheme 4.34 Synthesis of 4.68 and 4.114 using dithiane chemistry ...... 144

Scheme 4.35 A comparison study of the SNAr reaction in substituting F with diallylamine or azide...... 145

Scheme 4.36 A nucleophilic substitution reactions of 4.88 with pyrrolidine as nucleophile (a) pyrrolidine, DMSO, pTsOH ...... 146

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Scheme 4.37 Attempt in the synthesis of hydroxyketone 4.119; ...... 147

Scheme 4.38 Attempt in the synthesis of hydroxyketone 4.124; ...... 148

Scheme 4.39 A proposed syntheses of hydroxyketone 4.139 ...... 152

Scheme 4.40 attempt on synthesis of 4.142 ...... 153

Scheme 4.41 An attempt to synthesized 4.144 by Vilsmeier Haack reaction (a) DMF,

POCl3 ...... 154

Scheme 5.1 Synthesis of Nile red derivatives ...... 176

Scheme 5.2 Attempt to synthesize a Nile red incorporating the julolidine substructure 177

Scheme 5.3 hydrolysis of alkylated umbelliferone by β-lactamase which releases the fluorophore umbelliferone...... 179

Scheme 5.4 Proposed scheme of cephalosporin incorporated oxy-DCDHF undergoing hydrolysis by β-lactamase which releases the fluorophore oxy-DCDHFs anion ...... 180

Scheme 5.5 Synthesis of oxy-DCDHF ...... 182

Scheme 5.6 Esterification of oxy-DCDHF by refluxing in pyridine ...... 182

Scheme 5.7 Synthesis of unsymmetrical tetrazine; (a) Zn(OTf)2, NH2-NH2 (anhydrous);

(b) acetic acid / NaNO2 ...... 199

Scheme 5.8 Synthesis of unsymmetrical tetrazine 5.25; (a) NH2-NH2 hydrate; (b) acetic acid / NaNO2 ...... 199

Scheme 5.9 Synthesis of tetrazine 5.27; (a) NH2-NH2.H2O; (b) acetic acid / NaNO2 (c)

MeI, KOH, DMF...... 200

Scheme 5.10 Synthesis of DCDHF derivatives; (a) 4-fluorobenzaldehyde, ethanol, (b) N- methylaminoethanol, DMSO, 160°C; (c) 7, ethanol; (d) N-methylaminoethanol, neat . 201

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Scheme 5.11 Synthesis of tetrazine-linked fluorophore by Mitsunobu reaction; (a) Ph3P,

DIAD, THF ...... 201

Scheme 5.12 Synthesis of asymmetric tetrazine 5.37 (a) CuCN, NaCN, DMF; (b) 2- cyanopyridine, NH2-NH2 (anhydrous), Zn(OTf)2, (e) NaNO2, CH3CO2H ...... 202

Scheme 5.13 Synthesis of functionalized tetrazine 5.40 (a) Boc anhydride, EtOH; (b)

5.37, NaH, DMF ...... 203

Scheme 5.14 Synthesis of unsymmetrical tetrazine 5.44; ...... 204

Scheme 5.15 Synthesis of functionalized tetrazine 5.40 (a) DIAD, Ph3P, 5.39, THF ... 204

Scheme 5.16 An alternate route to synthesize 5.41; (a) 5.39, NaH, DMF; (b) 2- cyanopyridine, Zn(OTf)2, anhyd. NH2-NH2; (c) NaNO2, CH3CO2H; (d) CF3COOH,

DCM, ...... 205

Scheme 5.17 Synthesis of DCDHF derivative 5.50 (a) HCl, 110°C, 3h; (b) 4- fluorobenzaldehyde KOH, H2O, 110°C, 3d; (c) 7, EtOH; (d) N-hydroxysuccinimide,

DCC, DMAP) ...... 206

Scheme 5.18 Synthesis of tetrazine-linked DCDHF 5.51; (a) N,N-diisopropylethylamine,

DMF ...... 207

Scheme 5.19 Synthesis of 5.54 (a) 2-cyanopyridine, Zn(OTf)2, anhyd. NH2-NH2; (b)

5.39, NaH, DMF; (c) 4N HCl, dioxane, in DCM, 30 min, rt; (d) NaNO2, CH3CO2H ... 207

Scheme 5.20 Synthesis of tetrazine-linked Nile red; (a) dihydrofuran-2(3H)-one,

NaOMe, MeOH, reflux, overnight; (b) DCC/DMAP, DCM, rt ...... 208

Scheme 6.1 The synthesis of perfluorophenazine 6.2 with side product perfluoroazobenzene 6.3 ...... 214

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Scheme 7.1 Synthesis of 1,2,3,4-tetrafluorobenzo[b] acridine (7.3) ...... 250

Scheme 7.2 Synthesis of 1,2,3,4-tetrafluorobenzo[b] acridine (7.3) using different intermediates to avoid the possible isomer formation. (a) neat, 2,6-dimethylaniline (b) 2- naphthylamine, xylenes, 140°C ...... 251

Scheme 7.3 Synthesis of partially fluorinated dibenzophenazine derivatives...... 252

Scheme 7.4 Synthesis of 6-nitroperfluoroaniline (7.12)...... 253

Scheme: 7.5 Synthesis of perfluoroaniline-1,2-difluorobenzene (7.7) ...... 253

Scheme 7.6 Synthesis of 10,11,12,13-tetrafluorophenathro[4,5-abc]phenazine (7.14) (a)

RuCl3.2H2O (10 mol%), 4 equiv NaIO4, DCM, MeCN, H2O, rt (b) glacial acetic acid,

120°C ...... 255

Scheme 7.7 Synthesis of 10,11,12,13-tetrafluorophenanthro[4,5-abc]phenazine-4,5-dione

(a) RuCl3.2H2O (20 mol%), 8 equiv NaIO4, DCM, MeCN, H2O, rt (b) glacial acetic acid,

120°C ...... 260

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LIST OF TABLES

Table 1.1 Some conventional fluorescent dyes (structures are in Figure 1.4) with photoswitching properties with the addition of redox reagents in buffer...... 14

Table 2.1 List of compounds synthesized by Scheme 2.16 ...... 47

Table 3.1 The UV-Vis-absorption of rhodamine spirolactams in dichloromethane...... 73

Table 3.2 Some property notations and their meanings used in the study of the photophysical properties of the rhodamine spirolactams in acid/base experiments...... 84

Table 3.3 Rhodamine spirolactams bulk characterization in acetonitrile and 1/1 acetonitrile/water (The notation at the top of the table are described in Table 3.2...... 88

Table 3.4 Some pKa measurement of selected rhodamine spirolactams...... 90

Table 4.1 Reaction data between azido-Ph-DCDHF-CF3 and norbornene as monitored by

HNMR...... 123

- Table 5.1 UV-is absorption of NileRed-SO3 , Nile Red and NL03008 in DMSO and water ...... 164

Table 5.2 a comparison of the fluorescence area of the Nile red-SO3K, NL03008 and

Nile red ...... 172

Table 5.3 a comparison of the photophysical properties of the PAINT fluor candidates

...... 173

Table 5.4 Photophysical characteristics of the four oxy-DCDHFs ...... 184

Table 5.5 Second order rate constant k2 of in reactions between different strained alkenes and alkynes with various tetrazines...... 192

Table 6.1 Intermolecular parameters ...... 238

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Table 6.2 Intermolecular close contacts in co-crystals per PFP ...... 239

Table 7.1 Intermolecular close contacts in 10,11,12,13-tetrafluorphenathro[4,5- abc]phenazine molecule...... 258

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LIST OF ABBREVIATIONS

µ micro, 10-6 Å Angstrong, 10-12 AcOH Acetic acid anhyd. anhydrous ATA Azide To Amine

Ba(OH)2 Barium hydroxide BCN Bicyclononyene BG benzylguanine

Boc2O Di-t-butyl dicarbonate C. crescentus Caulobacter crescentus CCD Charge Coupled Device

CFCl3 Trichlorofluoromethane

CH3SO3H Methyl sulfonic acid CHO Chinese Hamster Ovary

CNCH2CN Malanonitrile cP Centipoise

Cs2CO3 Cesium carbonate CSDS Cambridge Structural Database System

Cy Cyanine d doublet DA Diels Alder DBU 1,8-Diazabicycloundec-7-ene DCC N,N'-Dicyclohexylcarbodiimide DCDHF 2-Dicyanomethylene-3-cyano-2,5-dihydrofuran

DMAP 4-Dimethylaminopyridine xxvii

DMF Dimethylformamide DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DSC Differential Scanning Calorimetry DTT Dithieno[3,2-b':2',3'-d]thiophene e.Coli Escherichia Coli EDG Electron donating group EI-MS Electron Ionization-Mass Spectroscopy

Et2NH Diethylamine

Et2O Diethylether

Et3N Triethylamine

Et3SiH Triethylsilane EtOH Ethanol eV electron Volt EWG Electron withdrawing group FP Fluorescent Protein Fluorescent Photoactivation Localization FPALM Microscopy FRET Fluorescence Resonance Energy Transfer FTIR Fourier Transform Infrared Spectroscopy g grams GC Gas Chromatography HeLa Henrietta Lacks HMPA Hexamethylphosphoramide HOMO Highest Occupied Molecular Orbital Hunig's base N,N'-diisopropylethylamine HVZ Hell-Vollard-Zelinsky xxviii

Hz Hertz IBX 2-Iodoxybenzoic acid IC Internal Conversion ISC Inter system crossing l, L Liter LDA Lithium diisopropylamine

LiAlH4 Lithium aluminium hydride LUMO Lowest Unoccupied Molecular Orbital LUV Unilamellar vesicles m meter MHz Mega Hertz mM millimolar MS Mass Spectrometry MW Mega Watt N Normal NA Numerical Aperture n-BuLi n-Butyllithium

NH2-NH2 hydrazine NHC N-Heterocyclic Carbene NHS N-Hydroxysuccinimide NIR Near InfraRed nm nanometer NMP N-Methyl-2-pyrrolidone OTf triflate p para PAINT Point Accumulation for Imaging in Nanoscale xxix

PALM Photoactivation Localization Microscopy PBS Phosphate Buffered Saline

Pd(OAc)2 Palladium acetate

Pd(PPh3)4 Tetrakis(triphenylphosphine)palladium(0) Pd/C Palladium / activated carbon PEG Polyethyleneglycol PFP perfluorophenazine PMMA Poly(methyl methacrylate) PSF Point Spread Function p-TsCl para-toluenesulfonyl chloride Py pyridine

PyHBr3 pyridine bromohydrogenbromide q quartet QD Quantum Dot QY Quantum Yield R Alkyl

Rf Retention Factor RhD101 Rhodamine 101 RNA Ribonucleic Acid

RuCl3 Ruthenium trichloride s singlet, sec

S0 ground state singlet

S1 excited state singlet SMACM Single Molecule Active Control Microscopy SR Super resolution SSIM Saturated Structured Illumination Microscopy xxx

STED Stimulated Emission Depletion STORM Stochastic Optical Reconstruction Microscopy T-ATA Temperature-Azide-To-Amine TBAB Tetra-n-butylammonium bromide TBAF Tetra-n-butylammonium fluoride t-BuNO2 tertiary-butylnitrite t-BuOH tertiary-butylalcohol t-BuOK Potassium tertiary-butoxide TDA-1 Tris[2-(2-methoxyethoxy)ethyl]amine TEAB Tetraethylammonium bromide TFA Trifluoroacetone TFAA Trifluoroacetic acid TGA Thermal Gravimetric Analysis THF Tetrahydrofuran TLC Thin layer chromatography TMP Trimethoprim

TMSCF3 Trimethylsilyltrifluoromethane TMSCN Trimethylsilyl cyanide

TMSN3 Trimethylsilyl azide UNAA Unnatural amino acid UV-Vis Ultra violet - visible W Watt ε molar extinction coefficient, l.mol-1.cm-1

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ACKNOWLEDGMENTS

First and foremost I would like to express my gratitude and admiration to my advisor Professor Dr. Robert J. Twieg, who gave me opportunities, support, encouragement, patience and understanding over the years. With constant support of my wife and moral support of my parents, this work has been possible. I would like to thank

Marissa Lee from Stanford University who has been providing me all the photo-physical data and constant feedback about my research.

I would like to thank Dr. Bunge, Dr. Ellman, Dr. Sampson and Dr. Seed, for their precious input in many occasions during my research. I would like to thank both current and past members of our research group, especially Melati and Na who had been like mentors in my first year of study here in Kent state.

I would like to thank Dr. Mahinda Gangoda for his assistance and support in all kinds of instruments in our lab which was extremely invaluable during my research. I would also like to thank numerous members of the chemistry department support staff, especially Erin Michael, Lisa Stamper, Arla Dee McPherson, late Erica Lilly, and

Rochelle Gray.

I would like to thank Larry who has been so helpful in making glassware of my design that I needed. His exceptional skill was very useful in my entire research. I also would like to thank people from Writing Commons for correcting my English in many instances.

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CHAPTER 1

Introduction

CHAPTER 1 Introduction

1.1. A brief introduction of Super Resolution Imaging

Fluorescence microscopy is one of the most versatile and powerful tools for studying structural organization and intra/intermolecular processes within biological systems1-4. It has enabled noninvasive visualization of cellular components through molecule-specific labeling as well as the imaging of the dynamic processes in living organelles. However, the use of conventional fluorescence microscopes is limited by the relatively low spatial resolution due to the diffraction of light. Any object smaller than

~250 nm along the x-y axis (lateral direction) and ~500 nm in the z-axis (axial direction) can be seen only as a blurred or diffracted spot1,5. The size of this diffracted spot results in a fundamental limit on the minimal distance at which two or more objects can be resolved6,7. The intensity distribution of the images of this diffracted spot in three dimensions is represented by the point spread function (PSF) of the microscope3. In optical microscopy, the PSF of a microscope is the observed ‘peak’ from a single point source of the light8. The limitation of visible light to observe objects beyond the nanoscale was first recognized by Abbe (1894) and it is known as Abbe’s diffraction limit. He noted that a light microscope can distinguish two parallel lines only when their spacing is greater than λ/2NA, where λ is the detection wavelength, and NA is the numerical aperture of the microscope’s objective.

In order to overcome Abbe’s diffraction limit and to improve the spatial resolution of microscopy two conceptual strategies were introduced (1) sharpening the

PSF, and (2) redefining the problem to one of the individual fluorophore localization temporally rather than separating two or more fluorophores which appear to overlap at that level3. The first strategy to overcome the Abbe’s diffraction limit was proposed by

S.W. Hell (1994) which is based on the illumination and quenching of the probes using nonlinear optical approaches that finely tunes the PSF to the smallest possible shape9. He introduced Stimulated Emission Depletion (STED) fluorescence microscopy that achieved nanoscopic resolution 9 and later other microscopy techniques emerged such as

Saturated Structured illumination microscopy (SSIM)10. At present, STED and SSIM are capable of resolving fine structural details of biological structure on a nanoscopic level down to ~ 30 nm in the x-y direction in the case of STED and ~ 100 nm in the case of

SSIM. As examples, images of mitochondria membranes 11, chromosomal and nuclear envelope organization 12, and recently, movement of histone H2B core proteins in the nucleus of living HeLa cells13 have been achieved.

The second strategy for overcoming Abbe’s diffraction limit is the sequential localization of sparse subsets of individual photoswitchable/photoactivatable molecules with super resolution. The basic principle of this approach involves the stochastic activation of the fluorescent probes to turn on the individual photoactivation / photoswitchable molecules, then image it and bleach it / turn it off which temporarily separates molecules since at that level the emitting molecules are spatially indistinguishable3. This process was repeated several times. A final super resolution image is obtained by consolidating all the images of the single-molecule obtained by photoactivation / photoswitching cycles. Three major probes based super resolution

techniques have been independently developed by Betzig (2006), Photoactivated

Localization Microscopy (PALM)14, Hess (2006), Fluorescence Photoactivated

Localization Microscopy (FPALM)15, and Rust (2006), Stochastic Optical Reconstruction

Microscopy (STORM)16. These three methods basically use similar features in which the experiments are performed under active control of the concentration of emitting single molecules in a very low levels through-out the experiment; therefore these three super resolution techniques may be termed as ‘Single Molecule Active Control Microscopy

(SMACM)17.

1.2 Single molecule detection and its application in super resolution imaging

In optical microscopy, the light emitted from a single fluorescent molecule about

1-2 nm in size has provided a solution to overcome Abbe’s diffraction limit. The first detection of a single pentacene molecule doped in p-terphenyl was selectively detected in the co-crystal by Moerner and Kador (1989) 18 at liquid helium temperature. After this report on absorption by a single pentacene molecule, Orrit and Bernard reported that a single molecule of pentacene can be detected on the same pentacene doped p-terphenyl by monitoring its emission intensity as a function of excitation frequency rather than absorption 19. However, Orrit and Bernard concluded that certain optimizations are required for an emitter to be efficiently utilized in single molecule detection by fluorescence excitation: (1) In order to increase the interaction of the emitter with light, an emitter must have a sufficiently large absorption cross section, (2) as the overall

photon detection is rather low, a molecule must be able to emit as many photons as possible prior to its resonance frequency shifting irreversibly and, (3) the rate at which photons are emitted must be greater than those of the dark count of the detector. These three major optimizations in an emitter would be ideal for single molecule detection.

After the Moerner and Orrit imaging of single molecules in the solid state at cryogenic temperatures, it was further extended to ambient temperature in the mid-1990s with the advent of different microscopic techniques such as Near-Field Scanning Optical

Microscopy (NSOM)20 and Confocal Fluorescence Microscopy21,22 which was eventually extended to the broad range of biological studies occurring since the year 2000 23,24.

1.3 Single molecule imaging

Single molecule localization can be performed by a variety of microscopic configurations, including wide-field epifluorescence, total internal reflection, confocal imaging, multiphoton microscopy and near-field microscopy 17,23,25 with the aid of a fluorescent emitter. A typical wide-field epifluorescence microcopy set up possesses the following components as shown in Figure 1.1 in a simplified diagram25,26. The fluorescent probe labeled object can be excited by a certain wavelength laser beam. The input excitation laser beam from the source is reflected toward the microscope objective by a dichroic beam splitter and is absorbed by the labeled object. The fluorescence emission from the labeled object is collected back through the same microscopic objective and transmitted through the dichroic beam splitter which filters out the residual

laser excitation light. The light is then focused via the microscope tube lens to a CCD camera for recording.

Figure 1.1 Epifluorescence microscopy: a schematic of a representative experimental set up.

In an experiment, Moerner et al 27 schematically illustrated the use of single molecule detection and obtained the image of a cell (Figure 1.2). In this imaging process, a focused pumping green laser beam basically bathes the cell (object) to pump the fluorescent molecule (for example, single transmembrane proteins have been labeled with fluorescent dyes) inside the cell and the cell itself is essentially transparent and not fluorescent if the pumping wavelengths longer than about 500 nm are used.

A B C

Figure 1.2 Imaging single molecules (A) Illustration of single-molecule imaging. A diffraction-limited pumping region (green) illuminates a sample such as a cell containing fluorescent labels. For single-molecule imaging, only one molecule should be emitting (red) within a diffraction-limited pumping volume. (B) Example of wide-field epifluorescence imaging of single fluorescently labeled transmembrane proteins on a cell surface. Frame: 12× 12 μm. (C) Wide-field fluorescence image of a bacterial cell (red) containing a single protein fusion between the bacterial actin protein MreB and EYFP. Rendered in three dimensions with the z-axis as brightness, a single molecule looks like a mountain. A total of 100 ms acquisition time, bar: 0.5 μm27 [Adapted from PNAS, W.E.Moerner, 200717]

Each single molecule is a few nanometers in size and smaller than the focused laser spot. In a precise focusing only one molecule can be pumped in the pumping volume and the information related to one individual molecule and its local

‘nanoenvironment’ can be obtained by detecting the photons from that molecule alone.

This is possible only when the molecules are farther apart than about 500 nm3, i.e., ultralow concentrations of emitters are required. In terms of spatial resolution, when the emission from the fluorophore is scanned, a single peak is observed for each single nanoscale source of light, which approximately maps out the PSF of the microscope.

Basically a molecule is a nanoscale light absorber, smaller than the size of the PSF.

Figure 1.2C shows the approximate PSF shape for emission from a single molecule in

the bacterial actin protein MreB, “the White Mountain”, labeled by fusion to enhanced yellow fluorescent protein (EYFP) 27,28. It is this mountain-like image (PSF) from a single molecular emitter that forms one key element for current super-resolution efforts based on single-molecule microscopy. By measuring the shape of the PSF, the centroid position can be determined much more accurately than its width8 and the location of single emitter can be obtained precisely. This is the foundation of the super resolution imaging at the nanoscale.

1.4 Super-resolution imaging by single molecule localization

Betzig et al., Hess et al., and Rust et al. in 2006, separately modified standard wide field single-molecule fluorescence microscopy in an effort to overcome the optical diffraction limit14-16. There are some essential requirements that must be fulfilled to image beyond diffraction limits such as: (1) sufficient sensitivity to enable imaging of single-molecule labels, (2) determination of the position of a single molecule with precision better than the diffraction limit, and (3) the addition of on/off control of the molecular emission to maintain concentrations at low levels in each imaging frame.

When these requirements are met, sequential imaging of sparse subsets of single molecules yields many samples of the underlying structure, enabling reconstruction of a final image with resolution far below the optical diffraction limit, or super resolution imaging.

In wide-field microscopy an image of an object is seen like as in Figure 1.3A in which the closely spaced emitters cannot be resolved when all the emitters are activated at the same time. The basic idea behind getting a super resolution image is that not all the emitters are emitting at the same time. For example, a live cell tagged with photoactivatable fluorescent emitters is dark in the non-emissive precursor state illustrated in Figure 1.2A. A weak activating light is used to turn on only a very small number of emitters in such a way that their PSFs do not overlap (Figure 1.2B). These emitters can then be super-localized as described in Figure 1.3 and the positions of the individual single molecules are recorded. Next, these emitters are eventually photobleached, and then a new sparse subset is photoactivated.

Figure 1.3 A cartoon showing the key idea of super-resolution imaging of a structure by SMACM (A) The overlapping of images in densely located fluorescent labels does not allow resolution of the underlying structure in a conventional wide-field fluorescent image. (B) Using controllable fluorophores (photoactivatable/photoswitchable) only a sparse subset of emitting molecules can be localized with nanometer precision (a spiral is the underlying structure being sampled here). Once the first subset is photobleached/turned off, another subset is activated and the same process is repeated for the other remaining subsets and the resulting localizations were summed up to reconstruct the final super- resolution image of the underlying structure [adapted from M.A. Thompson and W.E. Moerner29].

After a series of sequential imaging of sparse subsets of emitters one is able to build a pointillist reconstruction of the underlying structure as shown in Figure 1.3C.

This idea was termed PALM (Photoactivated Localized Microscopy) by Betzig et al

200614. In the same year, Rust et. al. (2006) utilized photoswitching of a single photoswitchable fluorophore for super resolution demonstrated in fixed cells with immunofluorescence labeling termed STORM (Stochastic Optical Reconstruction

Microscopy)16. One of the most important aspects in super resolution imaging is the fluorescent probe properties because they report the different features of their local environment by emitting photons which possess a wealth of different information about the emitter and the specific environment, e.g. color (energy), polarization (orientation), rate at which they are being emitted (lifetime) and, finally, the position of the emitter.

Therefore it is important to collect as many photons as possible from the emitter, prior to the destruction of or turning off of the emitter. This allows obtaining the most detailed information about the emitter and its environment. An area of current research interest involves the applications of single molecule optical studies to the interior of the living cells 30.

1.5 Fluorescent probes for super resolution imaging

The most essential requirement of all the localization based super-resolution technologies is the availability of ideal photoactivatable or photoswitchable fluorophores.

All the super resolution microscopy techniques utilize different types of fluorescent probes for imaging according to their imaging approaches. So far it is known that these probes have been broadly classified into three major classes: fluorescent proteins, small organic fluorophores and quantum dots. Fluorescent proteins (FP) have been utilized as probes for super resolution imaging on several occasions1,2,31,32 since they have some advantages over the small organic fluorophores and Quantum Dots (QD). For example, they are genetically encoded and can be fused with the proteins of interest 33 which provides a direct access to specific labeling, therefore they are often the first choice of fluorophore for live cell imaging. However, an unavoidable problem that most FPs faces is their brightness. Dronpa, a well-studied FP34, is considered as the brightest FP but it is still 10 times dimmer than the small organic fluorophores. The development of brighter fluorescent proteins has been a challenging task due to the strict chromophore conformation demands inside the β-barrel35. In addition, FPs have slow photo response rates. For the fastest photo responding FPs such as Dronpa-monomeric variant36, KFP37, and EosFP38, the maximum frame rate that can be achieved (~25 second per frame) is still not fast enough to image most biological processes if a resolution of ~60 nm is required 1.

Quantum Dots (QDs), usually inorganic semiconducting materials, are popular for their brightness and photostability for imaging applications 39,40 but their emission often blinks in a complex pattern and they are physically large compared to organic fluorophores. When they are functionalized on the surface for their aqueous solubility and bioconjugation, they typically become even larger (12-20 nm) and as a result it may

hinder motion of the analyte and obscure the true dynamics. Although the blinking of some quantum dots has been corrected41, no water-soluble version of this QD has been reported yet42.

The use of photoactivatable/photoswitchable organic probes has become an integral part of the some single molecule SR techniques. They are brighter than the brightest fluorescent protein by 10 fold43-45 a critical property required for single molecule super resolution (SR) imaging2. They are smaller in size compared to any fluorescent protein so they can be delivered inside the live cell through the cell membrane. There is plenty of flexibility in functionalizing the organic probes. Therefore they can be conjugated to a broad range of biomolecules such as small receptor-binding peptides, short DNA/RNA fragments or hairpins, and small drug molecules with varieties of spacers and functionalities46.

1.6 Photoswitchable fluorescent probes utilized in super resolution imaging

There are several organic synthetic fluorophores that have been utilized in super- resolution imaging so far (Table 1.1) and a detailed evaluation of photoswitchable fluorophores for localization based super-resolution imaging was done by Zhuang et. al.47. Despite the problem of non-genetic specificity of the fluorophores, they can be bioconjugated with a number of peptides, enzymes, antibodies for the study of fixed cells and live cells.

Figure 1.4 Structures of some fluorescent dyes utilized in super resolution imaging.

class of Fluorescent dye Buffer activation excitation target fluorophore

enzymatic oxygen scavenging Double stranded DNA in YOYO 1 dimeric cyanine system and 50 mM β- - 488 nm fixed sample 48 mercaptoethylamine (MEA) Human histone H2B TMP-ATTO In presence of O2 with oxazine - 647 nm protein in living HeLa 655 glutathione (RS-) cells 13 Oxygen scavenger and 514 BG-Cy3-Cy5 carbocyanine - Fixed microtubules mercaptoethanol nm/633 nm Alexa 488, 532, phosphine-buffered saline (PBS; microtubules in COS-7 568 and ATTO Rhodamine - 496 nm pH 7.4) with MEA fixed cells 49 488, 590 ATTO 520, PBS, pH 7.4 in the presence of microtubules in COS-7 Oxazine - 516 nm 655, 700* 100 mM MEA fixed cells 50 49

Histone H2B proteins in a TMR Rhodamine glutathione 405 nm 554 nm COS-7 cell51

PBS, pH 7.4 in the presence of microtubules in COS-7 ATTO 565 Rhodamine - 516 nm 100 mM MEA fixed cells 50 49 phosphate-buffered saline (PBS, pH 7.4), containing F-Actin filaments in Alexa 647 carbocyanine 488/514 650 nm oxygen scavenger and 50 mm b- COS-7 cells mercaptoethylamine (MEA)

Table 1.1 Some conventional fluorescent dyes (structures are in Figure 1.4) with photoswitching properties with the addition of redox reagents in buffer. *Structure of ATTO 700 is not disclosed yet.

The photoswitching mechanism of the almost all of the fluorophores shown in

Table 1.1 involves taking advantage of their sensitivity towards reducing agents e.g. thiol compounds which are required for the formation of a stable non-fluorescent state after fluorescence quenching 52. There is another strict requirement for carbocyanine dyes such as Alexa Fluor 647, Cy5, Alexa Fluor 680 (structure not disclosed yet) and Cy 5.5 that are susceptible to molecular oxygen. In these cases oxygen scavengers play a critical role for their photostability and stabilize the reduced OFF state of the fluorophore53,54. Such

chemical cocktails may be toxic to the live cell and their use is not ideal for in vivo imaging.

Another best-known approach of photoswitching of organic fluorophores is governed by the nonradiative fluorescence resonance energy transfer (FRET) mechanism55. The operation of FRET requires two fluorescent dyes, one donor and the other acceptor, separated by 2-8 nm of distance that is referred to as the Förster distance.

The effective energy transfer process can take place when the donor’s emission spectrum overlaps the absorption spectrum of the acceptor molecule. The excited state donor molecule releases energy by a non-radiative process to the ground state and this released energy is transferred to a nearby acceptor chromophore in a nonradiative fashion through long range dipole-dipole interactions (Figure 1.5). The more overlap of spectra there is, the better a donor molecule can transfer energy to the acceptor molecule and better the emission from the acceptor molecule would be expected56. The acceptor can reemit the energy as emission, so the detected emission peak of the FRET pair should be bathochromic compared with the donor fluorophore.

1 2

Figure 1.5 The basic principles of FRET operation. (1) The two blue solid lines represent the absorption of the donor and acceptor fluorophores. The two red peaks represent the emission of donor and acceptor. The yellow area represents the excitation of the donor fluorophore whereas the red area represents the emission from the acceptor. “r” represents the distance between two fluorophores and the green area represents the overlap of the emission of the donor spectrum with the absorption of the acceptor spectrum. (2) A Jablonski diagram showing the transfer of resonance energy, the green solid line represents donor absorption (excitation), the red solid line represents the donor fluorescence emission, the blue solid line represents resonance energy transfer, red broken line represents the non-radiative energy transfer, the green broken line represents the non-radiative acceptor (excitation), and yellow wavy line represents the vibrational relaxation. (Source: Olympus Microscopy Resource Center and Wikipedia)

Pairs of fluorescent covalent heterodimers such as Cy3/Cy5, Cy3/Cy7, when properly positioned, exhibit optical switching phenomena; such as switching on and off the fluorescence of the dyes53,57. Such combination of dyes working with FRET was successfully utilized in high-resolution imaging techniques such as stochastic optical reconstruction. In Figure 1.6, the Cy2/Cy5 and Cy3-Alexa 647 pair attached to antibodies has been able to visualize microtubules and clathrin-coated pits in fixed mammalian cells respectively using STORM 58,59. The R group, mentioned in Figure 1.7,

is the binding site of the probes with the antibody via a functional group such as amine, thiol and other functionality.

Figure 1.6 Antibody conjugated with Cy3/Cy5 pair which communicates eachother

Figure 1.7 Structure of Cy3, Cy5, Alexa-647 with R group as the binding site with antibody 59

Although carbocyanine and Alexa fluor are dependent on other fluorophores for the photoswitching process, the presence of thiol-reagent (~ 100 mM) also plays critical role for their photoswitching mechanism54,60. Further they suffer from stringent requirements requiring an oxygen free environment. The use of a single fluorophore in any cell imaging greatly simplifies sample labeling and eliminates the necessary procedures to integrate two dyes into the required location61. Very recently photoswitchable ATTO 655 (rhodamine dyes) has been utilized for photoinduced

blinking in live cells13 (Figure 1.8). In this case the removal of oxygen is not necessary and reducing agents such as glutathione present in the cytoplasm of cells in the micromolar to millimolar range are the necessary condition for the switching mechanism and would be a relevant condition for single molecule super-resolution imaging even in living cells49.

Figure 1.8 Schematic representation of trimethoprim-dihydrofolate reductase protein from E. Coli (TMP-eDGHFR labeling system). TMP is covalently attached to ATTO655, binds the histone protein H2B-eDHFR. The reversible photoswitching is possible when there is presence of molecular oxygen and thiol enabling super-resolution imaging of live cells.13 (Adapted from Nature Methods, 2010, R. Wombacher et. al.13)

1.7 Problems faced by the existing photoswitchable probes.

The existing fluorescent probes can be photoswitched only in the presence of organic and inorganic buffers and the on and off processes are only possible by the use of redox reagents. In carbocyanine dyes such as Cy3, Cy5, there is a strict requirement for the presence of an oxygen scavenger in the assay. At the present time, many conventional synthetic ﬂuorophores can undergo reversible photoswitching in living cells under physiological conditions without addition of a redox cocktail since the cell compartment

itself possess those redox cocktails. However, one way to avoid the all these requirements for switching of molecule from on to off state and vice versa is the use of a natural photoswitcher which operates only in the presence of light, i.e. the turn on and off cycle should be triggered by the light. Amongst several fluorescent dyes, the rhodamine spirolactams are a class of photoswitchable dyes which may offer natural photoswitching properties and has ample possibilities of constituent structural modification according to our desire.

1.8 Scope of this thesis

The fundamental objective of this project is to design, synthesize, characterize, optimize fluorescent dyes with absorption wavelength ≥ 400 nm, water solubility, high fluorescence quantum yield, reduced photobleaching, quick photoresponse, and flexible functionalization for bioconjugation. This thesis in Chapter 2 and 3 describe the design, synthesis and photophysics of photoswitchable rhodamine spirolactams. The new photoswitchable rhodamine spirolactam probes, as biological markers may be suitable for single molecule SR imaging. Most important, photoswitchable dyes are mandatory for the study of dynamic cell organelles. A durable near infra-red (NIR) emitting photoswitchable probe that can survive many turn on and off cycles and provides quick photoresponse even at the single molecule level definitely has a special interest to the subdiffraction cell imaging world. The preliminary photophysics of the rhodamine spirolactams were done here in our lab. Beside the rhodamine spirolactams, this thesis in

Chapter 4 discusses some other fluorescent dyes that can be turned on to the bright state after a 1,3-dipolar cycloaddition reaction between an azide-Ph-DCDHF and strained alkene have been introduced.62 This chemical method of turning the dyes from dark to bright state is a new dimension in the bioconjugation arena. This thesis also describes in

Chapter 5 some other methods to turn the fluorescent dyes to a bright state from a dark state by collision on the cell surface more popularly known as Point Accumulation for

Imaging in Nanoscale Topography (PAINT) scheme63. For the PAINT scheme we synthesized some Nile red derivatives and DCDHF derivatives with improved properties than what was used in previous PAINT techniques. Beside the PAINT technique, some active control of emission by enzymatic cleavage of fluorescent dyes in the dark state to the bright state, which can be utilized in super resolution imaging64, are also described in

Chapter 5. Furthermore, similar to 1,3-dipolar cycloaddition reaction between azido-

DCDHF and norbornene described in Chapter 4, we discussed recently popularized tetrazine chemistry65 in Chapter 5. We synthesized some tetrazine derivatives and studied their properties. The photophysics of selected compounds described in Chapters

3 and 5 was performed by Marissa Lee from Stanford University.

Finally, in this thesis Chapters 6 and 7 describes x-ray crystallography of co-crystal and polynuclear heteroaromatic compounds respectively. The x-ray crystallography of these compounds was performed in Dr. Bunge’s lab at Kent State University.

CHAPTER 2

Design and Synthesis of rhodamine spirolactams

CHAPTER 2 Design and Synthesis of rhodamine spirolactams

2.1 A brief overview of Rhodamine Spirolactams

An important class of small organic fluorophores is the photoswitchable rhodamine spiroamide derivatives. They are belonged to the xanthene family along with fluorescein and eosin dyes. The general structure of xanthene chromophore and rhodamine derivatives are represented in Figure 2.166.

2.1 2.2 2.3

Figure 2.1 Molecular structures of xanthene (1), rhodamine dyes, and (3) rhodamine spirolactams

The photophysical properties of rhodamine spirolactams was first described by

Knauer and Gleiter 67 in the 1970s and their photokinetics were first investigated by

Willwhol, Wolfrum and Glieter 68. Rhodamine spirolactams undergo a reversible transition between an emissive and a dark state via light-induced isomerization in the absence of effector molecules69. The open photoisomer (emissive state) of a rhodamine spirolactam is photostable, bright, has high quantum yield, high extinction coefficient value, and provides ample modification sites for easy functionalization66,70 for tagging and water solubility. Therefore they are good candidates for the single molecule SR

imaging by controlling the emission of single molecules enabling temporal separation and localization. The basic photophysics of rhodamine lactam is described in Figure 2.2.

Isomerization Green absorption S1 (hv) pumps fluorescence 5µm Photobleached S0 Open isomer Isomerization (thermal) B

S0 Closed isomer

(Courtesy: Stanford University)

Figure 2.2 (A) simplified Jablonski diagram showing the photophysics of rhodamine spirolactam isomerization and representative bulk spectra. The dark closed isomer undergoes light-induced bond breakage to form a fluorescent open isomer. The open isomer can thermally return to the closed state, or it becomes irreversibly photobleached. (B) A montage of rhodamine lactam activation in a polymer film. Left box shows fluorescent molecules (imaged with 532 nm) before activation. Right box shows fluorescent molecules after 1s activation with 403 nm laser. Both boxes are summed intensities over 1s (10 frames), scaled to same contrast. Scale bar is 5µm

Upon absorption of the UV light by the closed isomer (non fluorescent), the C-N bond of the lactam connected to xanthene may be broken generating an open isomer

(fluorescent), which typically absorbs in the green region and emits in the red (λ~560-580 nm) for rhodamine B spirolactam. The two photoisomers have different spectral properties.

The open isomer then thermally reverts to the more stable closed isomer or irreversibly photobleaches.

Figure 2.3 Photoswitching properties of a rhodamine dye developed in Hell’s lab. Absorption spectra of 5-Li (closed isomer, black line, left axis) in water solution, and excitation and emission spectra of the open isomer in a PVA film after photoactivation with 366-nm light (red lines, right axis; detection: 620 nm; excitation: 530 nm).[Adapted from Angew. Chem. Int. Ed, 2007, Hell, SW et al69)

Hell’s lab has utilized the rhodamine spirolactams to obtain the super resolution images of tubulin in fixed cells69. The non-fluorescent (closed form) of rhodamine spirolactam can be photoswitched with 366 nm (UV) light to a fluorescent (open form) charge-separated isomer (Figure 2.3). The open isomer can be switched back to the thermally deactivated closed isomer.

Figure 2.4 Reconstructed images of the tubulin network in a PtK2 cell stained with 69 5-NHSS . A) Wide-field image; B) one-photon (375 nm) 1PA-PALMIRA image; C) two-photon (747 nm) 2PA-PALMIRA image; D, E) enlarged sections from (A) and (C) respectively (dotted-line boxes); F) Profiles across the x direction of two adjacent filaments, averaged in the y direction (full-line box in (A) and (C)), from the wide-field (black line) and 2PA- PALMIRA (red line) images. [Adapted from Angew. Chem. Int. Ed, 2007, Hell, SW et al69)

Hell’s lab has developed fluorogens (A, B, and C; Figure 2.5) and obtained SR resolution images of microtubulins of PtK2 fixed cells (Figure 2.4). There are still plenty of options available to further optimize rhodamine spirolactams for use in SR in terms of absorption, targeting, solubility, photostability and response time for live cell imaging.

A B C

Figure 2.5 A Few representative Rhodamine derivatives A, B71 and C72 applied in super resolution imaging by Hell's group.

2.2 Rhodamine spirolactam derivatives:

Since the two essential parts of a photoswitchable assembly - the switching and the fluorescent (reporter) groups - are incorporated in one chemical entity the rhodamine spirolactam is a suitable choice for SR technique. In our initial synthetic efforts to study the photophysical properties of rhodamine spirolactams the rhodamine base B has been used to make all the spirolactams. The photoswitching properties of these spirolactams are governed by at least three distinct but interacting substructural features in the molecule: the rhodamine chromophore, the anhydride unit and the chromophoric aryl amine, shown in Figure 2.6.

Figure 2.6 The structural features of the rhodamine spirolactams. (1) Red upper oval (right) highlights rhodamine chromophores. In the closed leuco isomer, this are two isolated rings on left (Gray upper oval), while the open emissive isomer has delocalization between the two rings. (2) Blue oval in lower region either isomer shows the anhydride portion and (3) Green oval in the leuco isomer shows the chromophoric aryl amines which is required for the absorption above 400 nm.

The xanthene chromophore in the open isomer (right red oval in Figure 2.6) has the two amine-substituted rings conjugated by an empty p orbital at the connecting carbon atom and charge delocalization through all three rings and both amine substituents which is responsible for the color of the rhodamines. In the closed form of the molecule

(left gray oval in Figure 2.6), the two amine substituted rings are now electronically isolated by the introduction of the spiro center. Synthetically, the components found in the left gray oval, except for the central carbon atom, are derived from 3-aminophenols.

The blue oval at the lower portion of the two isomers includes the aromatic lactam bearing substituent X (the spirocarbon and X bearing ring are derived from an X- functionalized aromatic anhydride). The green oval includes a Y group/substituent, an

aromatic ring and lactam nitrogen atom and both intersect the critical bond that is broken/made in the photoreactions that interconverts the open and closed isomers.

We are considering modification of the xanthene nucleus73 and chromophoric arylamide part which may significantly change the photophysics of the rhodamine derivatives. More importantly for the long range absorption by the rhodamine spirolactams in the closed leuco isomer, there is a substantial possibility of substructural modification in the amide portion. The aryl amine itself can be a chromophore and is also responsible for the fluorescence of the open isomers.

2.3 Synthesis of rhodamine derivatives:

2.4 Synthesis of tetra-N-ethyl-spiro[benzo[4,5]imidazo[2,1-a]isoindole-11,9'- xanthene]-3',6'-diamine (P-71)

Rhodamine spirolactams (dimethyl version of P-71) was first synthesized by

74 Gunzenhauser, S. et al. in 1979 . They investigated the complex proton equilibrium and determined the pKa of the compound. After their work, no record of this molecule was found. Rhodamine spirolactam P-71 can be prepared via intermediate 2.6 (Figure 2.7).

Furthermore, intermediate 2.6 can be prepared from the direct condensation between commercially available phthalic anhydride and 1,2-diaminobenzene. However, the synthesis of 2.6 is not as straightforward as one may think due to inconsistencies in the literature.

P-71 2.6 2.5 2.4

Figure 2.7 Retro-synthesis of rhodamine derivative P-71

2.5 Literature approaches to 1H-isoindolo<1,2-a>benzimidazole-1-one (2.6)

2.5 2.4 2.6

Scheme 2.1 Synthesis of 1H-isoindolo<1,2-a>benzimidazole-1-one by direct condensation between phthalic anhydride and phenylene-1,2-diamine (a) 1-pentanol, reflux

One approach reported in the literature for the synthesis of intermediate 2.6 involves the reaction of phthalic anhydride and 1,2-diaminobenzene refluxing in 1- pentanol (Scheme 2.1) affording 2.6 in a 39% yield as colorless solid as described by

Meegalla et al (1994)75. However, after following this procedure, the melting point and

NMR characterization of the substance we obtained did not match with what was presented in the literature. The reported melting point of 2.6 ranges from 209 to 227°C which is 70°C lower than what we obtained76. Due to this inconsistency, the compound was recrystallized and a crystal structure was obtained for further confirmation of the structure. From the crystal structure results, it can be concluded that the reported synthesis by Meegalla was compound 2.7 (Figure 2.8) and not structure 2.6.

2.7

Figure 2.8 Structure of 2.7 as determined by X-ray crystallographic study77.

Since the desired product was not obtained by using the synthetic procedure described by Meegalla et.al., we sought out other methods within the literature. A reaction procedure described by Kobrakov et al (2007)78 was carried out in aqueous acidic conditions with a reported 100% yield. However when this procedure was attempted for the synthesis of compound 2.6, a mixture of 2.7 and unreacted 1,2- diaminobenzene was obtained. Furthermore, changing the duration and temperature of the reaction was not productive.

There have been numerous reports in the literature that give compound 2.6 from compound 2.9 via two steps (Scheme 2.2 and 2.3). Therefore we decided to take this stepwise approach described in the literature. We attempted some methods for synthesizing compound 2.6. In one approach, a condensation reaction described by

Likhatchev et. al. (1998), a mixture of phthalic anhydride and 1,2-phenylenediamine (1:1 ratio) in DMF was stirred at room temperature to afford compound 2.8 after precipitation in toluene79. When this reaction procedure was carried out no precipitation of compound

2.8 was observed so the temperature of the reaction was increased to 40°C and it was run overnight. The reaction gave a mixture of products, which was difficult to separate.

2.5 2.4 2.8 2.9

Scheme 2.2 Synthesis of 2.9 by stepwise route (a) THF, rt, 4 hrs; (b) stirring THF

In another attempt by the same author Likhatchev (1999), DMF was replaced with

THF 80 to synthesize 2.9 (Scheme 2.2). The reaction began with stirring an equimolar mixture of 1,2-phenylenediamine (2.5) and phthalic anhydride (2.4) at room temperature for 4 hours, product 2.8 was separated as a white solid after washing several time with ether (52% yield with mp 147°C, reported yield 91%). Since the compound has the tendency of self-cyclization 79, the compound was left stirring in THF stirring overnight to produce 2.9 in 64% yield (reported 75%). The next step of the reaction is the cyclization of compound 2.9 to afford compound 2.6, which was reported to be done in a sealed tube with high temperature. However, the cyclization reaction can be done simply by heating 2.9 in NMP at 208°C for 24 hours to give 2.6 in 67% yield.

2.9 2.6 Scheme 2.3 Synthesis of 2.6 from 2.9 by ring closing condensation; (a) NMP, 200°- 210°C, 12hr

Although the reaction can be done in stepwise route, we also devised a method by which

2.6 can be synthesized in a one-step reaction with good yield by use of a microwave reactor within half an hour. This method has not been reported elsewhere.

2.5 2.4 2.6

Scheme 2.4 Synthesis of 2.6 using microwave reactor

In this method an equimolar solution of phthalic anhydride and 1,2- phenylenediamine in NMP was irradiated with microwave radiation with a setting of 300

W of electric power, at 205°C and 250 psi pressure and the holding time was set to 30 minutes. The mode of the microwave setting was chosen to “discover” which applies for closed vessel reaction. The yield was only 20%. When we chose to run the reaction in an

“Open Vessel” mode, the yield was improved to 67 %. In Open Vessel mode, the reaction was carried out at atmospheric pressure and 100°C. In this method the phthalic anhydride was slowly added via additional funnel. Not only did we increase the yield of the reaction but we also significantly reduced the reaction time from multiple days to half an hour.

This compound was recently synthesized by Mamada et al (2011) with 72% yield in water in a sealed tube at 300°C 76 but long reaction times are still needed.

In our next step, Rhodamine spirolactams P-71 can be prepared via 2.6 in a number of ways as reported earlier. In one approach as described by Gunzenhauser

(1979), when 2.6 was fused with 3-(diethylamino)phenol in 1:2 ratio at 200-210°C in a

81 sand bath for 90 minutes in the presence of anhydrous ZnCl2 , it produced a mixture of several compounds as indicated by Thin Layer Chromatography (TLC). We separated the mixture using gradient column chromatography by using hexanes and ethyl acetate mixtures to slowly increase the polarity. All the spots that appeared on the TLC were separated in trace amounts. The desired compound was obtained in the fourth fraction as colorless solid and it was halochromic compound when tested 81. Unfortunately the yield obtained was very poor (around 2%).

2.6 P-71

Scheme 2.5 Synthesis of rhodamine spirolactam P-71 by literature procedure.

Halochromism, or change in color due to change in pH, is observed in rhodamine spirolactam compound P-71. When compound P-71 is treated with acid, it becomes red and fluorescent and when the acidified compound P-71 is treated with base it becomes colorless again.

Figure 2.9 Halochromic properties of rhodamine spirolactam P-71.

In acidic conditions, the spirolactam ring opens up allowing benzene rings A and

B to become conjugated. This greater delocalization of electrons results in a color change of compound P-71. Furthermore, with the addition of base, the conjugation is lost producing the original colorless and non-fluorescent, state of compound P-71.

We attempted another method described by Corrie et. al. (1995) in his patent to synthesize P-71 by dissolving 2.6 and trimethylsilylpolyphosphate (PPSE) under nitrogen but this reaction did not work at all 82. However in a stepwise reaction described by

83 Muthyla (1997) to synthesize rhodamine dyes using H3BO3 gave us the desired product but still the yield was very poor (5.6%).

In order to improve the yield and search for an efficient reaction route, we tried the microwave reactor to synthesize P-71 under the same conditions described by Corrie et. al. with H3BO3 described by Muthyla. We irradiated a mixture of 2.6 and 3-

(diethylamino)phenol in 1,2-dichlorobenzene as solvent for 30 minutes in a setting of power 300 W, temperature 210°C and pressure 150 psi. It gave us compound P-71 in

32% yield. Again the microwave reactor was found to be promising for the synthesis of

compound P-71. The synthesis of compound P-71 using a microwave reactor and reagents mentioned in Scheme 2.6 has not been reported elsewhere.

2.6 P-71

Scheme 2.6 Synthesis of P-71 in a microwave reactor

We wanted to see whether P-71 would undergo photoswitching upon irradiation.

The UV-Vis study showed that the closed isomer absorbs below 340 nm and very hard to undergo ring opening under UV light. It favors the colorless state (ring closed state) rather than color fluorescent state (ring opened state)

P-71'

1.59

1.39

1.19

0.99

0.79

Absorption 0.59

0.39

0.19

-0.01

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700

WavelengthWavelength (nm) (nm)Absorbance

Figure 2.10 UV-Vis of rhodamine derivative P-71 (the peak at 560 nm is due to the open form P-71)

Figure 2.11 Photoswitching of the rhodamine spirolactams favors the ring closed colorless state.

However, after exposure of P-71 for two days in the daylight, it turned pink. It can be concluded that compound P-71 is photosensitive but its ring opening is very slow and upon keeping in the dark for days did not return to the colorless state. Since compound P-71 did not undergo easy photoswitching mechanism, we designed and attempted to synthesize other derivatives in which the open form would be stabilize by

addition of electron withdrawing groups on the phenylbenzimidazole side (and stabilizing the negative charge there) Scheme 2.7 and further it may absorb above 350 nm. The ring may be more prone to open due to the presence of electron withdrawing group. Cogne-

Laage et. al.84 and Poon et. al.85 have successfully demonstrated the electron withdrawing tendency of BF2 (β-diketonate) in the photoswitching of a dithienylethene system. We assumed similar idea can be applied in the switching mechanism of rhodamine spirolactam.

R = it can be a bioconjugation site

Scheme 2.7 A proposed synthetic route of synthesizing derivative of P-71 which may have longer absorption.

We attempted to synthesize a better donor amine (as shown in the figure below) that may reinforce a push effect in the photoswitching process to open the lactam ring.

For this purpose, we used 8-julolidinol (2.12 in Scheme 2.8). Rhodamine dyes incorporating the julolidine donor have better emission (rhodamine B.HCl salt has λmax

86 552 nm and rhodamine 101. HCl has λmax 578 nm and also further supported by P-80 with λmax 561 nm and JCW-03-073 with λmax 576 nm Figure 3.1 and Figure 3.8) compared to dyes containing the free rotating alkylamine. The 8-julolidinol (2.12) can be made from 3-methoxyaniline by refluxing with 1-bromo-3-chloropropane at exactly

145°C for 16 hrs 87,88 with 98% yield with no column chromatography. The outcome of the reaction is dependent on the temperature. If the temperature changes from 145°C to below or above we always ended up with a mixture of products requiring column chromatography. The removal of the Me group from 2.11 was carried out by using

HI/HCl and the work up with saturated NaHCO3 which is different from the one that is described in the literature. The synthesis of 2.13 was attempted using 2.12 and 2.6,

H3BO3 in 1,2-dichlorobenzene in a microwave reactor using discover mode with a set-up of power 300W, pressure 150 psi, and temperature 152°C for half an hour but it did not work at all. Only reactants were obtained even when run for an additional hour. When we attempted to change the pressure to 250 psi, and temperature to180°C no change was observed.

2.10 2.11 2.12 2.13

Scheme 2.8 Attempted synthesis of rhodamine derivative 2.13 from 2.12 and 2.6 (a) 1-bromo-3-chloropropanol, 145°C, 16 h;(b) HI, HCl, H2O and work up with NaHCO3 (c) 2.6, H3BO3, 1,2-dichlorobenzene, MW

2.6 Attempts on synthesis of rhodamine spirolactams (2.17) based on 7H-

Benzimidazo[2,1 a]benz[de ] isoquinolin-7-one

Rhodamine derivative 2.16 may be prepared via intermediate 2.15 (Scheme 2.9).

The intermediate 2.15 can be prepared from the direct condensation between commercially available 1,8-naphthalic anhydride (2.14) and 1,2-diaminobenzene (2.4).

2.16 2.15 2.14 2.4

Scheme 2.9 Retrosynthesis of 2.16 via 2.15 with starting material 2.4 and 2.14.

2.7 Synthesis of 7H-Benzimidazo[2,1 a]benz[de ] isoquinolin-7-one (2.15)

The synthesis of 2.15 was completed by heating an equimolar mixture of naphthalic anhydride and 1,2-phenylenediamine in toluene in presence of stoichiometric amounts of imidazole. It was obtained as a yellow highly fluorescent compound with a yield of 51% (reported yield 90%)89. The compound purified by column chromatography.

2.14 2.4 2.15 2.16

Scheme 2.10 Synthesis of 2.15 by microwave reactor (a) MW, NMP (b) 3-diethylaminophenol, H3BO3, 1,2-dichlorobenzene, MW

The synthesis of 2.15 can be carried out in a microwave reactor, which proceeded in quantitative yield and no need for column chromatography to separate it. We irradiated a mixture of naphthalic anhydride and 1,2-phenylenediamine with no base in NMP as solvent for 30 minutes with a setting of power 300 W, temperature 100°C in an open vessel mode. In our next step, we first used conventional method of heating 2.15 with 3- diethylaminophenol using anhydrous ZnCl2, and also used H3BO3 attempted microwave reactor to synthesize 2.16 but did not react at all. All we got was the starting materials back.

2.8 Attempts on synthesis of rhodamine spirolactams (2.20) based on 12H- phthaloperin-12-one

Rhodamine spirolactam 2.20 based with 12H-Phthaloperin-12-one may be synthesized via 2.19 starting from phthalic anhydride (2.18) and 1,8-diaminonaphthalene

(2.17).

2.19 2.18 2.5 2.17

Scheme 2.11 Retrosynthesis of 2.19 via 2.18 with starting material 1,8 naphthalic anhydride and 1,2-phenylenediamine.

2.9 Synthesis of 12H-phthaloperin-12-one (2.18) following the literature

The intermediate 2.18 can be synthesized by a condensation reaction between an equimolar mixture of phthalic anhydride, 1,8-diaminonaphthalene and imidazole in THF under reflux with a yield of 18% 89. There was a solubility problem with the starting material in THF.

2.5 2.17 2.18 2.19

Scheme 2.12 Synthesis of 2.19 by microwave reactor (a) MW, NMP (b) 3-diethylaminophenol, H3BO3, 1,2-dichlorobenzene, MW

Alternatively, this reaction was performed in the microwave reactor, which afforded 2.18 in quantitative yield. We irradiated a mixture of phthalic anhydride and 1,8- diaminonaphthalene in NMP as solvent for 10 minutes in a setting of power 300 W, temperature 100°C in open vessel mode.

Rhodamine spirolactams 2.16 and 2.19 synthesis did not work for all the highly conjugated benzimidazole derivatives. Possibly the reaction is hindered by the steric effect displayed by additional benzene ring so that the electrophilic carbonyl may not be well exposed for a nucleophile to attack.

2.6 2.15 2.18

Figure 2.12 Steric hindrance may a reason for the unsuccessful synthesis of hypothetical rhodamine derivatives 2.16 and 2.19

2.10 Synthesis of Rhodamine salts following literature procedure

Rhodamines are some of the oldest known dyes and can be synthesized in a number of ways. One of the oldest and most common methods of synthesizing rhodamine dyes is the direct fusion of phthalic anhydride and 3-dialkylaminophenol at a temperature above 160°C90 (Scheme 2.13). The perchlorate salt of rhodamine dyes may be obtained when the molten mixture of phthalic anhydride and 3-dialkylaminophenol was treated with perchloric acid. The dye was collected as a green solid in 25% to 30% yield after cooling overnight in the refrigerator.

2.5 2.20 R = CH3 (25%) 2.21; CH2CH3 (35%) 2.22

Scheme 2.13 Synthesis of N-(9-(2-carboxyphenyl)-6-(dialkylamino)-3H-xanthen-3- ylidene)-N-methylmethanaminium perchlorate

Another attempt to synthesize the rhodamine salt by direct fusion of naphthalic anhydride and 3-dialkylaminophenol as mentioned by Hammond90 did not work. We wanted to see the effect of extended conjugation in the anhydride part as well.

2.14 2.23 2.24

Scheme 2.14 An attempt on synthesizing rhodamine salt (2.25) from 1,8-naphthalic anhydride (2.16) (a) 160°C

In another attempt to synthesize a rhodamine salt (2.26) from 2,3-pyrazine carboxylic anhydride (2.25) and 3-diethylaminophenol (2.23), we tried a fusion reaction at 160ºC for

3 hours and overnight according to the literature procedure91. However, this reaction did not work even at higher temperatures around 220°C. This reaction was first mentioned by

De et. al in 193191. After this publication no one has ever worked on this according to the literature.

2.25 2.23 2.26

Scheme 2.15 Attempts in synthesizing rhodamine salt from 2,3-pyrazine carboxylic anhydride by fusion.

2.11 Synthesis of Rhodamine spirolactams from N-(9-(2-carboxyphenyl)-6-

(dialkylamino)-3H-xanthen-3-ylidene)-N-methylmethanaminium perchlorate

Rhodamine spirolactams can be prepared by activating the acid group present in rhodamine salts. In a literature procedure, the acid group is activated by making an acid chloride and successive addition of aromatic amine affords the rhodamine spirolactams

92,93. We followed this procedure of making the spirolactam from aniline but the yield was poor (20%). In order to improve the yield, we tried some other methods. In one approach, we used DCC/DMAP in dry DCM 94 to activate the acid but this reaction did not yield any product.

We used another method to activate the acid part of the rhodamine salt. In this method we used p-toluenesulfonyl chloride and DMAP in dry DCM at room temperature under nitrogen as described by Funasaka et. al. 95. In this method, Funasaka originally used p-toluenesulfonic anhydride. The p-toluenesulfonyl chloride makes a mixed anhydride with the carboxylic acid, which subsequently reacts with DMAP to make a super leaving group. In a subsequent step, the addition of aryl amine affords an amide, which undergoes cyclization to make a spirolactam. In our first attempt, the yield was an acceptable 56%. Then the reaction was tried using a number of other amines which afforded 56-86% of the rhodamine spirolactams depending on the nature of the amines.

- - 2.22 (ClO4 ) or 2.27 (Cl ) 2.28

Scheme 2.16 A general route for synthesis of Rhodamine spirolactams; aromatic/aliphatic amines (Y); (a) p-TsCl, DMAP, DCM/CH3CN, Y

We adopted the method shown in Scheme 2.16 to make all our rhodamine spirolactams. We began this reaction in an oven dried two neck flask cooled under nitrogen. The flask was charged with rhodamine salt (1 equiv.) and anhydrous DCM. To this stirred solution was added p-toluene sulfonyl chloride (1.1 equiv.) and DMAP (2.18 equiv.) at room temperature. After stirring it for 10 min, the aromatic amine (0.9 equiv.) was added and monitored by TLC. As TLC indicated the complete consumption of the starting material, the reaction was stopped. There was a typical spot observed in TLC, which was colorless and visible only under UV. However when the TLC plate was exposed in the light for some time, the colorless spot turned to a bright pink spot. We used a heat gun for fast conversion of the colorless spot to the pink spot. This technique was a regular practice for checking whether a rhodamine spirolactam formed or not.

Furthermore, this pink spot is halochromic. When the TLC plate was dipped in a solution of triethylamine in ethyl acetate, it becomes colorless and the pink spot reverts back as we dipped the TLC plate in a solution of acetic acid in ethyl acetate. We synthesized a series of rhodamine spirolactams shown in Table 2.1 employing the method shown in

Scheme 2.16.

Table 2.1 List of compounds synthesized by Scheme 2.16

Compounds Amines (Y) Solvent MP in °C Yield %

P-82 DCM 230 56

P-81 DCM 197.5 68

P-88 DCM 168 57

P-100 DMF 189 3

P-86 DCM 168 58

P-91 DCM 172-178 61

P-80 DCM 207 49

P-92 CH3CN 144-148 43

P-84 DCM 238 55

P-101 DCM >250 79

P-109 - 244-247 22

P-85 CH3CN 195 4

P-83 DCM - -

Compounds Amines (Y) Solvent MP in °C Yield %

P-89 CH3CN 154-158 86

P-145 - 250 24

P-149 DCM 155 82

P-154 DCM 280 29

P-155 CH3CN 95

P-93 CH3CN >260 94

P - 140 CH3CN 75-78 88

P - 146 CH3CN 124 86

P - 135 CH3CN 222 72

P - 137 CH3CN 198-200 99

Compounds Amines (Y) Solvent MP °C Yield %

P - 115 - 220 33

P - 150 - - 0

P - 152 - - 0

P - 157 - - 0

P - 166 DCM 199 85

P - 167 MeOH 197-199 74

The yields of P-81, 82 and 88 were acceptable but the yield of P-100 was extremely low.

The yield of P-85 was extremely low (1.45%). We believe this is due to the presence of

the dicyano group at the para position to amine which dramatically reduces the

nucleophilicity of the amine. To improve the yield we increased the temperature to 80°C

but surprisingly the dicyano group underwent hydrolysis to an aldehyde which was

confirmed by the presence of aldehyde proton in the 1H NMR at 10.2 ppm and the

absence of the C≡N peak at 2200-2100 cm-1 in FTIR and also the 12C NMR showed the

presence of an aldehydic carbon. The hydrolysis of dicyanomethylene group to the aldehyde is simply a reversible Knoevenagel reaction 96 in the presence of base. The reaction was carried out in an excess of DMAP and the presence of moisture probably made this hydrolysis possible. Therefore we attempted to synthesize P-406 and later convert it into the dicyano group.

2.22 P-406 P-85

Scheme 2.17 An alternate scheme of synthesizing P-85

We used a commercially available mixture of monomer/polymer 4- aminobenzaldehyde to synthesize P-406 following Scheme 2.18 but this reaction did not produce any product. We decided to use another route to make P-406. The chemistry began with the protection of 4-nitrobenzaldehyde (2.29) with ethylene glycol catalyzed by toluene sulfonic acid affording 2-(4-nitrophenyl)-1,3-dioxolane (2.30) (99%)97. The next step (reduction) was carried out using Pd/C with hydrogen 98. There are ample references for this reduction but all using Pt2O as catalyst in this system. The reduction of nitro did not work at all and finally we realized that dioxolane is sensitive to catalytic amount of water, acid or base 99 and undergoes polymerization with the loss of ethylene glycol.

2.29 2.30 2.31 2.32 2.33

Scheme 2.18 Attempt on synthesis of 2.31 (a) ethylene glycol, pTsOH, toluene (b) H2, Pd/C MeOH (c) cat. H2O

Then we used 4-aminobenzyl alcohol to make rhodamine spirolactam so that we

can oxidize the alcohol later to the aldehyde. The NH2 of 4-aminobenzyl alcohol is nucleophilic enough to carry out the lactam formation reaction using Scheme 2.16 but the yield of the reaction was not good (17-20%); even using 1.5 equivalent of the 4- aminobenzyl alcohol did not help. The poor yield may be due to its sensitivity with light and air. It might have reacted with air before the actual reaction. The poor yield became an obstacle for this desired goal. We decided to use 1,3-propanedithiol to generate, a rather robust dithiane aldehyde protecting group, which survives in both strong acid and base conditions 100. The aldehyde in 4-nitrobenzaldehyde (2.29) was protected with 1,3- propane dithiol using a catalytic amount of silica-sulfuric acid to afford 2-(4- nitrophenyl)-1,3-dithiane (2.34) (94%) 101. The silica-sulfuric acid was prepared according to literature procedures102. This reaction was tried previously with anhydrous

103 NiCl2 but the reaction failed .

2.22

Scheme 2.19 An alternate route for synthesizing P-150 (a) 4-aminobenzyl alcohol, pTsCl, DMAP, DCM (b) 1,3-propanedithiol, SiO2-H2SO4, CH3CN (c) SnCl2.2H2O, NaBH4 THF/EtOH (d) 2.22, pTsCl, DMAP, DCM (e) Py, DCM/H2O, PyHBr3, (n-Bu)4NBr (f) CNCH2CN, Piperidine, isobutanol, MW

The reduction of the nitro group in 2-(4-nitrophenyl)-1,3-dithiane (2.34) to the

104 amine 2.35 was performed by using NaBH4/SnCl2 in THF/ethanol mixture . The yield obtained was 39%, which proved to be better than the yields reported in literature. The rhodamine spirolactam P-405 was synthesized according to Scheme 2.16 (56%). The dithiane was removed by using pyridinium tribromide which was prepared referring to a literature procedure 105 (quantitative). The next step of the synthesis was the reaction of aldehyde P-406 with malononitrile to afford P-150 (85%), which was far better yield than previously prepared by the direct amidation reaction (4%).

An attempt to incorporate amino DCDHF (2.36) into a rhodamine spirolactam to synthesize P-83 (structure in Table 2.1) completely failed. As shown in Figure 2.13 the lone pair electrons of the amine nitrogen are less available due to the electron withdrawing nature of the DCDHF acceptor group.

2.36 2.37

Figure 2.13 A resonating structure of aryl amino version of DCDHF

In order to synthesize P-89, we first synthesized (E)-4-(2-(pyridin-4- yl)vinyl)aniline (2.40) by the Jeffery modification of the Heck reaction between 4- iodoaniline (2.38) and vinylpyridine ( 2.39) 106, and then followed Scheme 2.16.

2.22

Scheme 2.20 Synthesis of P-89 by two different routes (a) TDA-1, K2CO3, Pd(OAc)2, DMF (b) p-TsCl, DMAP, DCM

However, the yield was poor (18.2%). The product obtained after column chromatography was not very clean so we ran another reaction by Route 2. In Route 2 we reversed the order of intermediate formation, which proved to help. The intermediate

P-91 was obtained in good yield as shown in Scheme 2.16. The final cross coupling between P-91 and 4-vinyl pyridine was completed by a Jeffrey Heck reaction in which an

86.5% yield was obtained. In this Route 2, the yield not only increased significantly but also no column chromatography was required to separate the product. After completion of the reaction, addition of water precipitated out the desired product. Finally, washing the precipitate with water afforded the pure product. The cross coupling reaction was first tried with ligand tri-o-tolylphosphine but the reactions failed so we ran the reaction with tris(2-(2-methoxyethoxy)ethyl)amine (TDA-1) and the reaction worked with good yields.

Once the preparation of P-89 was accomplished, the synthesis of P-93 salt was straightforward. P-89 was dissolved in acetonitrile and treated with methyl iodide 107. The precipitate was washed several times with ether to afford a yellowish iodide salt.

Scheme 2.21 Synthesis of iodide salt (P-93) of P-89.

The synthesis of P-101 was carried out in a two step reaction which is more convenient than to introduce 4-(biphenyl-4-ylethynyl) aniline as described in Scheme

2.16. We therefore used a Sonogashira reaction in the second step after making P-91 to make P-101 which worked well (yield 79%) 108.

Scheme 2.22 Synthesis of P-101 by Sonogashira reaction.

The completion of the reaction was determined by the disappearance of the alkyne proton from HNMR since the Rf values of the starting material (P-91) and the final product (P-101) in TLC were same and it was difficult to distinguish between them as they both were halochromic.

P-109 was synthesized by Sonogashira chemistry between P-91 and methyl 4- ethynylbenzoate with only 22% yield (Scheme 2.23). The synthesis began with protection of the acid group of 4-iodobenzoic acid by esterification with methanol using catalytic amount of concentrated sulfuric acid (87% yield)109. A cross coupling reaction between methyl 4-iodobenzoate with trimethylsilyl acetylene via a Sonogashira reaction afforded methyl 4-((trimethylsilyl)ethynyl)benzoate in good yield (78%)108,110. The deprotection of the acetylene group was done by TBAF. We tried first KF to remove the

TMS group 111 but it did not work well i.e. it did not completely remove the TMS group, then we tried TBAF which worked well (74%)112. The next step was another Sonogashira cross coupling between P-91 and methyl 4-ethynylbenzoate which afforded P-109 with low yield (22%)110.

Scheme 2.23 Synthesis of P-109 by Sonogashira reaction between P-91 and 2.45 (a) MeOH, H2SO4; (b) ethynyltrimethylsilane, Pd(OAc)2, PPh3, CuI, Et2NH; (c) TBAF, THF

In an attempt to synthesize P-145 (Scheme 2.24), we first synthesized by refluxing 4-nitronaphthalene-1,8-dicarboxylic anhydride (2.46) with aniline in glacial

113 acetic acid (65%) . It was further reduced to 2.48 using SnCl2/HCl in hot glacial acetic acid (52%)114. An attempt to prepare a highly conjugated P-145 by Scheme 2.16 (Route

1) with 2.48 completely failed. A probable reason could be the weak nucleophilicity of the NH2 group in 2.48 due to high conjugation with electron withdrawing group. We attempted Route 2 in which there is a C-N bond formation between the parent N-H lactam (P-142) and an aromatic halide 2.50 115 using copper mediated Buchwald Hartwig chemistry. This reaction represents a new method to prepare the lactam fluorogen.

Rhodamine lactam P-142 was synthesized by using ethanolic ammonia but the yield was very low. It is probable that the ethanol may not have been fully saturated with ammonia although ammonia was bubbled over the surface of the calculated amount of ethanol with

constant stirring for 4 hours. (An ethanol solution saturated with ammonia can hold 16% by mass of ammonia).

The synthesis of P-142 was carried out overnight with a controlled supply of NH3 with rhodamine salt with Scheme 2.16 apparatus fitted with a gas trap. The percentage yield of the final product P-145 in cross conjugation reaction by copper chemistry was very low (24%). However it may be improved by the use of other amine ligands such as

N-methyl-2-(methylamino)ethylamine and trans-N,N'-dimethylcyclohexane-1,2- diamine115.

2.22

Scheme 2.24 Synthesis of P-145 using Buchwald Hartwig chemistry (a) aniline, AcOH; (b) SnCl2/HCl, AcOH; (c) p-TsCl, DMAP, DCM; (d) CuI, K2CO3, Toluene, N1-ethylethane-1,2-diamine

2.22

Scheme 2.25 Synthesis of P-137 (a) p-TsCl, DMAP, DCM, 4-aminopyridine; (b) MeI, Et2O

As soon as P-135 was treated with MeI, the spirolactam ring opened and the molecule resided only in the fluorophoric open state. Even in the dark P-137 could not revert back to the original fluorogen closed state. We followed Scheme 2.16 for the preparation of P-135 in a reaction of rhodamine salt with 4-aminopyridine (73%).

We synthesized rhodamine spirolactam P-146 containing a bioconjugation moiety. The intermediate 2.51 required for making P-146 was synthesized by using DCC and DMAP with 3-iodopropanoic acid and N-hydroxysuccinimide 116. Although the pyridinium nitrogen in the stilbazolium has a possibility of attacking two possible sites of

2.51 either iodine or activated carboxylic site, it exclusively displaced iodine 116 to obtain

P-146.

We synthesized the Rhodamine spirolactam P-140 with water soluble properties resulting from incorporation of an ethylene glycol oligomer. The intermediate 2.52 was

synthesized by a reaction between 2-(2-(2-ethoxyethoxy)ethoxy)ethanol and toluene sulfonyl chloride in aqueous THF using sodium hydroxide following a literature method)117. Due to two phase solvent system this reaction was easy to handle and process than the one we tried using pyridine as a base which gave us a mixture of the product 118.

Scheme 2.26 Synthesis of P-146 with NHS and P-140 with water soluble moiety

2.12 Rhodamine Spirolactam incorporating other dyes

We attempted to incorporate other dyes into the rhodamine spirolactam so that it would potentially make the rhodamine moiety itself a photoswitcher for the other dye. By doing this, we expected that two dyes might work simultaneously.

P-157

Scheme 2.27 A proposed rhodamine dye incorporating Nile red in a possible switching mechanism.

We assumed that a rhodamine spirolactam incorporated with another dye in its closed state may absorb above 400 nm which is one of our objectives for example, rhodamine incorporated Nile red P-157. In the closed state of P-157, the push pull system in Nile red is not fully operated as the lone pair of the donor amine is trapped by the amide subunit. When a flash of 403 nm was utilized to activate this two dye system, the rhodamine closed form undergoes photoisomerization to the open form as described in

Jablonski diagram (Figure 2.2 Chapter 2). Now the open form of rhodamine and Nile red can be excited by 541 nm. We assumed that emission of one dye would reinforce the emission of the other dye, which makes the whole system more brighter.

With this idea we attempted to synthesize rhodamine dye incorporated with several dyes. We attempted to synthesize P-150 by using a Buchwald-Hartwig reaction between P-142 and (E)-2-(4-bromostyryl)-3,3-dimethyl-3H-indole (2.53) utilizing CuI and N1,N1,N2-trimethylethane-1,2-diamine but the reaction failed. We tried another route in which the aldehyde previously made can condense with 2,3,3-trimethyl-3H-indole

(2.54) by simply refluxing in ethanol with no other bases or reagent. The same chemistry was utilized in the synthesis of P–460 but it did not react as the starting material was obtained.

Scheme 2.28 A proposed synthesis of P150 and P-460 by direct condensation with P-406 1 (a) EtOH, reflux; (b) CuI, K2CO3, Toluene, N -ethylethane-1,2-diamine

Another attempt to incorporate the 2-hexyl-3,6-diphenylpyrrolo[3,4-c]pyrrole-

1,4(2H,5H)-dione (2.57) dye with P-91 by a similar Buchwald-Hartwig reaction using

CuI and 1,10-phenanthroline as a ligand was tried but it failed. This reaction was tried again with another ligand, N,N’-dimethylethane-diamine which equally works well for this reaction under similar conditions. However this reaction also did not work. The dye

2.57 (2-hexyl-3,6-diphenylpyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione) utilized in this

Scheme 2.29 was synthesized by Alexander N. Semyonov.

Scheme 2.29 Attempt on synthesis of P-152 by Buchwald Hartwig reaction 1 (a) CuI, K2CO3, Toluene, N -ethylethane-1,2-diamine

We attempted to introduce amino-Nile red in our Rhodamine spirolactam but this was not successful. The nitro-Nile red P-153 was synthesized by literature procedure

(yield 12.50%) 119. The reduction of the nitro group afforded P-156 by hydrogenation using Pt/C catalyst 119. We attempted to increase the yield of P-153 by using the microwave reactor but the yield was not improved. We performed the next step to synthesize P-157 using Scheme 2.16 with a reaction between rhodamine B and P-156 but failed.

2.22

Scheme 2.30 Attempt to synthesize P-157 incorporating Nile red; (a) AcOH, 120°C (b) H2 Pd/C, MeOH; (c) p-TsCl, DMAP, DCM

2.13 Synthesis of Rhodamine derivatives with different amine donor groups:

The pyrrolidine group is a better donor compared to a simple dialkylamino group present in the xanthane part of the Rhodamine

2.13.1 Synthesis of (1-(9-(2-carboxyphenyl)-6-(pyrrolidin-1-yl)-3H-xanthen-3- ylidene) pyrrolidinium chloride)

We synthesized P-168 in quantitative yield by direct fusion reaction between 3’,6’- dichlorofluoran (2.60) and pyrrolidine at 140ºC 120. After treatment with hydrochloric acid, it afforded P-168. After following the Scheme 2.16 we obtained P-169 in 68.5%.

With Jeffrey-Heck reaction of P-169 and 4-vinylpyridine, P-253 may be obtained.

2.60

Scheme 2.31 Synthesis of P-253 (a) pyrrolidine, 140°C; (b) p-TsCl, DMAP, DCM, 4-iodoaniline (c) TDA-1, K2CO3, Pd(OAc)2, DMF

2.13.2 Synthesis of Rhodamine 101 spirolactams

RhD 101 possesses two fixed rings on either side of the amine donor in the xanthan part which makes it much better donor than free rotating dialkylamines such as diethylamine. Therefore we synthesized RhD 101. The starting material 8-julolidinol

(2.12) was prepared by following the literature method 88 as described previously. In a fusion reaction between 2.12 and phthalic anhydride, RhD 101 was synthesized which was precipitated out as a perchlorate salt 96. The Rhodamine spirolactam P-485 may synthesized by a Jeffery Heck reaction between P-201 and vinyl pyridine. Whereas P-

201 may be synthesized by Scheme 2.16 with iodoaniline.

2.12

Scheme 2.32 Synthesis of P-485 from RhD101 salt (a) phthalic anhydride 160°C, HCl; (b) p-TsCl, DMAP, DCM, 4-iodoaniline; (c) TDA-1, 4-vinyl pyridine, K2CO3, Pd(OAc)2, DMF

2.13.3 Attempt in synthesizing Rhodamine spirolactam incorporated with 7- azanorbornane as donor group

The alkylamino group present in the xanthane substructure suffered a severe problem of photostability because electron rich N-alkylated amino groups tend to undergo stepwise photo-oxidative dealkylation reaction in most push-pull system causing a blue shift in the absorption maximum of the dye121. Photo-oxidative dealkyation is believed to follow an α-elimination reaction via an initially formed aminium radical

∙+ + 122 cation (RCH2-NR2 ) to afford a reactive iminium (RCH=NR2 ) salt which eventually in presence of moisture leads to the formation of aldehyde and amine (RCHO + HNR2).

In our preliminary bulk study as well as in a single molecule detection study, when the dialkylamino rhodamine spirolactams were irradiated with 405 nm radiation, most of the molecules undergo irreversible photobleaching and there was hardly any photoswitching.

To reduce the photobleaching of rhodamine spirolactams, we attempted to incorporate the

7-azanorbornane (which is more resistant to photodegradation) in our rhodamine

spirolactams as reported by Song et al 123. The idea behind the introduction of the 7- azanorbornyl donor would be to avoid the formation of iminium salt because for dealkylation the formation of a highly energetic anti-Bredt iminium salt is required124.

2.13.4 Synthesis of 7-azanorbornane hydrogen chloride by Mitsunobu reaction following literature procedure:

The chemistry began with the protection of amine functional group of trans-4- aminocyclohexanol with a toluenesulfonyl group to afford 2.62 (quantitative) 117,125-127. In the next step, a Mitsunobu reaction of 2.62 proceeded via an intramolecular cyclization

(Scheme 2.32) to afford 2.63 (65%) 128-130. The removal of tosyl group was done by reduction with sodium naphthalenide (64.5%)131. When we scaled up the reaction to a gram scale the yield decreased (affording insufficient material to proceed to the next step). Several attempts were made to get this compound but none were successful.

Scheme 2.33 Synthesis of 7-azanorbornane HCl (2.64) by Mitsunobu reaction (a) p-TsCl, NaOH, THF, H2O; (b) PPh3, DIAD, THF; (c) Sodium naphthelenide, THF, NaHCO3, K2CO3

We pursued another method in which the amino group is protected by a benzyloxycarbonyl group to afford 2.65 in 90.0% yield 126 without column

chromatography (Scheme 2.34). The next tosylation step afforded 2.66 in quantitative yield117. An attempt to remove the benzyoxycarbonyl group was made using glacial acetic acid and 47% HBr. We tried several attempts to get the solid salt 2.67 but we always ended up with a mixture of a sticky substance.

Scheme 2.34 Synthesis of 2.64 by another route (a) benzyloxycarbonylchloride, NaOH; (b) TsCl, Py; (c) AcOH, HBr;(d) NaOH, HCl

Since the yield of 2.64 by this method was not good, we found another method of making it (Scheme 2.35). The chemistry began with the protection of the amino group with di-tert-butyl-dicarbonate anhydride 132 to afford 2.68 and next the OH group was activated by a mesyl group to afford 2.69 117. The amine was then deprotected with TFA to afford 2.70. In the successive step, the cyclization took place by addition of NaOH and stirring overnight at room temperature and work up with HCl afforded 2.64 (86.4%).

Scheme 2.35 Alternate synthetic route for 2.64 (a) EtOH anhyd, 0°C; (b) TFA, DCM; (c) TFA (d) NaOH/HCl

After the synthesis of 2.64, we attempted to synthesize the Rhodamine dye with

7-azanorbornane donor by irradiating the a mixture of 3',6'-dichlorofluoran (2.60), 2.64, anhydrous ZnCl2 and Hunig’s base in a microwave reactor with a setting of power 300W, temperature 200°C, and pressure 250 psi. There was no indication of formation of characteristic red Rhodamine color. However this reaction was reported elsewhere in a patent performed at 190°C in high pressure reactor in the presence of anhydrous ZnCl2 for 12 hours with 40% yield133. This reaction with a typical secondary amine such as pyrrolidine worked perfectly well (Scheme 2.31).

Scheme 2.36 Synthesis of 43 in a microwave reactor; (a) Hunig’s base, ZnCl2 anhyd.

CHAPTER 3

Photophysical properties of rhodamine spirolactams

CHAPTER 3 Photophysical properties of rhodamine spirolactams

3.1 Photophysical properties of rhodamine spirolactams

In our initial physical characterization of rhodamine spirolactams, we set a goal of quantifying how the structural variations affect the photophysical properties of these fluorophores, and using the results to iteratively design rational rhodamine spirolactam systems more suited to super-resolution imaging in living cells. Maximizing the efficiency and increasing the absorption wavelength of the closed isomer is desirable because UV light is well known to damage cells. Background is minimized if the open isomer emits at longer wavelengths and with a larger Stokes shift. Beside these criteria, the rhodamine spirolactams require some degree of water-solubility while working in cells so characterization was attempted in biologically relevant aqueous environments whenever possible.

3.2 UV-Vis absorption of the rhodamine lactam in their closed state

In our preliminary study, we took the UV absorption of all the rhodamine spiroamides we prepared and collected them as found in Table 3.1. We were focused on the rhodamine spirolactams that absorb above 400 nm. With that demand in mind, first we wanted to see how the simple alkyl and aryl groups in the amine part of the spiroamide affect the absorption of the rhodamine spirolactams in their closed state.

Compounds P-80, 81, 82, 86, 88, 91, 92 and 100 all have comparable UV-Vis absorption in their closed state (315-320 nm). The absorption in the dark (off) state can be shifted well towards the longer wavelength by certain structural modification in arylamine

(lactam unit) such as incorporation of more conjugated systems and electron withdrawing groups. With this intention some spirolactams P-84, 85, 89, 101, 109, 145, 149 and 154 were synthesized but we achieved only a little improvement in the absorption. Aromatic amines with more than two condensed aromatic ring (e.g., anthracen-2-amine) were not tried because they may quench the fluorescence 134. We decided to use more electron poor units in the spirolactam substructure. The stilbazolium group (zwitterionic derivatives; P-93, 140 and 146) present in Table 3.1 has been found to be particularly valuable since all of the three structures deliver the required absorption, enhanced water solubility due to the ionic content and provide a convenient site (alkylated pyridine) for attachment of further functionalization (as in P-146 which has a NHS ester attached).

Closed form Closed form Long Closed Amines (Y is the aromatic Extinction Compd wavelength form Cut amine part of the lactam ring) Coefficient ε = absorption off [nm] [M-1 cm-1] peak λabs [nm]

P - 82 326 350 1.28 x 104

P - 81 320 350 1.28 x 104

4 P - 88 315 350 1.16 x 10

P - 100 320 340 0.7 x 104

P - 86 320 343 1.4 x 104

P - 91 320 342 1.9 x 104

P - 80 280 400 1.4 x 104

P - 92 320 370 2.4 x 104

P - 84 350 430 2.5 x 104

P - 101 320 375 2.7 x 104

P - 109 326 385 5.7 x 104

P - 85 370 450 2.13 x 104

P - 89 320 425 1.21 x 104

Closed form Closed form Amines (Y is the substituent Long Closed Extinction Compd attached to the first benzene wavelength form Cut Coefficient ε = ring shown) absorption off [nm] -1 -1 [M cm ] peak λabs [nm

P - 145 360 395 1.43 x 104

P - 149 367 430 4.53 x 104

P - 93 395 495 2.6 x 104

P - 140 385 480 2.6 x 104

P - 146 396 490 2.66 x 104

Table 3.1 The UV-Vis-absorption of rhodamine spirolactams in dichloromethane.

The photophysics of some selected rhodamine spirolactams were studied further in more detail. For our convenience, we categorized the rhodamine spirolactam into three groups as short, medium, and long wavelength absorbing units from Table 3.1.

3.3 Effect of substitution on the absorbance of the closed form

] Courtesy: Stanford University

-1 80000

cm P80 -1 P81 [M P82

 60000 JCW03075 JCW03074 JCW03-075 40000 P-80 JCW-03-074

P-81

20000 P-82

0 Molar extinction coefficient ( extinction Molar 200 250 300 350 400 450 500 550 600

Wavelength (nm)

P-82 P-81 P-80 JWC03074 JWC03075

Figure 3.1 Rhodamine lactams with short wavelength absorbance in acetonitrile (278 nm - 317 nm); The blue arrow indicates the absorption of JCW03-075, black arrow indicates the absorption of P-80, purple arrow indicates the absorption of JCW-03-074, green arrow indicates the absorption of P-81 and the red arrow indicates the absorption of P-82.

The UV-Vis absorption of the rhodamine lactam of P-80 (280 nm), P-81 (315 nm), P-82

(326 nm), JWC-03-074 (316 nm) and JWC-03-075 (303 nm) ranges from 280 to 326 nm which indicated that the absorption is affected only little by the nature of alkyl group present in the amine donor unless otherwise some other conjugation substructural units

are added (Figure 3.1). We assumed that the ionic character in the lactam subunit as found in JWC-03-075 would display an absorption at longer wavelength due to the interaction with the solvent. However, it was found to absorb at even a little shorter wavelength absorption than its parent structure (JWC-03-074). This suggests that ionic character may or may not contribute for the absorption towards long wavelength.

]

-1 80000

-1 P84

[M P145

 60000 P149

P-149 40000 P-84

P-145 20000

0 Molar extinction coefficient ( coefficient extinction Molar 200 250 300 350 400 450 500 550 600 Wavelength (nm)

P-149 P-145 P-84

Figure 3.2 Rhodamine lactams with medium wavelength absorbance in acetonitrile (325nm - 360 nm); The light blue curve represents the absorption of P-145, the blue curve represents the absorption of P-84 and black curve represents the absorption of P-149. The extended π conjugation in P-149 is responsible for its absorption in longer wavelength.

]

-1 1e+5

-1 P 93

[M 8e+4 P 140



6e+4

4e+4

2e+4

0 Molar extinction coefficient ( 200 300 400 500 600 Wavelength (nm)

P-93 P-140

Figure 3.3 Rhodamine lactams with long wavelength absorbance in acetonitrile (385 - 396 nm); The brown curve represents the absorption of P-93 and red curve absorption represents the absorption of P-140.

Figure 3.4 Relationship between unsaturation and abs > 400 nm of closed rhodamine lactams. (Courtesy: Marissa Lee, Stanford University)

From Figure 3.2, it is demonstrated that the absorption towards longer wavelength is possible when there is an increase in conjugation. The extended conjugation involving an electron withdrawing group (P-84) increases the absorption towards longer wavelength compared to compounds only with extended conjugation as in

P-145 and P-149. From Figure 3.3, compounds with high conjugation and ionic character have the longest wavelength absorption in this series. The relationship between the extended conjugation and the absorption is explained in Figure 3.4. As the degree of unsaturation increase, the absorption towards long wavelength increases. This is in agreement from the plot B, when the area under the absorption curve is plotted against the degree of unsaturation.

3.4 Preliminary study of photoswitching efficiency of rhodamine spirolactams

In a typical experiment to evaluate the switching efficiency of the rhodamine spirolactams possessing absorption close to 400 nm (such as P-93 and similar compounds), the lactams were doped in 10% PMMA film and irradiated continuously with an imaging laser beam wavelength of 532 nm. It was also pumped with an activation laser of wavelength 405 nm as shown in the Video Clip 1.

P93-Activation-movie_in_rhodamines_oct2009.avi

Video Clip 1 Photoactivation-movie of rhodamine spirolactam P-93 pumped by 405 nm radiation. (Courtesy: Marissa Lee, Stanford University)

The polymer spectrum was obtained from a very concentrated sample in 10%

PMMA films. When the sample was irradiated with a 532 nm imaging laser, it was in the dark state. When it was pumped with the 405 nm laser for varying amounts of time, the dye fluoresced and returned to the dark state when the pump laser was turned off. The absorption/emission spectrum of P-93 is shown in Figure 3.5. Only P-93 and similar

derivatives gave reasonable looking spectra from this procedure. Many of the remaining rhodamine spirolactams were found to be difficult to switch.

0.7 35000

0.6 30000

0.5 25000

0.4 20000

0.3 15000

Absorbance Abs P-93 0.2 10000 closed, In (cps) Fluorescence 0.1 toluene 5000

0.0 0 300 350 400 450 500 550 600 650 Wavelength (nm)

Figure 3.5 The absorbance of closed form and excitation/emission of the open form of rhodamine spirolactam (P-93). The experiment was conducted in very concentrated samples in 10% PMMA film and dried in thick layer on top of glass slide. The slide was kept in front of 403 nm laser for a few minutes to open rhodamine spirolactam. The background was subtracted and the excitation and emission were measured with a fluorimeter. The polymer stabilizes the open form. Black is the absorption curve in the solution, and this goes with the left y- axis. The four colored curves are the spectra of the open form in PMMA and match the right y-axis. The blue, green and yellow curves are the excitation spectra and the red curve is the emission. The absorption/excitation of the open form of Rhodamine (P-93) is red shifted from the closed form. (Courtesy: Marissa Lee, Stanford University)

1-p146-polyLys-g255-OD1-neutral-100ms-100x_Colors.avi

Video Clip 2 Photoactivation-movie of rhodamine spirolactam P-146 pumped by 403 nm radiation. Scale bar is 2 µm. The green bar in the upper left shows that the 532 nm laser is on, the purple bar means the 403 nm laser is on. (Courtesy: Marissa Lee, Stanford University)

The P-146 sample was irradiated using the same procedure as was done for P-93 in a poly-lysine polymer (neutral). The sample was bathed continuously with an imaging laser at 561 nm and also irradiated with purple pulses (activating laser) at 405 nm. After the purple pulse, more single molecules appeared. Some single molecules remained in the on state and did not revert back to the closed dark state. It may be assumed that the

PMMA may play a role in the slowing down the rate of closure forming the lactam ring.

Figure 3.6 A representative imaging experiment for the rhodamine lactam (P-82) after activation by 403 nm irradiation (10% PMMA). (Courtesy: Marissa Lee, Stanford University)

In a representative imaging of the rhodamine spirolactam (P-82), it was continuously irradiated with an imaging laser 532 nm which shows a very weak fluorescent or no fluorescence (Figure 3.6, image 1). The sample was activated with 403 nm laser flash (image 3) and the sample immediately shows an intense fluorescence which slowly faded away due to photobleaching or chemical changes. The photoswitching of rhodamine spirolactam was not an easy process because the ring opening process of spirolactams hardly occur under 405 nm of flash.

3.5 Rhodamine spirolactams under the influence of pH

The majority of compounds underwent light driven isomerization to the open form with a very short life time and with difficult which was not long enough time to get the bulk spectra. Consequently, poor spectra were obtained which were not worth

analyzing. Since rhodamine spirolactams are acid sensitive compounds, and although it was beyond our immediate interest, we had to use a chemical method to trap the open form to get the bulk spectra of the open form.

Figure 3.7 Spectra showing reversible stabilization of open isomer at low pH. The solid lines are absorption (left axis) and the dashed lines are fluorescence emission (right axis). Rhodamine lactam (P-93) was dissolved in 1:1 acetonitrile:water mixture. The absorbance and fluorescence emission was measured after HCl (1M) added (pH~2). The change in both absorbance and emission could be reversed by neutralizing the solution with KOH. Acid stabilizes the open form. The stability of the open form in acidic may be attributed to the protonation of the amide anion and gain of conjugation of the middle ring with two side benzene ring in the xanthene part. (Courtesy: Marissa Lee, Stanford University)

This experiment was carried out with a solution of P-93 as a representative compound in

50/50 (v/v) acetonitrile and water (Figure 3.7). The solid curves show absorption (left y- axis) the dashed curves are fluorescence (right y-axis). The green line is the initial

(neutral, no acid or base) absorbance and fluorescence. The red lines are after the addition of acid (40 µL of ~1M HCl) and the blue lines represent the absorption and fluorescence spectra after titrating back with KOH. During the addition of acid, there is a mixture of fluorogen and fluorophore and the open form dominates as the acid concentration increases and when the base was added all the opened form completely turns back to the closed form as is indicated by blue line (Figure 3.7). There is a perfect match of absorption spectra of the closed form in neutral condition and the spectrum obtained after neutralization. Furthermore, the open spectra obtained from the pH method matched in shape to the polymer spectra.

Property On Details

Maxima in UV-vis absorption, measure in 50/50 (v/v) acetonitrile/water mixtures and PMMA or PVA.  To measure open form in solution, Open and closed stabilize with acid addition. λ abs forms  To measure open form in polymer, use excitation spectrum after irradiation with UV or blue light.

Maxima in fluorescence emission, measure in 50/50 (v/v) acetonitrile/water mixtures and PMMA or PVA.  To measure open form in solution, Open and closed λ stabilize with acid addition. fl forms  To measure open form in polymer, irradiate with UV or blue light.

Molar extinction coefficient, calculate from UV-vis ε Closed form spectra of dilutions of known concentration Fluorescence quantum yield, compare to Rhodamine B standard. Measure in 50/50 (v/v) Φ F Open form acetonitrile/water mixtures with acid stabilization and in PMMA or PVA after exposure to blue/UV irradiation. + Effective acid dissociation constant, calculate from Closed + H  (Open- pKa + titration with known amounts of acid, calibrate for H) solvent mixture Total emitted photons, histogram emitted photons N tot Open form from single molecule traces in PMMA and/or aqueous gelatin, fit to exponential.

Table 3.2 Some property notations and their meanings used in the study of the photophysical properties of the rhodamine spirolactams in acid/base experiments. (Courtesy: Marissa Lee, Stanford University)

In a series of other rhodamine spirolactams such as P-80, 81, 82, 84, JCW03073, 074,

075 and P-93, the open form was trapped with acid and studies of their properties were

undertaken (Figure 3.18). In most situations the red curves have substantial absorption in the 350 nm range relative to the 550 range such as in P-80, the red peak at 550 is much larger than the peak at 350 while in comparison for P-93, the red peak at 550 is significantly smaller than the peak at 350. These differences in peak height (for the solid absorption lines) are coming from different relative amounts of the open from (different efficiency of reaction with acid) and /or from different extinction coefficients of the open form. Some compounds with a large amount of conjugation in the lactam region will have large (red curve) ~350 nm contribution to the absorption that will not change

(much) between open and closed forms. Another reason could involve the solubility of the open isomers in the given solvent. All the absorption/emission studies were done in

DCM. It would be useful to study their absorption/emission and extinction coefficients in more polar solvents such acetonitrile/water. However, a preliminary study showed that there is no significant change in any of the parameters in polar solvent mixture of acetonitrile/water.

Emission, H+

Absorbance, neutral Absorbance, KOH Absorbance, neutral Absorbance, H+ Absorbance, KOH

Emission, neutral

Emission, KOH + Emission, neutral Absorbance, H Emission, KOH

+ Absorbance, neutral Emission, H + Absorbance, neutral Absorbance, KOH Emission, H Absorbance, KOH

+ Emission, neutral Absorbance, H Emission, neutral Absorbance, H+ Emission KOH Emission KOH

Emission, H+ Emission, H+ Absorbance, neutral Absorbance, KOH Absorbance, neutral Absorbance, KOH

Emission , neutral + + Emission , KOH Absorbance, H Absorbance, H Emission, neutral Emission, KOH

Emission, H+

Absorbance, neutral Emission, H+ Absorbance, neutral Absorbance, KOH Absorbance, KOH

Absorbance, H+ Absorbance, H+ Emission, neutral Emission, neutral Emission, KOH Emission, KOH

+ Emission, H Absorbance, neutral Emission, H+ Absorbance, KOH Absorbance, neutral Absorbance, KOH

Absorbance, H+

Emission, neutral Emission, KOH Emission, neutral + Absorbance, H Emission, KOH

Emission, H+ + Emission, H

Absorbance, neutral Absorbance, neutral Absorbance, KOH Absorbance, KOH + Absorbance, H+ Absorbance, H Emission, neutral Emission, KOH

Emission, neutral Emission, KOH

Figure 3.8 The absorption/emission properties of a number of rhodamine spirolactams as a function of pH. (Courtesy: Marissa Lee, Stanford University)

1/1 CH3CN/water CH3CN 1/1 CH3CN/water

Closed λ Molecule Φ pKa abs ε Closed λ Open λ Open F cut-off Closed abs Fluor

P80 0.507 ------350 278 39380 561 587

P81 0.492 ------344 315 12790 560 585

P82 0.397 ------336 317 9581 560 583

P84 0.428 ------413 325 35900 560 585

JCW-03- 0.363 ------341 292 59930 562 585 075

JCW-03------073

JCW-03- 0.388 ------345 316 13700 562 587 074

P93 0.438 ------452 365 32860 560 585

P140 0.205 3.62±0.02 460 369 31160 559 583

P149 0.142 3.57±0.03 404 360 56770 558 584

P145 0.646 ------376 352 15670 563 586

P146 ------3.20±0.02 ------

Table 3.3 Rhodamine spirolactams bulk characterization in acetonitrile and 1/1 acetonitrile/water (The notation at the top of the table are described in Table 3.2. (Courtesy: Marissa Lee, Stanford University)

Figure 3.9 Spectral changes in rhodamine spirolactam P-146 with acid titration (acetonitrile/H2O 1:1 mix). The operational pH, “paH*” is defined as: paH* = pHwater only- Δ, where Δ is -0.28 pH units for 50/50 (v/v %) acetonitrile/water solution. (Courtesy: Marissa Lee, Stanford University)

In this representative acid base titration of the P-146, the absorption spectrum of the closed isomers and the open isomers behaves as a function of paH*. The absorption peak height of the closed isomers started decreasing with the decrease in paH* value and started increasing the absorption peak height of the opened isomers (Figure 3.9). The brown solid absorption curve representing closed isomer has no opened isomer at paH*

7.29. When the paH* decreases to 4.97, the orange solid absorption curve representing

closed isomer decreases its height with the appearance of absorption peak for the opened

isomer. At paH*2.63, the absorption peak of the opened isomers attained the highest level.

A series of studies was conducted on the rhodamine spirolactams P-140, 145, 146 and

149 and the results shown in Table 3.4 were obtained.

Compound Structure pKa

P-140 3.62±0.02

P-145 Not enough data points

P-146 4.20±0.02

P-149 3.57±0.03

Table 3.4 Some pKa measurement of selected rhodamine spirolactams. (Courtesy: Marissa Lee, Stanford University)

For P-140, the pair of open and closed isomers are in equilibrium at pKa 3.62. As soon as

the pKa becomes less than 3.62, the open isomer starts dominating and if the pKa

becomes more than 3.62, the equilibrium starts favoring the closed isomer. This behavior

also applies to the rest of rhodamine lactams with their respective pKa values (Table

3.4). The change in absorbance of the rhodamine spirolactams (P-146) with decreasing pH was conducted by adjusting the pH to 50/50 acetonitrile/water solution. As it is clear from the spectrum, the two closed and open isomers intersect at pH 3.46. At 337 nm these two isomers have same molar absorptivity. Only two species, an open isomer and a closed isomer are contributing the UV Vis absorption.

0.14

0.12

0.10

0.08 OPEN - 560nm 0.06 CLOSED - 320nm

0.04

A bsorbance

0.02

0.00

8 7 6 5 4 3 2 pH (adjusted for acetonitrile)

Figure 3.10 Change in absorbance of rhodamine lactam (P-146) as a function of pH. The isosbestic point is at 337 nm for open and closed isomers. (Courtesy: Marissa Lee, Stanford University)

3.6 Single molecule detection in neutral and acid conditions

One of the important parts in our study is the efficiency of the single molecule detection of the rhodamine spirolactams. For this study we utilized P-146 as a representative molecule. The rhodamine lactam was immobilized on poly Lys coverslips and the single molecule activation cycles were measured in a neutral condition. After one photoswitching there was no activation occurred most likely the molecules were photobleached (Figure 3.11 (B)). From the photon count histogram in the neutral condition was found to be similar with that of the acidic condition. There exists a problem of complicated blinking behavior.

Figure 3.11 (A) photon counts of rhodamine lactam P-146 in neutral and acidic condition (B) single molecule detection. (Courtesy: Marissa Lee, Stanford University)

3.7 Summary

We have quantified the switching efficiency (on the bulk or single molecule level). However, the switching process was complicated by the rapid thermal isomerization back to the closed state and photobleaching from both the activation and imaging laser. In polar solvents, the open isomer persists for ~ms time scales, and fluorescence turn-off from photobleaching is ~6x faster than thermal closure (5.9±1s vs.

40±15s), as measured on P-93. One reason for rapid photobleaching may be the primary alkyl unit attached to the nitrogen of donor amine undergoes rapid photo-oxidative dealkylation122. Due to this rapid photobleaching, it was difficult to observe multiple turn-on events. The rhodamine spirolactams those were mentioned in our introduction part, synthesized in Hell’s lab have comparable photobleaching rate with the rhodamine spirolactams those we synthesized, particularly rhodamine lactams with diethyl amine as a donor. The dyes we synthesized have absorption close to 400 nm and can be excited by

403 nm radiation which is harmless to living cells but the dyes synthesized in Hell’s lab can be photoswitched only in 325 nm UV radiation which were not suitable for the live cell imaging.

We have shown that protonation of the negatively charged lactam nitrogen with strong acid selectively stabilizes the open isomer allowing the bulk spectral measurements of this normally transient species (Figure 3.7). The addition of base

restores the original spectra of the closed lactam. The spectra from acid stabilization compared favorably with the spectra obtained by stabilizing the open form in a poly

(methyl methacrylate) polymer matrix. Molecular interaction between PMMA film with the open form of the dye makes may be a reason for the stability of the open form in

PMMA film. Varying the substitution did not significantly change the bulk spectral properties of the open isomer—all derivatives measured have absorption maxima ~560 nm and all emit in the ~580 nm range, with a small Stokes shift (~23-26 nm). From acid titrations, the pKa of the rhodamine lactam derivatives were determined to lie in the range from 3.57±0.03 (P-149) to 4.20±0.02 (P-146).

We have also made efforts to combine the PEG content with reactive functionalization and the introduction of amine donors other than diethylamine (for NR2) with the overall goal to improve both water solubility and photostability in the same molecule. The problem of photobleaching may be prevented somewhat by using 7- azanorbonane as an amine donor since this moiety is more resistant to oxidative photodegradation123 at least in some systems.

CHAPTER 4

Modifications of DCDHF asides and strained alkenes for efficient 1,3-dipolar cycloadditions

CHAPTER 4 Modifications of DCDHF azides and strained alkenes for efficient 1,3 dipolar cycloadditions

4.1 Introduction

The study and imaging of the microscopic world in vivo or vitro through the super resolution (SR) technique has become a regular practice among researchers in the world.

As described already in the previous chapter, significant advances have taken place in optical imaging beyond the diffraction limit and several nanoscopic labeling techniques including PALM 14, FPALM 15, STORM 16 and STED 9 have been developed which can detect and image a single molecule with great precision on a nanoscopic scale providing detailed structure and dynamics at the molecular level. At the same time, however, it has demanded some stringent requirements of new labeling techniques and fluorescent probes. For the single molecule SR technique, the organic fluorophores are required to switch on to the bright state from its dark state at will (actively controlled) to ensure that only one fluorescent emitter is switched on at a time in a diffraction-limited region1,17.

The most common activation process usually involves the use of light (UV or visible).

Super resolution techniques utilize photoactivatable or photoswitchable probes for the imaging process. Besides photoactivatable and photoswitchable fluorophores, there are other SR techniques which utilize chemical reactions to activate the fluorophores from a dark state to a bright state, but relatively few studies have been done in this class of activation.

In a chemical reaction based technique, the fluorogen can be triggered to a fluorophore after a chemical reaction. In this technique basically a fluorogen contains a highly reactive functional group that reacts with targeting biomolecules conjugated with certain functional group or structure that forms covalent bonds with a certain distinctive change in the photophysical properties especially in their absorption and emission of radiation. One example of such chemical activations of a fluorogen to fluorophore is a chemical reaction involving an azide undergoing dipolar cycloaddition with strained alkynes or alkenes.

4.2 A brief introduction to azido functionalized chemistry

Fluorophores incorporating an azido group have been widely studied due to their highly selective reactivity and metabolic stability for application in single molecule imaging135,136. The azido group has a highly selective reactivity with strained alkenes and alkynes structure which can undergo a cycloaddition reaction [1,3-dipolar cycloaddition] reaction137. This reaction is popular with the name Huisgen cycloaddition reaction138 which was later popularized with “Click chemistry” under mild conditions with the use of a copper catalyst by Sharpless and co-workers139. After the introduction of Click chemistry140,141, it has become an essential tool for different aspects of chemistry such as polymer synthesis as well as for the modification of surfaces and nanometer- and mesoscale structures142, bioimaging incorporating dyes with an azido group in a

spacer136, carbohydrate chemistry135, application bioconjugation and drug delivery143 and many more.

Click chemistry has been exploited in the bioimaging area on many occasions, taking advantage of the weak fluorescent properties of the fluorogen due to the poor donor azido group which is switched to a strong fluorescent state after Click chemistry.

For example, a weakly-fluorescent compound (3-azido-7-hydroxycoumarin) and propargyl alcohol transformed to strong fluorescence compounds (1,2,3-triazole compounds) were utilized in mutational p53 DNA sequence detection as described by

Qiu144. The mandatory use of copper is not strictly bioorthogonal due to its slow kinetics145 and cytotoxicity effect to both bacterial146 and mammalian cells147,148. The

Click chemistry was further extended to Cu free Click chemistry using an activated alkyne such as a strained alkyne instead of terminal unstrained alkyne by Agard and group147. For example, a strained alkyne (cyclooctyne) undergoes fast and smooth 1,3- dipolar cycloaddition with an azido group in the absence of any auxiliary reagents under physiological conditions.147,149-151

Scheme 4.1 (A) Click chemistry catalyzed by Cu using unstrained alkyne with azide (B) Bioorthogonal Click chemistry using strained alkyne in the absence of Cu catalyst with azide

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After the introduction of copper free Click chemistry using a strained alkyne and azide (Scheme 4.1 B), a lot of attention has been paid in this chemistry to adapt in the real time single molecule imaging applications. Although the reaction between strained alkyne-cyclooctyne and azido group is bioorthogonal, it is not fast enough in imaging cell dynamics. However, Bertozzi and her co-workers incorporated an electron withdrawing group (CF2) next to the alkyne structure which reacted at significantly fast pace comparable to Cu catalyzed Click chemistry152.

A B

Figure 4.1152 (Top)152Strategy for metabolic labeling of cell-surface glycans with bioorthogonal chemical reporter. Cells were first incubated with Ac4ManNAz, which is metabolically converted to cell- surface SiaNAz residues, and subsequently reacted with difluorinated cyclooctyne probes for visualization. (bottom)153 Live-cell imaging of cell-surface glycans difluorinated cyclooctynes. CHO cells were metabolically labeled with (A, B) 25 μM Ac4ManNAz. [Adapted from QSAR & Combinatorial Science, Baskin et. al. 2007(top) and PNAS, Baskin et.al. 2007 (bottom)]

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Bertozzi and her co-workers successfully demonstrated Click chemistry between N- azidoacetylmannosamine (Ac4ManNAz) conjugated with Chinese Hamster Ovary (CHO) and difluoromethylene cyclooctyne (DIFO) incorporated with a fluorescent dye-Alexa

Fluor 488 to image the dynamics of glycans (Figure 4.1). A severe disadvantage encountered in this strategy was the washing of unreacted difluoromethylene cyclooctyne incorporated Alexa Fluor 488 was not successful. As a result, there was an interference of unlabeled dye with targeted fluorophores. Prior to imaging, the unlabeled DIFO could be washed away, but this strategy made observation of the dynamic events more difficult.

Also, due to the activation of alkyne by introduction of CF2 group, the alkyne group is more prone to Michael type addition with the bionucleophiles such as thiols154.

In an alternative approach, the kinetics of the Click chemistry between an azido group and alkyne was further increased by additional ring strain caused by incorporation of two phenyl rings next to the cyclooctyne developed by Boon and co-worker154,155 but the problems associated with the washing step to remove unlabeled fluorophores remained the same.

In an alternative approach, the kinetics of the Click chemistry between azido group and alkyne was further increased by additional ring strains caused by incorporation of two phenyl rings next to cyclooctyne developed by Boon and co-worker154,155 but the problems associated with the washing step to remove unlabeled fluorophores remained the same.

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4.3 A different approach of azide chemistry with strained alkenes

We were interested in similar chemical transformations but with different strategies, which may avoid some of the problems faced by azide/cyclooctyne 1,3-dipolar cycloaddition reaction. The azide-based fluorogens, reported previously, require short wavelength (UV) activation and the resultant photolysis products are not photostable enough or do not have emission at a sufficiently long wavelength to be applied in single molecule imaging. Our group has designed some DCDHF fluorophores based azide compounds, which can be activated with longer wavelength light >400 nm and the photoactivated products are even stronger emitters at wavelength >500 nm and are photostable enough to various single molecule imaging156. However, our current interest involves the activation of the fluorophores concomitant with the bioconjugation by a chemical process as an alternative to a photochemical process Figure 4.2. In this approach, azide-based DCDHF fluorogens can undergo chemical reaction by reacting with a strained alkene such as norbornene without any catalyst. The activation process always involves the conversion of an azide to amine (ATA Azide To Amine). Since this is a thermal activation, we can abbreviate the thermal route as T-ATA. The covalent bond formation between azido-DCDHF with the strained alkenes incorporated with the biological specimen is a kind of new bio-conjugation technique so far. The [2+3] cycloaddition reaction of norbornene with an azido group proceeds in a considerable rate since angle strain can greatly enhance the rate of 1,3-dipolar cycloaddition compared to unstrained alkenes137,157. An additional essential step in this strategy (Figure 4.2) is the

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rearrangement of the dihydrotriazole product, which can afford secondary amine that turns on the fluorogen from dark to bright fluorophore.

Since a washing problem of untargeted fluorophores existed in the bioconjugated azide and fluorophore incorporated cyclooctyne, the bioconjugation technique we are introducing will not have such problems because dark molecules become fluorescent only after a bioconjugation chemical reaction which means untargeted fluorophores remain dark and do not appear in the imaging process.

Figure 4.2 A general scheme showing the transformation of the fluorogen to fluorophore by chemical process.

The existing DCDHF derivatives must be modified or tuned to adapt in this new conjugation technique. For a chromophore to fully functionalize through a push-pull

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mechanism requires three critical components (Donor – π system – Acceptor) (Figure

4.3). The DCDHF derivatives, which we are considering in our case, lack one of the three critical substructures. As a result it is in the dark state. The azido-DCDHF derivative possesses an azido group in the donor position and it is not a very good donor. It requires short activation wavelengths and is not photostable enough to be applied in super resolution imaging.

Figure 4.3 DCDHF possessing three critical components; Donor – π system – Acceptor

For the creation of fully functionalized push-pull system, a better donor is required which can be possible only when the azido group undergoes a chemical transformation to an amine. In order to have this transformation, the azido group undergoes 1,3-dipolar cycloaddition reaction readily whenever strained alkenes are found in the nearby milieu. The cycloaddition reaction affords a dihydrotriazole intermediate and in a subsequent rearrangement affords the secondary amine (Scheme 4.2) with the loss of a nitrogen molecule. The stereochemistry of the secondary amine rearranged product 4.7 can be explained by a possible mechanism described in Scheme 4.2. Since

4.3 is under the influence of strong electron withdrawing group DCDHF, it is not very stable and it subsequently loses the nitrogen molecule accompanied by Wagner-Meerwin

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rearrangement to afford a zwitterionic intermediate 4.5. The anion picks up a proton that is spatially in proximity and subsequent electron pair transfer affords the rearranged product 4.7.

Scheme 4.2 A plausible mechanism in a T-ATA process between norbornene 1 and azido-Ph-DCDHF4.2 in a dipolar cycloaddition reaction.

Na Liu from our group investigated a reaction between norbornene 4.1 and azido

DCDHF 4.2158. The formation of dihydrotriazole 4.3 took place at a very slow rate. In 12 hours of standing, only 12% of the 4.3 was formed and after 23 hours, 21% of 4.3 was formed. As such, modifications are needed in either, and more likely both, of the reacting parties in order to enhance the overall rate of the two-step process. For example, the electron withdrawing group present in the π-system may have some effect in the

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cycloaddition between the azido moiety and the strained alkene. Therefore, the tetrafluoro derivative of azido 4.9 was reacted with norbornene 4.1159 (Scheme 4.3).

Scheme 4.3 Cycloaddition between semifluorinated azido Ph-V-DCDHF 4.9 and norbornene 4.1.

With the same concentration that was applied in the previous study, the cyclization reaction rate was significantly enhanced, but it now suffered from two serious drawbacks:

(1) the reaction took different route to form an aziridine and not the desired secondary amine. The aziridine and azido groups both absorb at shorter wavelengths. (2) The extinction coefficient decreased compared with the non-fluorinated compounds.

Some modification in strained alkenes may help in enhancing the rate of reaction to be a useful reaction. In a kinetic study of the cycloaddition reaction by Huisgen (1980), among a series of strained alkenes, only norbornene (Figure 4.4) exhibited a superior rate in cycloaddition reactions which suggested that the strain is not the only factor that

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controls the rate of reactions 160. Therefore, for our purpose we chose norbornene and its derivatives as strained alkenes in our cycloaddition reactions.

Figure 4.4 A series of strained alkenes(a) norbornene (b) bicyclo[2.2.2]octane (c) bicyclo[2.1.1]hexane (d) Tricyclo[3.3.0.0]oct-3-ene

For the overall reaction to be useful, the cycloaddition step and rearrangement must be fast at ambient temperature. In order to make the reaction go fast, we redesigned certain substructural features in the DCDHF-acceptor and π-system. The π-system can be made more electron deficient by introducing some fluorine atoms in the benzene ring and further addition of CF3 group in place of CH3 in the DCDHF head (Figure 4.5).

Figure: 4.5 A proposed modification in azido-Ph-DCDHF by incorporating electron withdrawing groups in π-system and DCDHF head.

4.4 Some Preliminary Studies on functionalized strained alkenes.

We performed some cycloaddition reactions between norbornene derivatives and azido-DCDHF 4.2. In a 1,3-dipolar cycloaddition reaction between 4.12 and 4.2 (Scheme

4.4), there was no reaction product observed even in 24 hours of standing as monitored

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by HNMR. This reaction should work since there is a precedent of 1,3-dipolar cycloaddition between phenyl azide and norbornene 4.12161.

4.12 4.2 4.13

Scheme 4.4 A dipolar 1,3-cycloaddition between 4.12 and 4.2

In a similar cycloaddition reaction between 4.14 and 4.2 with same concentration as in

Scheme 4.4, there was also no sign of reaction product (Scheme 4.5). In the case of cycloadditions of 4.12 and 4.14 the strain of the alkene will be nearly identical to that found in parent norbornene but the reactions are slowed by the electronic influence of the respective diester and anhydride groups.

4.14 4.2 4.15

Scheme 4.5 A dipolar 1,3-cycloaddition between 4.14 and 4.2

The kinetics of the 1,3-dipolar cycloaddition reaction on phenyl azide was studied with several substituents present such as nitro and methoxy161-163 reporting the formation of triazole at room temperature on standing for 10 days and more.

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The dipolar cycloaddition between various derivatives of bicyclo[2.2.1]heptadiene and phenyl azide has been extensively studied by Halton and Woolhouse (1972)164. The study was concentrated in the formation and photolytic decomposition of dihydrotriazole derivatives. The dipolar cycloaddition took place in the dark for 9 days which exclusively afforded exo-isomer 4.18 (76% yield) and no-endo isomer were detected. In the subsequent photolytic decomposition of dihydrotriazole (4.18) afforded aziridine (4.19) derivatives in good yield (85%) Scheme 4.6. The reaction was directed to form aziridine only.

4.16 4.17 4.18 4.19

Scheme 4.6 A 1,3-dipolar cycloaddition between strained alkene (4.16) and phenylazide (4.17).

The 1,3-dipolar cycloaddition reaction is a type of Diels Alder reaction in which the reaction in many instances correlates well with polar effects, and a complementary principle follows: Electron rich dipolarophiles react fastest with the electron-poor azides and vice versa137. We attempted to observe a cycloaddition between a strained alkene incorporated with electron rich moiety silicons and electron poor azido DCDHF 4.2. We observed no reaction at all. There is no precedent for cycloaddition reaction between 4.20 and any azide moiety. We wanted to extend this reaction in Scheme 4.7 by replacing

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165 chlorine atom further by CH3 to make norbornene more electron-donating so that the reaction may go faster.

4.20 4.2 4.21

Scheme 4.7 A cycloaddition between electron rich strained alkene 4.20 and azido DCDHF 4.2.

We attempted 1,3-dipolar cycloaddition reaction between 7- oxabenzonorbornadiene 4.22 with azido-DCDHF 4.2 as mentioned in the literature a reaction between 4.22 and phenyl azide166. The formation of adduct was much faster than the previous reactions. In 20 minutes, 23.07% of the dihydrotrazole was formed (4.23).

4.22 4.2 4.23

Scheme 4.8 A 1,3-dipolar cycloaddition between 4.2 and strained alkene 4.22

The formation of dihydrotriazole began as soon as the reaction was started which can be identified with the peaks indicated by 8,8’(Figure 4.6). Within 20 minutes, 23% of the dihydrotriazole was formed. After the reaction, the symmetry of the 7- oxabenzoborbornadiene is lost, as a result all the individual aromatic peaks are observed and each CH3 showed individual peak (14 and 14’).

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8 7 6 5 4 3 2 1 0 ppm

After 29.5 hours, 57% of dihydrotriazole was formed.

8 7 6 5 4 3 2 1 0 ppm

After 77.5 hours, 82% dihydrotriazole was formed.

8 7 6 5 4 3 2 1 0 ppm

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After 173.5 hours, 90% dihydrotriazole was formed.

8 7 6 5 4 3 2 1 0 ppm

After 317.5 hours, 95% dihydrotriazole was formed.

8 7 6 5 4 3 2 1 0 ppm

Figure 4.6 A kinetic study of 1,3-dipolar cycloaddition between azido-DCDHF 4.2 and strained alkene 4.22.

The dihydrotriazole formation from 7-oxabenzonorbornadiene proceeded faster than the other 1,3-dipolar cycloaddition previously discussed but no rearrangement of the dihydrotriazole was observed.

We synthesized benzonorbornadiene (4.24) and ran the same cycloaddition reaction with azido-DCDHF167 (Scheme 4.9) but the reaction rate was not better than the

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cycloaddition between 7-oxabenzonorbornadiene 4.22 and azido-DCDHF 4.2 (Figure

4.6). The rearrangement of 4.25 to 4.26 was not observed even for 24 hours (Figure 4.7).

4.24 4.2 4.25 4.26

Scheme 4.9 A cycloaddition between strained alkene 4.24 and azido-DCDHF 4.2

At the beginning of the reaction:

8 7 6 5 4 3 2 1 0 ppm

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After 24 hours at room temperature

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

Figure 4.7 A kinetic study of 1,3-dipolar cycloaddition between azido-DCDHF 4.2 and strained alkene 4.24.

In another experiment, we wanted to see the strain and/or electronic influence of a cyclopropane ring connected to the norbornene for 1,3-dipolar cycloaddition reaction between strained alkene 4.27 and azido-DCDHF 2 (Scheme 4.10). The norbornene derivative 4.27 was synthesized by a reaction between norbornadiene (~5 equiv) and

168,169 ethyl diazoacetate (1 equiv) in presence of catalyst Pd(OAc)2 . The product was a mixture of four isomers in a ratio of exo/endo (1.19:1); [exo, syn and exo, anti isomers:endo, syn and endo anti]. The reaction on this substrate containing a cyclopropyl ester proceeded very slowly since two small new aromatic peaks were observed after nine hours. After ten days, there is an indication of formation of dihydrotriazole 4.28 protons, but still there was difficulty in determining the alkene protons of the rearranged product

4.29 due to the possibility of formation of different product. There was difficulty in determining the kinds of by products formed due to spectra of four isomers in the

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aliphatic region. One possibility, the reaction may have stopped in intermediate 4.28. In any case, this process is too slow to be useful.

4.27 4.2 4.28 4.29

Scheme 4.10 A 1,3-dipolar cycloaddition between strained alkene 4.27 and azido- DCDHF 4.2

We synthesized strained alkene 4.30 and its isomers by reduction of esters 4.27 into alcohols 4.30 using strong reducing agent LiAlH4 and performed another 1,3-dipolar cycloaddition reaction with azido-Ph-DCDHF 4.2. The formation of dihydrotriazole 4.31 was slow. After 8 hours’ standing at room temperature, small aromatic peaks were observed, but due to four isomers of 4.30, it was indeed difficult to figure out which isomer reacts fast. The dihydrotriazole could not undergo rearrangement step to form

4.32 as indicated by the absence of new alkene protons. Since the starting material was a mixture of four isomers it is difficult to determine what other product could have formed.

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4.30 4.2 4.31 4.32

Scheme 4.11 A 1,3-dipolar cycloaddition between strained alkene 4.30 and azido- DCDHF 4.2

Our colleague, Alan Grubb synthesized some norbornene derivatives (4.33, 4.36,

4.39, and 4.42, and carried out 1,3-dipolar cycloaddition reaction between norbornene and azido-Ph-DCDHF170. The cycloaddition reaction showed a slow formation of dihydrotriazole intermediate 4.34 and it undergoes rearrangement also at a slow rate to form the product 4.35 (Scheme 4.12). In 48 hours, as HNMR indicated, 0.4% of the fluorescent secondary amine 4.35 was formed whereas dihydrotriazole 4.34 was formed in 12% in 48 hours.

4.33 4.2 4.34 4.35

Scheme 4.12 A derivative of strained alkene 4.33 in a 1,3-dipolar cycloaddition reaction with azido-Ph-DCDHF 4.2.

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A similar norbornene system as 4.33, 4.36 undergoes 1,3-dipolar cycloaddition reaction with azido-Ph-DCDHF 4.2 (Scheme 4.13). In 48 hours, as HNMR indicated,

7.2% of the fluorescent secondary amine 4.38 was formed whereas 4% of dihydrotriazole

4.37 was formed in 48 hours. This is an encouraging result compared to the norbornene.

The dihydrotriazole 4.37 started to undergo rearrangement to the secondary amine as soon as it was formed. This is of particular interest for our objective therefore further attention is necessary on this norbornene.

4.36 4.2 4.37 4.38

Scheme 4.13 A derivative of strained alkene 4.36 in a 1,3-dipolar cycloaddition reaction with azido-Ph-DCDHF 4.2.

In another norbornene derivative 4.39, the formation of dihydrotriazole 4.40 was good as 25% of 4.40 was formed in 24 hours, but the problem again was no formation of rearranged product even for 69 days. The dihydrotriazole 4.40 was stable and persisted for many days.

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4.39 4.2 4.40 4.41

Scheme 4.14 A derivative of strained alkene 4.39 in a 1,3-dipolar cycloaddition reaction with azido-Ph-DCDHF 4.2.

In an almost identical system as norbornene 4.39, the dimethoxy version 4.42 undergoes a 1,3-dipolar cycloaddition with azido-Ph-DCDHF 4.2, 35.6% of dihydrotriazole 4.43 was formed in 48 hours, but the problem again is that there is no formation of any rearranged fluorescent product such as 4.44.

4.42 4.2 4.43 4.44

Scheme 4.15 A derivative of strained alkene 4.42 in a 1,3-dipolar cycloaddition reaction with azido-Ph-DCDHF 4.2.

The Schemes 4.12, 4.13, 4.14 and 4.15 indicated that the dipolar cycloaddition speeds up somewhat due to the presence of electron donating group in the strained alkene

(norbornene), which is also in agreement with electronic demand for dipolarophiles for

1,3-dipolar cycloaddition. The dipolar species (azido) should have electron withdrawing group attached on it.

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4.5 A 1,3-dipolar reaction between azido functionality and norbornene and to understand the kinetics of the addition.

The changes in norbornene structure examined thus far have helped a little in the reaction rate between azido DCDHF and the strained alkene. In a kinetic study performed by Alan Grubb between bis(methoxyphenyl) containing norbornene 4.36 and azido-

DCDHF4.2, the initial cycloaddition was not speeded up but the rearrangement was better. From the plot it is evident that the rearrangement took place as soon as the dihydrotriazole was formed. In our attempt to speed up the 1,3-dipolar cycloaddition between strained norbornene and azido DCDHF and subsequent rearrangement, we worked simultaneously on the modification of azido-DCDHF to better understand what role this component had on the process. In a reaction between bicyclo[2.2.1]hept-5-ene and 2-(4-(4-azidophenyl)-3-cyano-5-methyl-5-(trifluoromethyl)furan-2(5H)- ylidene)malononitrile (4.45), the dihydrotriazole (4.46) was formed in a 1,3-dipolar cycloaddition reaction as an intermediate. The cycloadduct is unstable and undergoes subsequent rearrangement to afford secondary amine with the loss of nitrogen. The secondary amine possessing DCDHF (4.47) in its donor substructure is a fluorescent product which we were expecting. The reaction was carried out in a NMR tube and the time lapse reaction process was monitored and summarized in the Table 4.1 and Figure

4.8. The reaction did not occur as soon as they mixed together. So no new peaks were observed during the HNMR scanning. After 5 hours, small peaks of intermediate dihydrotriazole 4.46 and rearranged product 4.47 were detected following careful

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observation of the change in the proton absorption peaks. For convenient comparison purposes, the HNMR chemical shift was chosen in between 10 ppm to 3 ppm where major changes in starting materials, dihydrotriazole 4.46 and rearrangement product 4.47 can be observed; the chemical shifts of respective protons are labeled.

4.45 4.46 4.47

Scheme 4.16 A kinetic study of norbornene 4.1 and azido-Ph-DCDHF-CF3 (4.45)

At the beginning of the reaction

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 ppm After 5 hours at room temperature

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 ppm

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After 10 hours at room temperature

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 ppm After 27 hours at room temperature

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 ppm After 83 hours at room temperature

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 ppm After 130 hours at room temperature

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 ppm

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After 298 hours at room temperature

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 ppm

Figure 4.8 1,3-dipolar cycloaddition reaction between norbornene and azido-Ph- DCDHF-CF3 monitored by HNMR with consumption of the starting material and formation dihydrotriazole and rearranged product.

The time lapse reaction process was monitored by HNMR and is summarized in Table

4.1 and Figure 4.8. The reaction rate was slower than we expected. The formation of

dihydrotriazole 4.46 is significantly faster than the rearrangement to the product-

secondary amine 4.47 with the loss of nitrogen as indicated in Figure 4.2. In Figure

4.9, there is comparison of the 1,3-dipolar reaction between azido-Ph-DCDHF-CH3

(4.2) vs norbornene (4.1) and azido-Ph-DCDHF-CF3 (4.45) vs norbornene (4.1). When

both types of DCDHF-CF3 and DCDHF-CH3 were consumed by 50% of their

concentration, it indicates that 50% of the azido-Ph-DCDHF-CF3 reacts three times

faster than azido-Ph-DCDHF-CH3 to form their respective dihydrotriazole. The

dihydrotriazole of azido-Ph-DCDHF-CH3 is twice as stable than that of azido-Ph-

DCDHF-CF3 (considering 50% of the both species consumed). Due to this stability the

rearranged final secondary amine formed by azido-Ph-DCDHF-CF3 rearranged four

times faster than that of the azido-Ph-DCDHF-CH3.

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Table 4.1 Reaction data between azido-Ph-DCDHF-CF3 and norbornene as monitored by HNMR

Norbornene Rearranged Reaction run Dihydrotriazole concentration product time (hrs) formation % in % with time formation % 0 100 0.00 0.00

5 90 7 3

10 69 16 5

27 52 35 8 53 35 52 10

83 25 60 11

107 14 72 14

130 13 69 16

250 6 76 17

298 5 75 19

Reaction condition: Standing at rt without spinning (concentration 0.058M)

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[2+3] cycloaddition reaction process for Azido-Ph-DCDHF-CF3 and norbornene and successive rearrangement

Figure 4.9 Reactants (norbornene), intermediate (dihydrotriazole) and final rearranged product (secondary amine) distribution curve with time in T-ATA process

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Figure 4.10 A comparison of rate of norbornene consumption, dihydrotriazole formation and rearranged product (secondary amine) formation between norbornene vs azido-Ph-DCDHF-CH3 and norbornene vs azido-Ph-DCDHF-CF3; The purple curve represents the concentration of azido-Ph-DCDHF-CH3, the blue curve with diamond represents the concentration of azido-Ph-DCDHF-CF3, light blue curve represents the concentration of dihydrotriazole azido-Ph-DCDHF-CH3, red curve with circle represents the concentration of azido-Ph-DCDHF-CF3, the orange curve represents the concentration of rearranged product secondary amine of azido-Ph-DCDHF-CH3, and green curve with triangle represents the concentration of azido-Ph-DCDHF-CF3

In the reaction of bis(methoxyphenyl) substituted norbornene 4.36 and azido-DCDHF-

CH3, the initial cycloaddition was not speeded up but the rearrangement was faster than the reaction between norbornene and N3-Ph-DCDHF-CF3 Figure 4.10. The good thing about 4.36 is that the rearrangement took place as soon as the dihydrotriazole was formed. If we can improve the reaction rate in the 1,3-cycloaddition stage, this reaction could be useful.

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Figure 4.11 A comparison of rate of norbornene consumption, dihydrotriazole formation and rearranged product (secondary amine) formation between norbornene 37 vs azido-Ph-DCDHF-CH3 and norbornene vs azido-Ph-DCDHF-CF3; The blue curve represents the concentration of azido-Ph-DCDHF-CH3, the purple curve with diamonds represents the concentration of azido-Ph-DCDHF-CF3, green curve represents the concentration of rearranged product secondary amine of azido-Ph- DCDHF-CH3, orange curve with squares represents the concentration of rearranged product secondary amine of azido-Ph-DCDHF-CF3, brown curve represents the concentration of dihydrotriazole of azido-Ph-DCDHF-CH3, and blue curve with triangle represents the dihydrotriazole of azido-Ph-DCDHF-CF3

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4.6 Synthesis and Result

The synthesis proceeded in two directions: One direction was to develop the strained alkene and the other direction was to optimize the azido-DCDHF dyes for the faster cycloaddition [2+3] reaction between azido group and strained alkenes then speed up the rearrangement reaction to make secondary amines.

4.6.1 Synthesis of norbornene with ester functional group

We synthesized some strained alkenes for our preliminary test reactions. Strained alkene 4.12 was synthesized by dissolving dimethyl fumarate in anhydrous DCM under nitrogen at 0°C with successive addition of cyclopentadiene and stirring at room temperature overnight171 and 4.16 was synthesized in a neat reaction between cyclopentadiene and dimethyl acetylenedicarboxylate with quantitative yield172. The cyclopentadiene used in this reaction was synthesized from endo-dicyclopentadiene by cracking173 (Scheme 4.17). We did not anticipate that these compounds would be more reactive than simple norbornene or norbornadiene but since they were so simple to make we gave them a try.

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Scheme 4.17 Synthesis of strained alkenes 4.12 and 4.16 from dimethyl fumarate 4.49 and dimethyl acetylenedicarboxylate 4.50 respectively.

4.6.2 Synthesis of norbornene with benzo-group:

We synthesized another strained alkene-benzonorbornene following a literature procedure174. A mixture of 1,2-dibromobenzene (4.51) and cyclopentadiene (4.48) was treated with n-BuLi by slow addition at 0°C under nitrogen and the mixture was stirred overnight to afford the desired product174. The reaction was monitored by TLC. The purification was a little problematic, however the final clean product 4.24 (42.3%) was obtained by Kugelrohr distillation after column chromatography.

4.51 4.48 4.24

Scheme 4.18 Synthesis of benzonorbornene 4.24

4.6.3 Synthesis of norbornene with alkyl group on a bridge head position

In our attempt to synthesize a more electron rich norbornene, we attempted the synthesis of-1-methybicyclo[2.2.1]hept-2-ene (4.58). We began the synthesis with the

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reduction of commercially available bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (4.52) to bicyclo[2.2.1]heptane-2-carboxylic acid (4.53) in quantitative yield (Scheme 4.19). The next step was a Hell-Volhard-Zelinsky (HVZ) reaction involving a rearrangement. In all of the reported reactions, PCl3 was utilized to make an acid chloride in situ, which undergoes bromination to produce an α-brominated acid chloride. In successive neighboring group participation a rearranged intermediate is formed then in a final work up the acid would be regenerated as a rearranged brominated product175-1804.54. The mechanism for brominative rearrangement may be considered as a Wagner –Meerwein rearrangement which involves the stereospecificity associated with a “non-classical” carbocation181.

Due to the strict supply of PCl3 we could not purchase it. Instead we used SOCl2

182 which is a better chlorinating agent than PCl3 . The reaction worked but with very low yields of only 2% and the rest unrearranged product was obtained. HVZ reaction can be

183 carried out in PBr3 and Br2 combination but the reaction did not occur at all and only starting material was obtained. We attempted another reaction in which bromine and red phosphorus was stirred at 70°-80°C for 10 h and additional bromine was added and the stirring was continued overnight184.

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Scheme 4.19 A proposed synthetic route of 4.58 H2/Pd, EtOAc; (b) Br2, red phosphorus, 70°-80°C (c) MeI, K2CO3, DMF; (d) t-BuOK, t- BuOH; (e) DIBAL-H, THF, -70C, (f) Wolf-Kishner reduction

Using this latter method, the intermediate 4.54 was obtained as a white solid after washing with heptane. The yield was 60.3% which is better than the reported yield (35% yield based on acids). There are two options for the next step: (a) direct elimination of bromine to the norbornene and (b) esterification followed by elimination185. The direct elimination of bromine from 4.54 ends up with two isomers whereas elimination from ester 4.55 only one isomer was reported 4.56. We first esterified 4.54 using MeI, K2CO3 in DMF (73.6%). The elimination of HBr from 4.55 was attempted in t-BuOK/t-BuOH185 but did not work. We were not successful in synthesizing our designated target compound.

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4.6.4 Synthesis of norbornene with cyclopropane bearing alcohol group

The target compound mixture of tricyclo[3.2.1.02,4]oct-6-en-3-methanol [exo-syn, exo-anti and endo-syn, endo-anti 4.27a and 4.27b] can be synthesized in a series of steps starting from the cheap starting material glycine. The glycine (4.61), when subjected to ultrasonication with freshly distilled SOCl2 in absolute ethanol, afforded 4.62

186 (quantitative) . In the next step 4.63 was obtained in a reaction using reagent NaNO2 in aqueous sulfuric acid in good yield (65.3%)187. To minimize the formation of dicyclopropanation of norborna-2,5-diene, excess of diene was used in a ratio of 5:1 with diazoacetate (4.63). The reaction was catalyzed by Pd(OAc)2 at room temperature to yield a mixture of four isomers168 (52%). The next step was the reduction of ester to alcohol by using LiAlH4 in THF. It was not possible to separate the four isomers in a regular column chromatographic technique but a mixture of the four isomers with no other compounds present was obtained after Kugelrohr distillation which was acceptable for preliminary kinetic studies.

Scheme 4.20 Synthesis of cyclopropane ring possessing norbornene with alcohol tail for possible bioconjugation (a). SOCl2, EtOH; (b) NaNO2/H2SO4, H2O; (c) Pd(OAc)2, norborna-2,5-diene (d) LiAlH4, THF

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4.7 Synthesis of DCDHF dyes with an azido group

For our preliminary study, we synthesized the azido-Ph-DCDHF 2 from already prepared starting material 4.63 by Na Liu. The displacement of F by diallylamine was a decent reaction to afford 94% of 4.64 62. In a subsequent reaction to get the free amine we used N,N’dimethylbarbituric acid and Pd(PPh3)4 to afford a decent yield (quantitative) of

4.65 188. In the next step, 4.65 was first converted to a diazonium salt in situ which was then used in a successive nucleophilic substitution with NaN3 to afford 4.2 (quantitative).

In an alternate approach (Route 2), 4.2 can be directly obtained from 4.63 in excellent yield (86%).

Route 1

4.63 4.64 4.65 4.2

Route 2

4.63 4.2

Scheme 4.21 Synthesis of 4.2; (a) diallylamine, py, rt, 24h; (b) N,N’dimethylbarbituric acid, Pd(PPh3)4 DCM; (c) NaNO2/HCl, NaN3; (d) NaN3, DMSO.

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The reaction rate of different strained alkenes that we synthesized with 2 did not show any significant difference according to our preliminary test study. We concentrated now more on the DCDHF dye part. A chromophore containing CF3 can shift the emission by 30 nm compared to the same compound with no fluorinated group. Although there is no direct correlation of the absorption with the cycloaddition reaction, we assumed that the presence of this inductive withdrawing group might enhance the cycloaddition reaction. Azides bearing electron withdrawing groups undergo faster cycloaddition and makes the product triazoles more heat sensitive137. Some classes of triazoles are unstable at room temperature which immediately loses nitrogen to make aziridine137. Our interest here does not concentrate on the formation of aziridine but rather on another process involving rearrangement to secondary amine with the loss of nitrogen. The factors that enhance or influence the aziridine formation or the dihydrotriazole rearrangement to transform the secondary amine are not well understood.

It is an established fact that electron withdrawing group possessing azido group enhances the 1,3-dipolar cycloaddition reaction with strained alkenes. With this concept, we decided to modify the π-system by substituting electronegative atoms and fluorine is one candidate. On the other hand, the DCDHF head may possess several possibility of modification in R and R’ (Figure 4.5). The strong electron withdrawing group such as fluorinated alkyl chain is expected to enhance the cycloaddition. The emission of the

DCDHF-CF3 version is more slightly towards red shift than the DCDHF-CH3 although the absorption pattern of both DCDHF-CF3 and DCDHF-CH3 is the same.

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4.8 Synthesis of DCDHF dyes incorporating CF3 group with azido group on it.

4.47

The DCDHF head incorporating CF3 can be synthesized from a reaction starting with trifluoroacetone (Scheme 4.22). The chemistry began with the reaction of trifluoroacetone (4.64) with TMSCN in presence of catalytic amount of n-BuLi in anhydrous THF which afforded an intermediate 4.65. The use of n-BuLi was to trigger the formation of cyanohydrin 4.65. The volatile nature and hygroscopic nature of trifluoroacetone made this reaction a little problematic. In a separate reaction, a Grignard reagent 4.66 was prepared from 1-bromo-4-fluorobenzene. In a successive step in a reaction between 4.65 and 4.66 an imine intermediate would form which is not showed in the Scheme 4.22. The acidic hydrolysis of the imine gives the ketone with simultaneous deprotection of the OH, removing TMS. It was surprising that TMS was not removed so we had to use TBAF to remove the TMS group112. Our main concern in this scheme is to make the π-system – DCDHF more electronegative which may enhance the cycloaddition reaction. An attempt to synthesize 4.69 was not successful since it was formed as an intermediate which immediately undergoes another aromatic nucleophilic substitution by

189 malononitrile to afford 4.70 affording a blue compound. When there is no CF3 incorporated in the molecule, the reaction gave 4.2 (Scheme 4.22) in a decent yield.

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4.47

In an alternate route, the installation of nitrogen for azido group can be done by displacement of fluorine atom by diallylamine – an amine protection group. The displacement of F of 4.68 afforded 4.71 with the highest yield obtained was 76%. The ring closing reaction with malononitrile to produce 4.72 from 4.71 was poor (10-14%).

The cyclization reaction to make the furan ring from 4.71 to 4.72 was a difficult since the cyclization never went to completion. The highest yield reported, similar to 4.72, in a previous reaction was 10-27% when F was substituted by dihexylamine189. There was a mixture of five closely spaced compounds as indicated by TLC. The desired compound was separated by column chromatography. In deprotection step 4.72 to 4.73, the highest

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yield ever achieved was (55%). The same reaction in the non-fluorinated version, the removal of allyl group tested to work well with 89% (Scheme 4.21). The conversion of amino group to the final product azide was 0.073g (60.4%). The non-fluorinated version of this reaction worked well in every step with good yield. We found the fluorinated version of DCDHF does not proceed accordingly.

The synthetic route of 4.47 was not efficient since we have to utilize a huge amount of starting materials to get a very small amount of the final product 4.47 (0.073 g). We started with TMSCN (25.2 mmol, 2.5 g, $39/5g), trifluoroacetone (3.4 g, 2.68 mL, 30.35 mmol, $33.2/5g), and 4-bromofluorobenzene (10.8 g, 61.5 mmol).

In an alternate route, the synthesis of compound 4.71 was attempted by converting 4.75 into a Grignard reagent (Scheme 4.23). However, an attempt to make Grignard reagent of

4.75 failed. Then we tried n-BuLi190 to make a metallated species but it could not afford

4.76 but rather at the end of the reaction after work up we got the diallylaminobenzene.

This result suggested that the lithiation of 4.75 was successful to product 4.76 but 4.76 could not attack efficiently at the CN of 4.65 group to make imine (not shown) which eventually hydrolyzes in aqueous acidic condition to hydroxyketone 4.71. We maintained the reaction condition as dry as possible by supplying nitrogen atmosphere and oven dried glassware. The synthesis of 4.75 was carried out by following a literature191 procedure using silica gel (Green Chemistry) but it showed a mixture of mono and diallylated product which never went to completion even when additional allylbromide

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was added and reaction was run for longer than that was reported. We then used another method, allylbromide and Na2CO3 in ethanol which afforded 84% of 4.75 after a column chromatographic separation of the mixture192.

4.65

4.74 4.75 4.76 4.71

Scheme 4.23 Attempt to synthesize 4.71 (a) allylbromide, Na2CO3, EtOH, H2O; (b) n-BuLi, THF; (c) HCl/NaHCO3 work up

Since the installation of diallylamine and deprotection of the amine by removal of the allyl groups took two steps and removal sometimes ended up with only monodeallylation, we attempted an alternative pathway to make directly 4.77 from 4.68 (Scheme 4.24).

Unfortunately, this conversion failed. The azide could not replace F so there is always the starting material at the end. The reaction did not occur at all so we recovered the starting material. This step would be very useful which would have avoided some steps of reaction to reach the target.

4.68 4.77

Scheme 4.24 Attempt to synthesize 4.77 (a) NaN3, DMSO, rt

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4.9 Synthesis of DCDHF incorporating two F atoms in π-system

After the synthesis of 4.47 (Scheme 4.22), the route involved in Scheme 4.22 for the synthesis of intermediate hydroxyketone 4.68 can be utilized in the synthesis of 4.82.

The intermediate 4.82 would have additional electron withdrawing F atom in the π- system but it ended up in a very low yield (14%). With this low yield we cannot proceed further. 444 4.80

4.78 4.79 4.81 4.82

Scheme 4.25 Synthesis of 4.82 following Scheme 4.22; (a) Mg, THF, rt followed by 4.80; (b) HCl (aqueous )

4.10 Synthesis of DCDHF incorporating CF3 and Ph ring.

We attempted to synthesize another DCDHF derivative with a CF3 and phenyl ring on the DCDHF head. The required 1,1,1-trifluoroacetophenone (4.83) was readily available and easier to handle than trifluoroacetone and in addition we wanted to see the effect of phenyl ring. We followed a similar sequence to that in Scheme 4.22. The intermediate 4.84 can be synthesized by two methods; we chose the cheap method of

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using NaCN and TMSCl, which can be isolated in pure state solid (66%) Scheme 4.26.

The next step proved a problematic since the hydrolysis never worked well. Also 4.86 was probably may not be a good nucleophile due to F so that it did not effectively react with CN group of 4.84 as a result after the work process, fluorobenzene was obtained in the first fraction. The hydroxyketone 4.88 was obtained only in 20.5% yield.

Scheme 4.26 Synthesis of 4.88, a precursor for DCDHF head possessing CF3 and Ph; (a) n-BuLi, THF; (b) 1. HCl, 2.NaHCO3; (c) TMSCN, n-BuLi, THF; (d) NaCN, DMSO, TMSCl

After several unsuccessful attempts to increase the yield of hydroxyketones 4.68,

4.82, and 4.88, we attempted a direct intermolecular cross benzoin condensation reaction using N-heterocyclic carbene-catalyzed coupling of aromatic aldehyde with trifluoromethyl aromatic ketones. First we synthesized the N-heterocyclic carbene moiety as a precursor for the catalyst utilized in the hydroxyketone synthesis. It can be synthesized from N-methyl-2-pyrrolidone and trimethoxonium tetrafluoroborate to form a salt 4.90 and this step was followed by addition of phenyl hydrazine and stirring for two days and in a successive step the salt 4.91 so obtained was further treated with triethyl orthoformate to afford 4.92 as yellow crystals after cooling in a refrigerator193,194 (52%).

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4.89 4.90 4.91 4.92

Scheme 4.27 Synthesis of precursor of catalyst (NHC); (a) trimethoxonium tetrafluoroborate, DCM; (b) phenylhydrazine, rt, 2d; (c) triethylorthoformate, 100°C,12h

The first reaction we tried using NHC catalyst precursor 4.92 was a reaction between 4.93 and trifluoroacetophenone in presence of base DBU in THF to afford 4.88.

Similarly 4.96 was also obtained in good yield whereas there was no reaction at all between 4.95 and trifluoroacetophenone. We synthesized 4.88 and 4.96 in gram quantities195.

X = Y = H (4.93), X = Y = H 4.88 (72%) X = H, Y = F (4.94), X = H, Y = F 4.96 (67%) X = Y = F (4.95) X = Y = F 4.97 (0%)

Scheme 4.28 Synthesis of hydroxyketone using intermolecular cross benzoin condensation reaction

The aldehyde 4.95 was synthesized by the reaction of the Grignard reagent of 5-bromo- 1,2,3-trifluorobenzene (4.78) with DMF196.

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4.78 4.95

Scheme 4.29 Syntheses of 92 (a) Mg, ether and later DMF and then work up.

Due to the expensive precursor required to make the catalyst 4.92, we sought another alternative to synthesize the hydroxyketones.

X = H (4.98) X = H (4.100), 100% X = H (4.102), 80% X = H (4.88) 65% X = F (4.99) X = F (4.101), 85% X = F (4.103), 36% X = F (4.96) 62%

Scheme 4.30 An alternate path to synthesize 4.88 and 4.96. (a) triethylphosphite, neat; (b) KCN, DMF, 1,1,1-trifluoroacetophenone; (c) Et3N, H2O

Acid chlorides 4.98 and 4.99 were treated with triethylphosphite and stirred at room temperature which afforded 4.100 and 4.101 in good yield197. The next step is the intermolecular coupling between acyl phosphonate esters and ketone catalyzed by CN- which is similar to a benzoin type condensation followed by a complex intramolecular rearrangement. The coupling reaction worked well in both reactions. The removal of phosphonate ester was reported in a patent with 33% yield198 using 164 equivalent of

Et3N. However when we used 10 equivalent of Et3N and 3 hours of stirring afforded an acceptable yield of 4.88 and 4.96. The precursor acid chloride 4.99 required for this reaction was synthesized by reacting 3,4-difluorobenzoic acid with thionyl chloride

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stirring at 75°C for four hours. However extreme care was taken to process this compound as it is moisture sensitive. Since the procedure explained in Schemes 4.28 and

4.30 are only for the aromatic ketone with no α-H since they all are a kind of extension of benzoin condensation. Therefore there will be only CF3 and Ph present in DCDHF head if DCDHF derivatives are synthesized.

Since we had gram quantities of 4.88, first we attempted to install an amino group by replacing F in the p-position by several methods but F could not be displaced. It is very difficult to rationalize why this reaction did not work at all since the similar compound 4.71 (Scheme 4.22), which we made from trifluoroacetone, worked well. The reaction was monitored by 1H NMR. The typical splitting pattern caused by F in p- position never disappeared and no symmetric disubstituted H peak caused by another group ever appeared.

4.88 4.104

Scheme 4.31 Attempt to displace F in 4.88 by diallylamine to afford 4.1.04; (a) diallylamine, CH3CN, Et3N (attempted microwave as well); or diallylamine, p-TsOH, DMSO; or diallylamine, pyridine; or neat; or diallylamine, K2CO3, DMF; or diallylmine, Cs2CO3, DMSO

From hydroxyketone 90, we synthesized 100 (84.6%). We wanted to reduce a three-step reaction to one step as described in Scheme 4.32; however in the next reaction step, the complete formation of DCDHF head (4.106) was not successful. After

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cyclization, the further step did not take place (which was confirmed by 12C NMR) and further the azido group was reduced to amine (which was confirmed by FTIR). The compound isolated could be 4.107. This substance was isolated and reacted with malononitrile but no changes occurred.

4.96 4.105 4.106 4.107

Scheme 4.32 Attempt to synthesize 4.106 from 4.96 (a) NaN3, DMSO; (b) malononitrile, py

In another route as described in Scheme 4.33, 4.108 was synthesized by nucleophilic substitution of F at the p-position (68.2%). The attempt to synthesize 4.109 from 4.108 did not work at all.

4.96 4.108 4.109

Scheme 4.33 Attempt in synthesizing 4.109 from 4.96 (a) diallylamine, pyridine; (b) malononitrile, py

Since the benzoin condensation type reaction (Scheme 4.28 and 4.30) afforded only CF3 and Ph in hydroxyketone (4.88 and 4.96), we tried another route to make CF3 and CH3 in hydroxyketone 4.68 (Scheme 4.22). We introduced dithiane chemistry to

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protect the aldehyde and then further react it with trifluoro carbonyl compound to afford the hydroxyketone. The chemistry began with the protection of aldehyde 4.93 and 4.88 by using 1,3-propanedithiol using silica-sulfuric acid as a catalyst in dry acetonitrile to afford 4.110 and 4.111 (100%)101 as previously described in Chapter 1. The next step was the H-metal exchange reaction with n-BuLi or LDA. The metal-H exchange was tried with LDA199 first but due to excessive steps and precautions for making it, we chose n-BuLi which afforded 4.112 (50%) and 4.113 (36%)200. The deprotection of carbonyl was carried out by using pyridine tribromide which afforded 4.68 in good yield105.

X=H, 4.93 X=H, 4.110 X=H, R=CH3 4.112 X=H, R=CH3 4.68 (100%) (50%) (100%) X=F, 4.94 X=F, 4.111 X=F, R=CH3 4.113 X=F, R=CH3 4.114 (100%) (36%) (65%)

Scheme 4.34 Synthesis of 4.68 and 4.114 using dithiane chemistry (a) SiO2-SO3H, 1,3-propanedithiol, CH3CN; (b) 1. n-BuLi, THF, 2. 1,1,1-trifluoroacetone or LDA, 1,1,1-trifluoroacetone, THF; (c) Py.HBr3, Py, TBAB, DCM, H2O

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Scheme 4.35 A comparison study of the SNAr reaction in substituting F with diallylamine or azide.

The presence of CF3 group adjacent to the carbonyl group may make the F more labile for nucleophilic aromatic substitution as a result, the reaction in equation 1 worked well (Scheme 4.35). We attempted this reaction with 4-fluorobenzaldehyde which did not work at all, however in a very harsh condition (Na2CO3, HMPA, hydroquinone, n-

Bu4NBr, for 150 hrs) 4-fluorobenzaldehyde can react with diallylamine in good yield

(95%)201. The reaction in equation 2 never worked, the displacement of F by

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diallylamine was not possible. However with the addition of one extra fluorine atom in aromatic ring, the reactivity dramatically changed as in equation 4 which worked well.

The process in equation 3 as compared to equation 1 should work and it indeed worked well. Although the azido group is a powerful nucleophile, it was not able to displace F as in equation 5 but it worked well when two fluorines are present in the benzene ring as in equation 6. All the reactions worked in accordance with the electronic factors that assist the nucleophilic displacement of F. When we attempted a reaction of type found in equation 2 with piperidine, it worked well affording quantitative yield (Scheme 4.36) but the question is still not answered how Ph has such a dramatic effect compared to CH3 for such nucleophilic substitution while comparing reaction rate in equation 1 and 2. The geometry of the molecule 4.68 and 4.88 was checked in HyperChem which gave almost similar structure so we could not assume the steric factors have some influence. It can be assumed that the diallylamine is not nucleophilic enough to carry out the substitution reaction as in equation 2 compared to other secondary amines.

4.88 4.115

Scheme 4.36 A nucleophilic substitution reactions of 4.88 with pyrrolidine as nucleophile (a) pyrrolidine, DMSO, pTsOH

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4.11 Attempts on the synthesis with two CF3 groups on DCDHF head

With the successful synthesis of hydroxyketone with one CF3 required for DCDHF synthesis, we further extended the synthesis of two CF3 group on hydroxyketone as in

4.119 (Scheme 4.37). The most critical part in DCDHF dye synthesis is the efficient synthesis of hydroxyketone so our first focus is the synthesis of hydroxyketone.

4.93 4.116 4.117 4.118 4.119

Scheme 4.37 Attempt in the synthesis of hydroxyketone 4.119; (a) dimethylhydrazine, toluene; (b) 2,6-lutadine, TFAA, CHCl3; (c) 5N HCl, THF; (d) TMSCF3

The chemistry began with the imine formation between 4-fluorobenzaldehyde and dimethylhydrazine affording 90.5% of 4.116. The next step was the acylation of the

4.116 with TFAA in which the acylation did not occur well as reported202. A mixture of four compounds closely spaced in TLC was observed. They were separated by column chromatography affording only 15% of 4.117 as yellowish solid. The hydrolysis of 4.117 to 4.118 failed when carried out in HCl or H2SO4. For Scheme 4.37, p- fluorobenzaldehyde is an important precursor because once 4.119 is made, substitution of

F can be done easily by diallylamino group readily in the next step. We need an amine at the p- position because we want to turn this amino group into an azido group.

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In a similar reaction, nitrogen can be introduced in p-position by changing the precursor. In our next attempt we used 4-nitrobenzaldehyde. The nitrobenzaldehyde underwent hydrazide 4.121 formation with 95% yield. The acylation step of 4.121 to

4.122 also afforded a good yield of 65.5%. The hydrolysis of 4.122 to 4.123 contained many compounds in the mixture, which was difficult to separate by column chromatography.

4.120 4.121 4.122 4.123 4.124

Scheme 4.38 Attempt in the synthesis of hydroxyketone 4.124; (a) dimethylhydrazine, toluene; (b) 2,6-lutidine, TFAA, CHCl3; (c) 5N HCl, THF; (d) TMSCF3

4.12 Synthesis of the DCDHF unit incorporating a thiazole ring:

We have been exploring several options of chemical structure which can undergo

[3+2] cycloaddition reaction efficiently and further promote a rearrangement reaction as well. It is indeed a challenging task to find a system which can promote both chemical changes efficiently. We explored the thiazole ring which can possibly replace aryl units in donor-π-system-acceptor system. The incorporation of the thiazole unit in the place of phenyl shifts the electronic absorption more towards red which is a property we are interested in. The thiazole unit possesses superior NLO properties than the corresponding aryl analogues203. As Breitung et al have studied in detail and compared the following

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compounds 4.125 and 4.126 in which compound 4.125 absorbs at 371 nm whereas in compound with the replacement of phenyl ring with thiazole unit dramatically shifts the absorption to 443 nm which is interesting indeed.

4.125 (λmax 371 nm) 4.1.26 (λmax 443 nm)

Figure 4.12 Comparing absorption of two compounds (E)-2-(4-(4-diethylamino) styryl)benzylidene)malononitrile (4.125) and (E)-2-((2-(4-diethylamino)styryl) thiazol-5-yl)methylene)malononitrile (4.126)

Further, unsymmetrical thiazole unit has inherent dipolar properties in which C2 is electron poor and C5 is electron rich. Therefore thiazole can act as both an auxiliary donor and acceptor depending on its orientation.

matched case mismatched case

Figure 4.13 Electron density distributions in thiazole unit and matched and mismatched case according the orientation of thiazole unit.

When C2 of thiazole is connected to the acceptor unit, it significantly shifts the absorption more towards the red as evident in 4.128 (λmax 443 nm) compared to 4.127

(λmax 497 nm). This is a “matched case” (Figure 4.13). The matched case is described as

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the electron poor C2 site is connected with electron deficient acceptor part which reinforces the electron pull mechanism in a push pull system. In mismatched case the C2 site is connected with electron rich donor part, which partly cancels out the electron pull mechanism.

4.125 (λmax 443 nm) 4.126 (λmax 497 nm)

Figure 4.14 A comparison of absorption for the different orientation of the thiazole unit.

Therefore our goal is to make 4.129, 4.130, 4.131 and 4.4.132 (Figure 4.15).

There is no precedent for an azido thiazole unit reacting with strained alkenes. We wanted to try these new molecules in our project. However, we are still not sure what the effect of thiazole in cycloaddition and subsequent rearrangement reaction might be.

4.126 4.130 4.131 4.132

Figure 4.15 Proposed structure of azido DCDHF incorporating a thiazole ring.

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In our synthesis, our first attempt always focused on how efficiently we can synthesize the starting material (hydroxyketone) for any DCDHF dye. Once the hydroxyketone is synthesized then we can proceed to DCDHF dye synthesis. We thought that the synthesis of 4.139 can be carried out following Scheme 4.39. The chemistry began with the protection of the amino group in 2-aminothiazole (4.136) by allyl unit192

(57%). The next step was the lithiation of 4.137. The precedents for such lithiation was not available in the literature since there are very limited literature reported where a single thiazole unit has diallylamine at the C2 position204,205. There are several literature examples for the lithiation of thiazole at C5 206. From our previous experience a species with diallylamine can be lithiated (Scheme 4.22) and this is also reported in literature for aryl diallylamine190 but we are not sure about the thiazole system. For α-hydroxyketone synthesis, we need an electrophile and we used the Weinreb amide (4.135) rather than a cyanohydrin of acetone in Scheme 4.39. This is because whenever a thiophene is lithiated, it is prone to attack on both TMS as well as CN of the cyanohydrin with OH protected by TMS207 (Figure 4.16). This situation reduces the yield as well makes the separation complicated due to presence of additional undesired compounds as reported208.

We used the same analogy that a thiazole would also behave the same for lithiation reaction. We synthesized Weinreb amide 4.135 starting from α-hydroxyacid 4.134. When

4.133 was activated with 1,1-carbonyldiimidazole at 0°C in DCM and sequential addition of imidazole followed by DMAP, N,O-dimethylhydroxylamine.HCl afforded 4.134209

(60%). Subsequently the OH of 4.134 was protected by TMSCl in presence of pyridine

(56.1%).

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In the next step, lithiation of 4.137 was carried out. As soon as n-BuLi was slowly transferred to the solution of thiazole 4.137 at -78°C, it started turning black which was not normal in our previous lithiation cases. The color of the solution usually changed to faint yellow.

Figure 4.16 The reactivity of lithiated thiophene with a TMS protected cyanohydrin

Scheme 4.39 A proposed syntheses of hydroxyketone 4.139 (a) allylbromide, Na2CO3, EtOH, H2O; (b) n-BuLi, THF; (c) 2-hydroxy-2- methylpropanenitrile; (d) malononitrile, pyridine, CH3CO2H; (e) IBX/TEAB, CH3CN, rt (f) 1,1-carbonyldiimidazole, N,O-dimethylhydroxylamine.HCl; (g) TMSCl, py

In a successive step, we added electrophile 4.135 to the lithiated species 4.138 and left it stirring overnight. The reaction was monitored by 1HNMR which did not indicate

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the change in the chemical shift of 4.135 and TLC was different for the thiazole. After processing we found the unreacted 4.135 and no traces of 4.137. We assumed thiazole itself not a very stable compound probably it did not tolerate the n-BuLi conditions.

In an alternate route, we attempted to convert 2-aminothiazole (4.136) to 4.141

(Scheme 4.40) using IBX synthesized210,211 in our lab but the bromination did not take place. We attempted another method in which we brominated 2-aminothiazole so that lithiation may work well with bromine rather than hydrogen. We synthesized 4.141 by following a literature procedure212 (36%). Compound 4.141 is considered as an unstable and light sensitive compound213. In the next step we attempted to protect the amine with an allyl group but unfortunately as soon as we heated the solution it completely turned black. We could not determine the structure of the products.

4.136 4.141 4.142 4.143

Scheme 4.40 attempt on synthesis of 4.142 (a) Br2, CH3CO2H; (b) allylbromide, Na2CO3, EtOH, H2O; (c) diallyamine, Et3N, THF

We attempted to synthesize 4.142 by an alternative route in which bromine at C2 position of 2,5-dibromothiazole (4.143) may be displaced by diallylamine214 but this reaction did not work at all. We further tried in a microwave reactor as well, it also failed.

In the actual literature dialkylamines were used.

Since we have successfully used NHC as a precursor for catalytic reaction in intermolecular cross benzoin condensation (Scheme 4.30) between aromatic aldehyde

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and aromatic trifluoroaromatic ketones, we attempted to apply a similar reaction herewith thiazole but there is no precedent for an NHC precursor utilized in heteroaromatic system. We first attempted to synthesize aldehyde 4.144 from 4.137 by Vilsmeier Haack formylation reaction215 (Scheme 4.41) but instead of a formylation reaction it removed one allyl unit from 4.137 in good yield (56%). It is difficult to rationalize its mechanism how it happened. However, this reaction can be a new deprotection method of diallylamine if it can be optimized.

4.137 4.144 4.145

Scheme 4.41 An attempt to synthesized 4.144 by Vilsmeier Haack reaction (a) DMF, POCl3

4.13 Conclusion:

The chemical activation of fluorogen Azido-Ph-DCDHF-CF3 to secondary amine fluorophore was very modestly successful but the reaction rate of the formation of secondary amine with the loss of nitrogen followed by rearrangement process was very slow. We attempted several modifications in strained alkenes with the incorporation of several functionalities which helped a little especially in the rearranged product fluorophore. At the same time, we modified DCDHF system to enhance the electron withdrawing tendency of the pull system by incorporating CF3 in DCDHF head and F in

π-system. This modification helped a little, but we could not speed up both the

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dihydrotriazole formation and subsequent rearranged product sufficiently. The syntheses of CF3 incorporated DCDHF remained problematic and a number of routes were pursued.

The key steps were the synthesis of α-hydroxyketone in gram quantity so that cyclization of DCDHF can be tried in different methods. Due to the presence of CF3, the nucleophilicity of OH significantly decreased and effective cyclization did not take place.

We attempted to synthesize some thiazole based DCDHFs, but were not successful. The potential in this cyclization-rearrangement still requires further optimization for it to find practical application.

CHAPTER 5

Some Cell Imaging Techniques Involving Chemical Activation

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CHAPTER 5 Some Cell Imaging technique By Chemical activation

5.1 Introduction

Stochastic super resolution methods such as PALM14, FPALM15 and STORM15 basically rely on the precise determination of the position of single emitters utilizing the photoactivatable or photoswitching character of the probes. In contrast to these approaches, point accumulation for imaging in nanoscale topography (PAINT)63 introduced by Hochstrasser et. al. follows a different approach to obtain high-resolution imaging which involves the concepts of intermittency caused by bimolecular collisions216, photobleaching, and PSF measurement for single molecule detection. The

PAINT experiment is useful for the extracellular membrane and it is compatible with live-cell experiments as well such as in reporting on the dynamics of membrane receptor by following single molecule movement and can provide diffusion and activity maps for membrane receptors217; binding of hotspots of enzymes on lipid layers218; and study of the dynamics of intracellular and membrane-bound biomolecules in combination with

PALM techniques. There is another technique, which is different from the PAINT technique, which utilizes enzymatic activity to cleave the fluorogen covalently bonded to the substrate in the dark state to the bright state. One such enzyme, which has those properties, is β-lactamase. Recently, tetrazine-linked fluorophores have drawn a lot of attention in the imaging arena due to their broad range of functionality tolerance, reactions which proceed in high yield in organic solvents, water, cell media or cell

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lysate219. The tetrazine quenched the fluorescence of the fluorophore which undergoes

“turn on” process when it undergoes inverse electron demand Diels Alder reaction220.

This chapter includes these three different kinds of approach for single molecule imaging by active control of emission.

5.2 A brief overview of Point Accumulation for Imaging in Nanoscale Topography

(PAINT)63:

In a PAINT experiment, the fluorescent probe is present in the solution and the object to be imaged is also contained in the solution. The fluorescent probes dissolved in the solution continuously target the objects to be imaged. The rate of fluorescent probe collision with the object depends on the diffusion coefficient and concentration of the probes. Once a probe binds and becomes (at least temporarily) immobilized with the object, a fluorescent signal appears as a diffraction-limited spot on the object. The signal from this probe decays when it dissociates from the object or if it becomes photobleached. The binding mechanisms of dyes with the objects may be explained by electrostatic coupling or a hydrophobic interaction. The collision rate of the probes with a single object can be controlled at will because it depends linearly on the concentration of the probes.

The concentration of the fluorescent probe should be low enough (one molecule/µm2) such that only a few binding events are located at a given time which

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basically leads to the detection of only a single localized fluorophore at a time. Single- molecule localization of these fluorophores provides accurate coordinates, and the image is reconstructed from an ensemble of the coordinates. Unlike a stochastic super resolution microscopic technique, which involves a stringent requirement for the fluorophores to be photoactivatable/photoswitchable, the PAINT techniques do not have much demand on the fluorophores, as no special buffer conditions or caged fluorphores are required.

However, as in every imaging technique, bright fluorophores are required which increase the localization accuracy, and high intensities allow faster image acquisition through rapid readout and photobleaching.

5.3 Factors Essential to PAINT fluors

Nile red was the first fluorophore utilized as PAINT fluor. It is a canonical viscosity sensitive dye. It becomes brighter when it is confined in a high viscosity environment since it is less prone to relax thermally from the first excited state by twisting and thus the fluorescence intensity increases. There are certain requirements a fluorophore should satisfy for the PAINT technique (1) it should have specificity in binding to the cell wall; (2) the emission must turn on as a result of this binding. This

“turn on” can occur due to suppression of twists by binding (viscosity effect) or due to the nonpolar nature of the lipid bilayer. In other words, there might be two turn-on mechanisms; and for both conditions mentioned in (1) and (2) should have a large

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contrast between dye bound to the cell surface vs. free in solution or bound to glass slide, and (3) obviously the dye should have high quantum yield.

The first in vitro imaging of cellular components was done in large unilamellar vesicles (LUVs)63. In this experiment, LUVs were attached on the glass layer and Nile red was used as the probe. The density of the probes is very low so that the probability of two molecules being co-localized within the diffraction limit is sufficiently low. The two vesicles in close contact can be resolved by the PAINT technique. The diffraction-limited spot in the regular image of LUVs in Figure 5.1c is observed as a single peak, while a synthetic image of LUVs in Figure 5.1d clearly shows two separate distributions of spots whose center-to-center distance is 202.4 nm.

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Figure 5.1 Fluorescence image of LUVs63 1(a) conventional fluorescence image, (b) high resolution reconstructed image of vesicle attached to a glass surface, (c) & (d) Fluorescence of synthetic image of two vesicles with a center-to-center distance of ≈ 200 nm; 2 (a) conventional fluorescence image, (b) high- resolution synthetic image obtained by locating 2,778 single Nile probes collected in 4,095 frames. (Adapted from: PNAS 2006, Sharonov, A. and Hochstrasser, RM)

A similar experiment was carried out with a lipid bilayer which is relatively large object maintaining a low density of the probe (0.23 probes/µm in every 20-msec time frame). A total of 6,494 fluorescent spots were collected in 4,095 frames at 50 frames/sec shown in image Figure 5.1b. The probe utilized in the imaging was Nile red because the collisional kinetics of Nile red with LUVs has been studied in detail221. Nile red is sensitive to polar solvents and hydrophobic environments222,223. Its typical characteristic

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is that it hardly emits in a hydrophilic environment while in a hydrophobic environment it emits more readily. All the photophysical properties of Nile red such as absorption, fluorescence, fluorescence lifetime, and fluorescence QY rely on the polarity of the solvent. The inability of Nile red to emit in a hydrophilic environment is associated with the twisted internal charge-transfer (TICT) configuration resulting from motions of the diethylamino group224,225.

With these experiments, it has been demonstrated that the PAINT method can be used to image objects such as cell organelles, cell membranes, and other lipids with almost any kind of probes. The only essential requirement of the proposed imaging method is that the probe molecules should interact and temporarily immobilize on or within the objects.

5.4 Rationale for screening PAINT fluors

We synthesized some Nile red derivatives and DCDHF fluors that can be utilized in generating a super resolution 3D structure of live Caulobacter crescentus cell surface.

For this experiment some requirements on the probes were tested such as the dye should have an aliphatic tail because the length of that tail may determine binding kinetics, should be water soluble which inhibits the aggregation of the probes and should possess some charge which should hopefully keep the dye from being endocytosed by the cell.

The probe should not be too water soluble because some hydrophobicity actually promotes binding with the membrane.

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The probes in Figure 5.2 were screened for possible use in PAINT. The DCDHF dye class is interesting because it has solvatochromic properties similar to Nile red.

Figure 5.2 Probes utilized in preliminary screening for PAINT technique.

5.5 Photophysical properties of some PAINT fluors

We first compared the absorption, emission and molar extinction coefficient of

Nile Red, P-193 and NL03008 (Table 5.1). Nile red shows very weak absorption and emission despite the different solvent polarity. However, P-193 shows absorption at 549 nm in DMSO with an extinction coefficient of 27100 M-1cm-1. P-193 has some water soluble properties as a result it showed absorption at 571 nm and with extinction

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-1 -1 coefficient 22400 M cm . P-193 has emission at 638 nm and 660 nm in DMSO and water respectively. NL03008 exhibited absorption at 607 nm in water with extinction coefficient of 124960 M-1cm-1 and there is weak absorption and emission in DMSO.

NL03008 shows similar behavior with Nile Red compared to P-193 so it may be a possible candidate for the PAINT scheme.

- Table 5.1 UV-is absorption of NileRed-SO3 , Nile Red and NL03008 in DMSO and water

Molecule λabs ε λabs ε λfluor λfluor (DMSO) (DMSO) (Water) (Water) (DMSO) (Water)

Nile Red– 549 27100 571 22400 638 660 - SO3 (P193)

Nile Red ------

NL03008 ------607 124960 ------631

(Courtesy: Marissa Lee, Stanford University)

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5.6 Spectra in Phosphate Buffer Saline (PBS) (~µM of dye concentration)

In another comparative study, the absorbance and emission of all the dyes were first examined in PBS (~µM of dye). All the dyes are suitable for 514 nm excitation and they were chosen based upon similar emission spectra to that of Nile red for easy

- comparison. The emission of DCDHF-tail-SO3 and rhodamine 6G are more towards the blue compared to the rest of the dyes. The absorption of all the dyes ranges from 460 to

585 nm. The blue curve represents the standard Nile red. As it is functionalized with

PEG, its water solubility increases and it drastically changes its absorption properties which absorbs at ~565 nm represented by red curve compared to Nile Red. A probable reason for this spectrum is attributed to the functionalized Nile red with PEG tail which exhibits specific solute-solvent interaction in the form of hydrogen bonding or bulk solvent properties226. In this screening process, we included rhodamine B and measured its absorption and emission in PBS which has the lowest emission compared to all other dye (Figure 5.3 B) under similar conditions. All the dyes have very good Stokes shifts

(ranging from 94-133 nm), which are essential for SR techniques. However, Rhodamine

B has a smaller Stokes’ shift (25 nm) compared to the rest of the dyes.

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A B

Figure 5.3 Absorption and emission spectra of PAINT fluor candidates in PBS (~µM of dye) (Courtesy: Marissa Lee, Stanford University)

5.7 Fluorescence of some individual PAINT fluor candidates:

5.7.1 Nile red sulfate (P193), NL03008, and Nile Red

Nile red-SO3K (P-193) exhibits similar absorption and emission spectra as unfunctionalized Nile red. Due to the highly polar sulfonate end group in P193, it is slightly soluble in an aqueous environment. A change in solvent polarity from DMSO to water has shifted the absorption slightly towards the red by 25 nm since water is slightly more polar than DMSO. The blue solid line represents the absorption of P-193 in DMSO and the red solid line represents the absorption in water. The blue broken line and red

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broken line represent the emission of P-193 in DMSO and water respectively (Figure 5.4

(1)). An increase in fluorescence was observed when the viscosity of the water based solution was raised by adding glycerol (Figure 5.4 (2)). There is a linear increase in the fluorescence with the increase of viscosity (in the range examined). Over the entire range of the wavelength, P-193 exhibits maximum emission of fluorescence as indicated by the purple solid line at viscosity of 0.0101 cP (88.5% glycerol) (Figure 5.4(2)).

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30000 1 5e+7 25000

) 4e+7

-1

cm 20000 -1 3e+7 15000 2e+7 10000

Fluorescence

Absorbance (M Absorbance 1e+7 5000

0 0 300 400 500 600 700 800 Wavelength (nm)

Absorbance, water Absorbance, DMSO Emission, water Emission, DMSO

2 2 e + 7

2 e + 7

1 e + 7

Fluorescence5 e + 6 (cps)

0 550 600 650 700 750 800 W avelength (nm )

0.000894 cP (0% glycerol (m /m )) 0.00100 cP (19.1% glycerol (m /m )) 0.00128 cP (43.8% glycerol (m /m )) 0.00149 cP (53.2% glycerol (m /m )) 0.00176 cP (60.8% glycerol (m /m )) 0.00304 cP (75.3% glycerol (m /m )) 0.0104 cP (88.5% glycerol (m /m ))

- Figure 5.4 (1) Absorbance and emission of Nile Red-SO3 (P-193) in DMSO and - water; (2) fluorescence of NileRed-SO3 with increasing viscosity (glycerol/water) (Courtesy: Marissa Lee, Stanford University)

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Fluorescence of nl03008 w ith increasing viscosity (glycerol/w ater) 1 . 2 e + 6

1 . 0 e + 6

8 . 0 e + 5

6 . 0 e + 5

4 . 0 e + 5

Fluorescence (cps) 2 . 0 e + 5

0 . 0 600 620 640 660 680 700 W avelength (nm )

0.000894 cP (0% glycerol (m /m )) 0.000945 cP (10.3% glycerol (m /m )) 0.0001102 cP (30.8% glycerol (m /m )) 0.0001271 cP (43.3% glycerol (m /m )) 0.0001765 cP (60.8% glycerol (m /m )) 0.0003830 cP (79.0% glycerol (m /m )) 0.0266 cP (92.9% glycerol (m /m )) NL03008

Fluorescence of N ile R ed w ith increasing viscosity (glycerol/w ater) 1 . 4 e + 7

1 . 2 e + 7

1 . 0 e + 7

8 . 0 e + 6

6 . 0 e + 6

4 . 0 e + 6

Fluorescence (cps) 2 . 0 e + 6

0 . 0 550 600 650 700 750 800 W avelength (nm )

0.000894 cP (0% glycerol (m /m )) 0.000945 cP (10.3% glycerol (m /m )) 0.0001099 cP (30.5% glycerol (m /m )) 0.0001102 cP (30.8% glycerol (m /m )) 0.0001235 cP (41.1% glycerol (m /m )) 0.0001765 cP (60.8% glycerol (m /m )) 0.0100 cP (88.2% glycerol (m /m )) 0.0266 cP (92.9% glycerol (m /m ))

Nile red Figure 5.5 Fluorescence of NL03008 and Nile Red with increasing viscosity (Courtesy: Marissa Lee, Stanford University)

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NL03008 and Nile red exhibit gradual increase of fluorescence with the increase of viscosity in similar experimental condition as it was performed for P-193.

5.7.2 Fluorescence QY increases with viscosity

Fluorescence QY of P193 with % glycerol 0.55

0.50

0.45

0.40

0.35

0.30

Fluorescence0.25 (QY)

0.20

0.15 0 20 40 60 80 100 % glycerol (m/m)

Fluorescence QY vs viscosity of P193 0.55

0.50

0.45

0.40

0.35

0.30

Fluorescence0.25 QY

0.20

0.15 0.000 0.002 0.004 0.006 0.008 0.010 0.012 Viscosity (cP)

Figure 5.6 A measurement of fluorescence (QY) with respect to viscosity. (Courtesy: Marissa Lee, Stanford University)

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As the viscosity of the medium was increased by the addition of glycerol in a solution of

P193, the fluorescence QY was increased (Figure 5.6). The fluorescence QY was increased only 3 fold with the increase of viscosity. This increase of fluorescence QY was not very encouraging as compared to DCDHF tail which has a 30 fold increase in fluorescence QY under similar conditions. The viscosity of the medium with the increase

% of glycerol was estimated using an empirical formula 227.

- 5.7.3 A comparison of fluorescence properties of Nile red-SO3 (P-193), NL03008 and

Nile red with respect to viscosity

- In a comparison of the fluorescence properties of NileRed-SO3 (P-193), NL3008 and Nile Red with respect to viscosity, it is clear from the plot explained in Figure 5.7 that the fluorescence of P-193 does not increase as much with viscosity as NL03008 and

Nile Red. The normalized fluorescence area (calculated by integrating under the fluorescence emission curve and the lowest curve was designated as 1 and other curves were divided by this area) from these three fluorophores increases with viscosity in which the experiment was carried out in solutions of varying viscosity resulting from the addition of glycerol.

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- Figure 5.7 A comparison of normalized fluorescence area of the Nile red-SO3 (P- 193), NL03008 and Nile Red with respect to viscosity of the medium. (Courtesy: Marissa Lee, Stanford University)

Molecule Ratio of integrated fluorescence area (viscosity = 0.266 / viscosity = 0) Nile Red-SO K 3.77 3 NL03008 12.2

Nile Red 33.2

Table 5.2 a comparison of the fluorescence area of the Nile red-SO3K, NL03008 and Nile red (Courtesy: Marissa Lee, Stanford University)

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5.8 Preliminary Comparison Results

In our preliminary comparison of the PAINT fluor candidates, there are strengths and weaknesses for each dye. Keeping Nile Red as our standard dye for comparison purposes, Nile red-SO3K and rhodamine B exhibit a fairly satisfactory level in terms of cell specificity, relative photon counts and shift in wavelength after binding with cells.

Nile Red-PEG, DCDHF-N-6 and DCDHF-tail exhibit good specificity to the cell but not as good as Nile Red. Another important requirement for any imaging purpose is the photon count.

Diffraction Nile Nile Nile Red DCDHF-N- DCDHF- Rhodamin Rhodamin Limited Red Red S0 K PEG 6 Tail e B e 6G Image 3

Specificity Very OK Good Good Good OK Very Good to cells Good

Relative Photons 0.3 xxx 0.25 1 1 0.56 n/a (same Filters)

Wavelength Shift small @514nm change in ~30nm n/a ~5nm n/a ~50nm No change with spectra addition of shape cells

Table 5.3 a comparison of the photophysical properties of the PAINT fluor candidates (Courtesy: Marissa Lee, Stanford University)

Both DCDHF-N-6 and DCDHF-tail exhibit better relative photon counts than Nile Red, which is good. Nile Red-PEG exhibits photon counts at a satisfactory level. Nile Red

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exhibits a shift in fluorescence wavelength by ~30 nm after interaction with cells. Nile

Red-PEG showed poor shift in wavelength (~5 nm) after interaction with the cells. The rhodamine salts examined were not good candidates as the PAINT fluors since they don’t have much change of their emission wavelengths after interaction with the cells.

5.9 Results and discussion

In our preliminary evaluations, no new molecules tested seem to be any better than Nile Red in terms of viscosity turns on for PAINT except DCDHF-tail. Nile red-

PEG works fairly well for PAINT as mentioned in Table 5.3 and was used in 2D imaging of e.Coli (Figure 5.9). DCDHF-tail was found to be an alternative to Nile Red for a variety of reasons. DCDHF-tail labels C. crescentus cell membranes228 with high specificity. DCDHF-tail has a strong viscosity dependent emission, which causes an increase in fluorescence intensity as large as 30-fold in glycerol vs water which is very useful for PAINT. Furthermore, DCDHF-tail displays characteristics of a good PAINT dye with additional properties compared to Nile Red as it has aliphatic region for membrane binding and charge allows molecule to remain outside of the cell and to impart water solubility. It has excellent photophysical properties such as uniform labeling in the membrane, a strong increase in fluorescence emission in more viscous environment, and an increased number of detected photons with the DH-PSF microscope comparable to

Nile Red.

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Diffraction Limited Image 2D e.Coli NR –PEG Super Res Paint

1µ Figure 5.8 2D image of e.Coli using Nile Red-Peg (Source: Stanford University)m

5.10 Synthesis of hydroxy-Nile red phenol:

Hydroxy-Nile red was synthesized by a two-step reaction (Scheme 5.1). The chemistry began with the nitrosation of 3-(diethylamino)phenol. The nitrosation reaction was carried out using sodium nitrite and HCl (12M) at 0°-5°C to afford 5.2229,230 but the product obtained was not the desired product as indicated by HNMR but rather it was the dinitrosylated compound. After changing the concentration of HCl (10M), the desired product 5.2 was obtained231 with 60% yield. This compound was used immediately without further purification since it was light sensitive and hygroscopic. The condensation of 5.2 with 1,6-dihydroxynaphthol (5.3) afforded Nile red phenol (56.0%).

In the next step, Nile red phenol (5.4) was reacted with 2-(2-(2-

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ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate to afford Nile red PEG (P-189)

(48.5%)232. In another separate reaction, Nile red phenol (5.4) was reacted with 1,4- butane sultone to afford Nile red SO3K (P-193) (60.0%)

Scheme 5.1 Synthesis of Nile red derivatives (a). NaNO2, HCl, H2O; (b) MeOH, HCl (10 M); (c) 2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4- methylbenzenesulfonate, KOH, DMF; (d) t-BuOK, t-BuOH, 1,4-butane sultone

We attempted to synthesize Nile Red 5.7 which could be an interesting addition to

Nile red family since amine in 5.7 cannot rotate due to ring constraints but the synthesis did not work at all. The starting material required for this (Scheme 5.2) was previously synthesized so we followed the similar procedure to synthesize 2.13. The nitrosation of

2.13 afforded 5.5 (56.0%)233. The next step was the condensation of 5.5 with 1,6- dihydroxynaphthol. The reaction failed since we retrieved unreacted 1,6- dihydroxynaphthol after 48 hours of run.

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2.13 5.5 5.3 5.6

Scheme 5.2 Attempt to synthesize a Nile red incorporating the julolidine substructure (a) NaNO2, HCl, H2O; (b) MeOH, HCl (10 M).

5.11 Conclusion

We synthesized and studied the photophysical properties of Nile red phenol derivatives Nile red SO3K (P-193) and Nile red PEG (P-189). During the screening of the potential dyes for PAINT, we compared their properties with Nile red since Nile red is a near ideal dye for PAINT. However neither of these dyes is comparable to Nile red in terms of their viscosity turn on properties, emission, and quantum yield. We modified the

Nile Red structure incorporating polyglycolic chain with phenol ring to increase the water solubility. This change in functionality did not help at all but rather the emission and QY became worse than Nile Red. We incorporated the charged moiety in Nile red so that it would prevent endocytosis but due to poor emission with the increase of viscosity, it was screened out for the PAINT application.

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5.12 Oxy-DCDHFs for potential application in β-Lactamase Active Control Scheme

5.12.1 Introduction:

As we described in previous chapters, we are interested in super resolution imaging of live cells. We synthesized and analyzed Rhodamine spirolactams, azido-

DCDHF and Nile red derivatives for potential use in super resolution imaging involving different mechanisms. In the Rhodamine spirolactams we expected that the fluorogen

(dark state) can transform to fluorophore (bright state) purely by light. For azido-DCDHF we expected that the switching process (dark state to bright) can be brought about by a chemical process (1,3-dipolar cycloaddition between azido-DCDHF and strained alkenes which in subsequent reaction step produce a secondary amine, an essential part required for push pull mechanism) and for Nile red derivatives we expected that the switching process (dark to bright state) can be brought about by diffusing the fluors onto the cell surface, where they light up due to various effects (hydrophobic binding, immobilization of intramolecular twist).

We are now interested in another switching process, i.e., dark to bright state conversion of the fluorophore with involvement of enzymatic actions. β-Lactamase is a family of bacterial enzymes which have the capability of cleaving penicillins and cephalosporins with high catalytic efficiency234. Cleavage of the β-lactam ring of a cephalosporin creates a free amino group which triggers the spontaneous elimination of any kind of leaving group previously attached.235 In an extension of this work, when the

OH group of umbelliferone is alkylated, the compound becomes practically non-

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fluorescent, and when β-Lactamase hydrolyzes an alkylated umbelliferone, there would be spontaneous release of umbilliferone creating extended conjugation with free negative charge, which consequently produces a fluorescent signal (Scheme 5.3). This scheme was first utilized to activate fluorophore on both umbelliferone and resorufin molecules attached to cephalosporin, by Rao, Tsein, and others64. With the cephalosporin quencher attached, the substrate was in a dark state (fluorogen) and when the β-lactamase cleaved the β-lactam bond to release the penicillin by enzymatic action, the fluorogen turns into a fluorophore in the visible region.

5.8 5.9 5.10 Cephalosporin– umberilliferone + β-Lactamase umbelliferone (blue fluorescent)

Scheme 5.3 hydrolysis of alkylated umbelliferone by β-lactamase which releases the fluorophore umbelliferone.

The process in Scheme 5.3 can be extended to include oxy-DCDHF as the fluorophore because we assumed the β-lactamase enzyme may cleave the oxy-DCDHF dyes from cephalosporin in the same way as it did for umbelliferone and resorufin. Cephalosporin could be attached to an oxy-DCDHF through phenolic oxygen. The cephalosporin could quench fluorescence, and after β-lactamase cleaves the penicillin, the oxy-DCDHF would possess negatively charged oxygen and could become fluorescent as in Scheme 5.4.

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Cephalosporin– oxy-DCDHFs + β-Lactamase oxy-DCDHFs anion

Scheme 5.4 Proposed scheme of cephalosporin incorporated oxy-DCDHF undergoing hydrolysis by β-lactamase which releases the fluorophore oxy-DCDHFs anion

Aspects of this general scheme need to be explored to determine their viability- principally, whether the “product-like” oxy-DCDHFs (DCDHF 202 and DCDHF 203 are significantly brighter than the “reactant-like” oxy-DCDHFs (P-221 and P-222). Any pH dependence would also be of interest. The following DCDHF derivatives were considered for our preliminary tests.

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Figure 5.9 List of oxy-DCDHF derivatives potential candidates for subdiffraction imaging by enzymatic action.

5.12.2 Result and discussion:

5.12.3 Synthesis of oxy-DCDHF

We synthesized oxy-DCDHF dyes by a simple condensation reaction and studied their potential application in our designated system (Scheme 5.5). We began the reaction with the synthesis of 2.55. Compound 2.55 can be prepared by Knoevenagel condensation of hydroxyketone 5.13 with malononitrile236. This reaction equally worked

237 238 well with Mg(OEt)2 and NaOEt as a base in EtOH. The next step was again a condensation in which aldehyde and 2.55 were involved. This condensation step was a regular practice in our lab208,239 using pyridine and most cases a column chromatography

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was required to separate the desired products from the mixture. We simply used ethanol and refluxed which afforded pure products with no column chromatography240,241.

Scheme 5.5 Synthesis of oxy-DCDHF (a) Py, CH3COOH, (b) EtOH, aldehyde (see the list), reflux

Since enzymes can also have reactivity to hydrolyze ester linkages64, we extended the system to include an ester linkage in oxy-DCDHFs. We therefore further esterified with acetyl group for preliminary studies.

Scheme 5.6 Esterification of oxy-DCDHF by refluxing in pyridine

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5.12.4 Photophysical study of oxy-DCDHFs for potential application in β-Lactamase cleavage.

To screen out an ideal fluorophore for our purpose that can fit in Scheme 5.4, some physical properties such as the extinction coefficient, fluorescent quantum yield, wavelengths of maximum fluorescence and absorbance, as well as the pH dependence of four DCDHFs were characterized and compared (Table 5.4). P-221 and P-222 have the highest molar extinction coefficient 16250 M-1cm-1 and 19600 M-1cm-1 respectively which means among the four, these two have the best absorbance. However they have very low fluorescence QY, at 0.0057 and 0.0048 (a sufficient fluorescence quantum yield is essential for any subdiffraction single molecule detection). They are very poor photon emitters. They also both emit light in the green range, which is shorter wavelength of light than is ideal for single molecule imaging making them less interesting candidates for β-lactamase cleavage. (For comparison; Umbelliferone emits 450-495 nm). More importantly, since the read out wavelength will be set to pump only the product of the enzymatic reaction and not these precursor forms, the short wavelength absorption as well as the low quantum yield is both beneficial. It will also be important to determine if adding penicillin will actually quench the small oxy-DCDHF fluorescence.

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Table 5.4 Photophysical characteristics of the four oxy-DCDHFs (Courtesy: Marissa Lee, Stanford University)

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A B

C D

Figure 5.10 The absorption and emission spectrum of oxy-DCDHFs which are of particular interest given their Stokes shifts (Courtesy: Marissa Lee, Stanford University)

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DCDHF 202 and DCDHF 203 have lower molar extinction coefficients at both peak wavelengths of absorbance, which means they are not as efficient as P-221 and P-222 in absorbance. However both DCDHF 202 and DCDHF 203 have higher QYs, 0.032 and

0.015 respectively, although they are still low for single molecule detection. We assumed they would probably have higher QY in rigid polymer or in a restricted environment in the cell, as most DCDHFs in general have higher QY in rigid environments242. DCDHFs

202 and 203 emit at 680 nm and 604 nm respectively (red-shifted emission) which is useful for filtering out auto-fluorescence and scattering in biological media. DCDHF 202 has an unusually large Stokes shift (176 nm) indicating the possible presence of multiple absorbing and emitting species. One possible explanation for multiple peaks is an excited state proton transfer243. DCDHF 202 has two hydroxyl groups that can be deprotonated, so it is possible as this molecule is pumped with light , one or more protons transfer to the solvent, creating oxy-anions which fluoresce at longer wavelength244

The pH dependence of DCDHF 202 was also investigated. The pKa was found to be 6.4 by measuring its absorbance curve in a series of solvents with different pH made from

Britton-Robinson buffer at a pH of 7 (Figure 5.10).

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Figure 5.11 Absorbance of DCDHF 202 depends on pH. (Courtesy: Marissa Lee, Stanford University)

The photobleaching QY of DCDHF 202 was determined to be 3.4±.9 x 10-5 and

8.4±.9 x 10-6 for DCDHF 203. These values correspond to the DCDHF family members245, suggesting that they could function as effective fluorophores. The photobleaching QY of DCDHF 202 is larger than that of DCDHF 203 so DCDHF 202 bleaches faster than DCDHF 203.

5.12.5 Conclusion

Both P-221 and P-222 emit at short wavelengths and have low fluorescence QY, which are not the desired properties for single molecule imaging. DCDHFs 202 and 203 have higher fluorescence QY and longer wavelength emission, making them better candidates for the β-lactamase Scheme 5.4. DCDHF 202 in particular has an unusually interesting large Stokes shift. However, unless their fluorescence QY is much higher in a

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rigid structure, they are probably not fluorescent enough to pursue for our designated goal. Furthermore, DCDHF 202 exhibits pH dependent absorption, due to the presence of two OH groups. Overall studies shows that oxy-DCDHFs may not be the best fluorophores for the β-lactamase scheme but are still worthy of further attention and possible further optimization.

5.13 Tetrazine based fluorophores for potential use in imaging applications.

5.13.1 Introduction:

Among several schemes of fluorescent ‘turn on’ by chemical method, the use of tetrazine-linked fluorophores has gained recently a tremendous popularity in bioorthogonal reaction schemes. It is a chemical reaction of a tetrazine-linked fluorophore with a strained alkene or alkyne by an inverse electron-demand Diels Alder

(DA) reaction219,220,246,247. The reaction of tetrazine and a strained trans-alkene was developed by Fox et al219,247 and Weissleder248. This cycloaddition reaction is fast enough for applying in biolabeling which has the second order rate constant of 2000 to 22000 M-

1S-1. The tetrazine based DA reaction undergoes rapid cycloaddition, selective, and chemically accessible coupling reactions that do not require a catalyst and tolerate broad range of functionality in high yield. One important aspect of this tetrazine based DA

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reaction is that it is compatible with aqueous environments and further the second order rate constants for this reaction are known to be enhanced up to several hundredfold in aqueous media in comparison to organic solvents249,250. Diels Alder reactions are sometimes reversible reactions251 which are not suitable for biological labeling. However tetrazines are voracious dienes for inverse electron demand DA reactions, and N2 is produced as the only byproduct upon subsequent retro [4+2] cycloaddition252.

There are now ample examples utilizing this system for imaging such as site specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic DA reactions between genetically encoded bicyclononynes and trans-cyclooctenes253, imaging of SKBR3 human breast cancer cells after pretargeting trastuzumab antibodies modified with rhodamine and norbornene and subsequent labeling with tetrazine-

VT680246, labeling of E. Coli protein in vivo utilizing genetically encoded unnatural amino acid utilizing cyclooctyene-tetrazine based fluorophore254 and many more examples have yet to come.

The reaction rate of this class of inverse DA reaction absolutely depends on the strain energy and the electronic factors of the substrates participating in the reaction. The reaction rate can be influenced by tuning at two possible structural modifications; (1) modification of alkenes and alkynes (2) modification of tetrazine. A tremendous effort has been put by chemists to develop strained alkenes and alkynes as described in Table

5.5 to speed up the reactions and the quest is still open.

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5.14 Combinations of strained alkenes and alkynes derivatives with tetrazine derivatives for fast cycloaddition DA reaction.

In most cases different tetrazines, spacer, solvent systems, measurement methods, temperature has made a quantitative comparison of reactivity of dienophiles with tetrazine of interest challenging. However we have gathered some information of cycloaddition of strained alkene and alkyne with tetrazine derivatives for qualitative comparison (Table 5.5) so that at least a basic idea of their reactivity can be understood.

Second order Entry Combination rate constant Solvent Ref -1 -1 k2[M s ] Methanol 1 0.94±0.0079 /water 253 (5/95)

Methanol 2 3.41 ± 0.066 /water 253 (5/95)

Methanol 3 9.46 ± 0.16 /water 253 (5/95)

Methanol 4 5.00 ± 0.096 /water 255 55:45

5 na

Methanol 6 0.15 /water 255 55:45

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7 3.1 ± 0.1 THF 219

Methanol 8 2000 ± 400 219 /water 9:1

9 6000 220

10 22000 ± 2000 Methanol 247

Methanol 11 5235 ± 258 /water 255 55:45

Methanol 12 17248 ± 3132 /water 255 55:45

13 9.5 256

Methanol 14 ~21850 /water 255 55:45

7.0 ± 0.7 x 15 Methanol 257 10-2

16 2.0 ± 0.3 Methanol 257

17 3.3 ± 0.4 Methanol 257

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18 44.8 ± 4.9 Methanol 257

19 40.9 ± 13.8 Methanol 257

Methanol 20 437 ± 13 /water 255 55:45

Methanol 21 1245 ± 45 /water 255 55:45

Methanol 22 80 /water 255 55:45

Methanol 23 2672 ± 95 /water 255 55:45

Water/D MSO (12% 24 13 ± 2 258 DMSO by volume)

Table 5.5 Second order rate constant k2 of in reactions between different strained alkenes and alkynes with various tetrazines.

The DA reactions of norbornene conjugated to unnatural amino acid (UNAA) with different types of tetrazine derivatives (Entry 1-6) do not show dramatic changes in the reaction rate with the change in electronic nature of the tetrazine derivatives but as

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soon as the strained trans- cyclooctene was used as the dienophile, there are dramatic changes in the reaction rates of cycloaddition. In Entry 7, a simple aromatic tetrazine was studied with trans-cyclooctene, the cycloaddition reaction was determined to be [k2

3.1 ± 0.1 M-1s-1]. When the pyridine ring is introduced in place of phenyl ring (Entry 8)

-1 -1 there is a dramatic shift in reaction to [k2 2000± 0.1 M s ]. Electron withdrawing groups in dienes favor the inverse electron demand DA reaction and this is in complete agreement with that notion. The study was done with the change in solvent system from

THF to methanol/water. As expected there is a significant hydrophobic effect for DA reaction in methanol/water system compared to pure methanol the reaction rate which is

-1 -1 -1 -1 [k2 1140 (± 40) M s ] and in pure THF [k2 400 (± 20) M s ] respectively. When an OH group in the dienophile (Entry 9) is introduced, there is another huge jump in the

-1 -1 reaction rate of [k2 6000 M s ] even though different tetrazine derivative was used which is less reactive than the one that was used in (Entry 8). With incorporation of cyclopropane ring in trans-cyclooctene (Entry 10) probably reinforce more strain in the

-1 -1 dienophile system, the reaction rate was further jumps to [k2 2200 (± 2000) M s ].

When dienophile 1 is conjugated to UNAA to make the bioorthogonal dienophile

6, the DA reaction between 6 and 12, and 13 (Entry 11 & 12), in the actual biological system do not change the reaction rate significantly compared to the reaction rate in the model test. In Entry 13, a reaction rate of tetrazine 17 with the absence of one aromatic

-1 -1 ring with dienophile 6 shows a dramatic decrease in reaction rate [k2 9.5 M s ] compared with the reaction rate of tetrazine 13 with the same dienophile 6 in Entry 12.

-1 -1 Comparing a model test (Entry 10 [k2 2200 (± 2000) M s ]) and an actual experiment in

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-1 -1 the biological sample (Entry 14 [k2 2200 (± 2000) M s ]), the reaction rate indeed remained consistent. In another study of cyclooctyne and tetrazine (Entry 15), it shows a

-2 -1 -1 slow reaction rate [k2 7.0 ± 0.7 x 10 M s ]. As expected from previous results, when the electron withdrawing tendency of diene (tetrazine) is increased by incorporation of

-1 -1 pyridine ring, the reaction rate jumps to ~29 time faster [k2 2.0 ± 0.3 M s ]. The incorporation of cyclopropane ring in cyclooctene as in 9 speeds up the cycloaddition by

22 times comparing Entry 16 and 18 in the same solvent system. As described earlier, the reaction rate is favored by the hydrophobic effect which is obvious while comparing

Entry 19 (methanol) and 20 (methanol/water) which shows the reaction rate of [k2 40.9 ±

-1 -1 -1 -1 13.8 M s ] and [k2 437 ± 13 M s ] respectively, 11 times faster in the latter case. A recent study utilizing a more strained ring system in the dienophile (Entry 24) for DA reaction with tetrazine did not deliver the expected result (Entry 24).

Reaction between (Entry 20) 8 &12 is ~1000 times faster than (Entry 1) 3 &

12255 and the reaction between (Entry 14) 20 & 12 is 50 times faster than (Entry 20) 8 &

12. The reaction between (Entry 11) 6 & 12 is 10-15 times faster than the reaction between (Entry 20) 8 & 12, In a qualitative comparison, the cycloaddition of 8

(bicyclo[6.1.0]non-4-yn-9-ylmethanol, BCN) with all the tetrazine (12, 13, 14, 15) is 500 to 100 times faster than 3 (norbornene-2-ol) with all the tetrazine, whereas the cycloaddition of 6 (trans-cyclooctene-4-ol, TCO) with all the tetrazine (12, 13, 14, 15) is

10 to 15 times faster than the one between BCN and all the tetrazines.

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Beside the fast reactivity of tetrazine-strained alkene/alkyne, it is possible to design and develop tetrazine based probes that exhibit ‘turn on’ fluorescence on their rapid reaction with strained alkenes or alkynes253,259 (Figure 5.12). Such activatable

“turn-on” probes would significantly increase the signal-to-background ratio, which is particularly relevant to imaging targets inside living cells since a stringent washout of unreacted probe is not possible259. A fluorophore-tetrazine pair shows a weak fluorescence. As soon as the tetrazine undergoes a cycloaddition reaction with alkenes / alkynes, the fluorescence of the fluorophore turns back on. This is an important practical intracellular bioorthogonal coupling scheme which avoid visualizing accumulated but unreacted imaging agent (i.e. background). A series of fluorescent dyes which have emission between 400-600 nm when covalently linked with tetrazine undergoes fluorescence quenching and their fluorescence is restored after reaction with dienophiles and quenching is particularly efficient for the probes emitting between 510 and 570 nm260. The quenching of fluorescent dyes by tetrazine is wavelength dependent259. The tetrazine-linked fluorescent dye such as coumarin which has short wavelength emission displays only a threefold enhancement in its fluorescence after cycloaddition reaction.

Tetrazine-linked green and red emitting dyes displayed approximately 15 to 20 fold enhancement of their fluorescence after cycloaddition reaction in PBS (phosphate- buffered saline). However near IR-emitting fluorescent dyes such as tetrazine-VT680 and tetrazine-BODIPY 650-665 were not quenched so no turn on process occurred.

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Figure 5.12 General Scheme for the process involving a tetrazine-linked fluorophore which undergoes ‘turn on’ after inverse DA reaction with the bioorthogonal dienophiles.

The mechanism involved in such quenching of the fluorescence of the dye by tetrazine has not been thoroughly studied; however, at least two possible mechanisms have been proposed for the quenching effect (1) Fluorescence Resonance Energy Transfer (FRET)

(as described in Chapter 1 Introduction) may occur between tetrazine and the fluorescent chromophore since tetrazine has a visible absorbance maximum at 515 nm;246,260 and (2) Photoinduced Electron Transfer (PET) may occur between excited

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fluorophore and a potential tetrazine acceptor. Tetrazine is a heterocyclic compound with electron deficit properties and it is assumed that PET based mechanism of quenching of fluorescence may be similar to the well-known quenching of fluorophores by strong electron-poor nitrogen aromatic compounds261,262.

5.15 Tetrazine-linked with DCDHF derivatives and Nile red derivatives

Following these concepts, we pursued a similar technique with DCDHF dyes since we have DCDHF dyes which can be tuned to emit with 450 to 600 nm with chemical substructural modifications. The easy synthesis of DCDHF derivatives and their bright fluorescence would be advantageous to study tetrazine-linked fluorophore chemistry and possible application in super resolution imaging of a protein superstructure in C. Crescentus. Therefore, we synthesized DCDHF linked tetrazine (Scheme 5.11). In our preliminary scheme, we first synthesized tetrazines with no pyridine ring(s) despite their reactivity issue since we wanted to see how the tetrazines behave more generally.

5.16 Synthesis of Tetrazine derivatives

Symmetric tetrazines can be synthesized by a number of literature procedures. In one procedure, tetrazine can be made from benzonitrile by using sulfur and hydrazine hydrate263. We used the same method to make asymmetric tetrazines in a reaction

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between 4-hydroxybenzonitrile and benzonitrile. The reaction worked but we did not get the desired product and the work up was complicate as we obtained a paste and there was bad smell of sulfur. All we obtained was the symmetric 5.21. We followed another procedure in which 4-hydroxybenzonitrile and benzonitrile (5 equiv) and hydrazine monohydrate were heated 90°-110°C overnight47. A heavy yellow precipitate of a mixture of dihydrotetrazines was obtained which was filtered under vacuum and dissolved in acetic acid at room temperature and dihydrotetrazine was further oxidized to tetrazine using NaNO2 but again we obtained only symmetric tetrazine 5.21. It seems like benzonitrile is more reactive than 4-hydroxybenzonitrile so all the benzonitrile undergoes reaction to form dihydrotetrazine. To synthesize the unsymmetric tetrazine, we followed

264 recently published literature using Zn(OTf)2 as catalyst . The reaction was reported to run in a sealed tube under high pressure. However, we found the reaction can be run under normal pressure and nitrogen environment. The desired unsymmetric tetrazine 5.20 was obtained with satisfactory yield of 30-35% along with the formation of symmetric tetrazine 5.21. Although there is possibility of formation of three products 5.19, 5.20, and

5.21, there was no formation of dihydroxytetrazine 5.19 observed.

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Scheme 5.7 Synthesis of unsymmetrical tetrazine; (a) Zn(OTf)2, NH2-NH2 (anhydrous); (b) acetic acid / NaNO2

In an attempt to synthesize asymmetric tetrazine 5.25 by the same procedure, we only got

5.26 as the sole product (Scheme 5.8) by a method without the use of catalyst253.

Scheme 5.8 Synthesis of unsymmetrical tetrazine 5.25; (a) NH2-NH2 hydrate; (b) acetic acid / NaNO2

We synthesized symmetric tetrazines and selectively functionalize the two ends.

The synthesis of 5.19 was carried out between 4-hydroxycyanobenzene 5.14 and hydrazine monohydrate in good yield (94%)253 but the yield of 5.27 via selective monomethylation was very poor (10.0%)265.

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5.14 5.16 5.19 5.27

Scheme 5.9 Synthesis of tetrazine 5.27; (a) NH2-NH2.H2O; (b) acetic acid / NaNO2 (c) MeI, KOH, DMF

5.17 Synthesis of tetrazine-linked-DCDHF derivatives

The synthesis of vinyl-DCDHF 5.28 is straightforward. DCDHF head 2.55 was refluxed with 4-fluorobenzaldehyde (4.93) in absolute ethanol which afforded 5.28 in

75% yield. No column chromatography was required to separate the desired product, which is pure enough to proceed to the next step. However, in the next step the fluorine cannot be displaced by N-methylamino ethanol as expected. Therefore we had to use an alternate route. In the alternate route, the 4-fluorobenzaldehyde is heated at 90°-110°C with N-methylamino ethanol with no solvent. This procedure is not reported elsewhere.

This type of reaction was performed by using K2CO3 in DMF as solvent with ~ 56 % yield. The product often obtained in a mixture of other compounds and was separated by a column chromatography.

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2.55 5.28 5.29 5.30 4.93

Scheme 5.10 Synthesis of DCDHF derivatives; (a) 4-fluorobenzaldehyde, ethanol, (b) N- methylaminoethanol, DMSO, 160°C; (c) 7, ethanol; (d) N-methylaminoethanol, neat

Once both DCDHF derivative 5.29 and tetrazine 5.20 were synthesized, the two compounds were connected by Mitsunobu reaction266. The yield was poor. We now believe that the poor yield may be due to a solubility issue of tetrazine in THF. The emission of DCDHF derivatives 5.29 is 560 nm. We expected that the fluorescence should be quenched after the reaction with tetrazine 5.20 but it was not. We wanted to see the quenching of the DCDHF dye fluorescence by the tetrazine. If the quenching mechanism occurs through FRET, then the distance between tetrazine and the DCDHF may play a crucial role.

5.20 5.29 5.31

Scheme 5.11 Synthesis of tetrazine-linked fluorophore by Mitsunobu reaction; (a) Ph3P, DIAD, THF

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5.18 Synthesis of pyridine containing tetrazines

Since the tetrazine 5.20 did not quench the DCDHF 5.31, we tried another tetrazine derivative. The cycloaddition between the dienophile and tetrazine is enhanced by increase in electron withdrawing character of the tetrazine, which is possible by introducing a pyridine ring instead of benzene ring (Table 5.5). We also wanted to improve the solubility of the tetrazine and so an ethylene glycolic spacer group was added. We first synthesized a tetrazine with a pyridine ring. We synthesized the pyridyl tetrazine 5.37 by using zinc triflate as a catalyst after oxidation with sodium nitrite in acetic acid. The yield was satisfactory (26%). The chemistry began with the preparation of 5.33 by replacing a bromine in 2, 5-dibromopyridine (5.32) with cyanide using CuCN and NaCN in DMF267. This reaction is extremely time and temperature sensitive. We ran the reaction at 150°C for exactly seven hours which afforded 64% of 5.33.

5.32 5.33 5.34 5.35 5.24 5.36 5.37 5.26

Scheme 5.12 Synthesis of asymmetric tetrazine 5.37 (a) CuCN, NaCN, DMF; (b) 2- cyanopyridine, NH2-NH2 (anhydrous), Zn(OTf)2, (e) NaNO2, CH3CO2H

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We further attempted to functionalize tetrazine derivative 5.37 for conjugation with a

DCDHF fluorophore. However, the reaction did not work as the color of the tetrazine turned to yellow suggesting it might have been reduced to dihydrotetrazine.

5.38 5.39 5.40

Scheme 5.13 Synthesis of functionalized tetrazine 5.40 (a) Boc anhydride, EtOH; (b) 5.37, NaH, DMF

To avoid this problem, we synthesized 5-hydroxypyridine-2-carbonitrile 5.42 to synthesize the unsymmetrical tetrazine 5.44 and intended to carry out a Mitsunobu reaction to functionalize it as this reaction was already tested in Scheme 5.11. However, the Mitsunobu reaction was not successful (Scheme 5.15). The chemistry began with the substitution of Br with 4-methoxybenzylalcohol to afford 5.41 and the alcohol is deprotected to afford 5.42 (Scheme 5.13). The next step was the tetrazine formation which used Zn(OTf)2 as a catalyst. A symmetrical tetrazine 5.28 was obtained as side product along with the desired product 5.44 (36%).

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5.33 5.41 5.42 5.43 5.24 5.44 5.28

Scheme 5.14 Synthesis of unsymmetrical tetrazine 5.44; (a) 4-methoxybenzylalcohol, NaH, DMF; (b) Et3SiH, DCM, and CF3CO2H; (c) 2- cyanopyridine, NH2-NH2 (anhydrous), Zn(OTf)2, (d) NaNO2, CH3CO2H

5.44 5.40

Scheme 5.15 Synthesis of functionalized tetrazine 5.40 (a) DIAD, Ph3P, 5.39, THF

In alternate route, the functionalization of 5.33 can be done prior to the tetrazine formation. With this scheme, 5.45 was synthesized (Scheme 5.16). In the next step, the dihydrotetrazine was synthesized using Zn(OTf)2 catalyst which was further oxidized to afford 5.40 along with heavy pink precipitate of symmetrical tetrazine of 2- cyanopyridine. The deprotection of the amine can be done by using HCl in dioxane253.

When we tried to isolate the free amine, tetrazine lost its color and became pale yellow

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color and later turned into a black sticky substance. It seems like tetrazine might have undergone some kind of photodegradation. Tetrazine is stable under acidic condition but under basic condition it is not stable. In any case it was found that the Boc group in 5.40 was removed by using trifluoroacetic acid in DCM affording salt 5.41 quantitatively.

5.33 5.45 5.40 5.41

Scheme 5.16 An alternate route to synthesize 5.41; (a) 5.39, NaH, DMF; (b) 2- cyanopyridine, Zn(OTf)2, anhyd. NH2-NH2; (c) NaNO2, CH3CO2H; (d) CF3COOH, DCM,

5.19 Synthesis of DCDHF derivatives with carboxylic end

The DCDHF derivative 5.49 with a carboxylic group as a linker can be synthesized by refluxing 2.55 with aldehyde 5.48 as previously mentioned. The aminoacid salt 5.47 was synthesized following a literature procedure by heating NMP with hydrochloric acid268,269 with quantitative yield. There was another method which directly can give a free amine with free acid using Ba(OH)2 but the work up was not

270 straightforward . The synthesis of 5.48 was attempted using bases such as Et3N to mop up the acid from aminoacid salt 5.47 to provide free amine so that it can act as a nucleophile to displace F from 4-fluorobenzaldehyde but this did not work271. We tried

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the same reaction in aqueous KOH and the reaction was heated for 3 days at 110°C which afforded 40% of the desired product 5.48 after work up with HCl272. We used water as a solvent since the aminoacid salt is soluble in water and 4-fluorobenzaldehyde is slightly soluble in water.

Scheme 5.17 Synthesis of DCDHF derivative 5.50 (a) HCl, 110°C, 3h; (b) 4- fluorobenzaldehyde KOH, H2O, 110°C, 3d; (c) 7, EtOH; (d) N-hydroxysuccinimide, DCC, DMAP)

The next step was the activation of the carboxylic acid using and NHS ester, which is done by reacting 5.49 with N-hydroxysuccinimide, DCC, and DMAP in DCM with good yield. In the final step, the functionalized tetrazine 5.41 and NHS ester of DCDHF 5.50 can be connected by stirring at room temperature in the dark in the presence of Hunig's base (N,N-diisopropylethylamine) and DMF to afford 5.51.

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5.41 5.50 5.51

Scheme 5.18 Synthesis of tetrazine-linked DCDHF 5.51; (a) N,N-diisopropylethylamine, DMF

5.20 Alternate proposed route for synthesizing 5.54

In an alternate proposed route for synthesis of 5.54, we can synthesize 5.52 and before oxidation to tetrazine, 5.52 can be functionalized with Boc-protected amine. Next, the dihydrotetrazine 5.52 can be oxidized to tetrazine 5.53 using NaNO2 in CH3CO2H and finally the amine can be protected to afford 5.54.

5.33 5.35 5.52 5.53 5.54

Scheme 5.19 Synthesis of 5.54 (a) 2-cyanopyridine, Zn(OTf)2, anhyd. NH2-NH2; (b) 5.39, NaH, DMF; (c) 4N HCl, dioxane, in DCM, 30 min, rt; (d) NaNO2, CH3CO2H

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5.21 A synthetic proposal for tetrazine linked Nile red

We have synthesized phenol-Nile red for PAINT scheme previously. Since Nile red emits around 500-600 nm, we assumed that it could be used in a tetrazine-linked fluorophore scheme. In a proposed synthetic route, phenolic Nile red can be derivatized to the carboxylic acid end by a known reaction with dihydrofuran-2(3H)-one 273 to produce 5.56. Since the tetrazine with amine functional group can be synthesized, it can be connected to carboxylic acid end of the 5.56 regular by a reaction in presence of

DCC/DMAP.

5.55 5.56 5.57

Scheme 5.20 Synthesis of tetrazine-linked Nile red; (a) dihydrofuran-2(3H)-one, NaOMe, MeOH, reflux, overnight; (b) DCC/DMAP, DCM, rt

5.22 Summary

Tetrazine linked DCDHFs were synthesized successfully. Their full photophysical properties are yet to be determined. There are still plenty of work remained in this project

(1) water solubility of the tetrazine linked dye because this kind of reaction is compatible with aqueous environments and further the second – order rate constants for this reaction

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are known to be enhanced up to several hundred – fold in aqueous media in comparison to the organic solvent and (2) the dye must have emission below 600 nm for the quenching process by tetrazine to occur. We are working on suitable dye for this project.

The final goal of this project is to install tetrazine-linked fluorescent dye unit inside the live cell.

CHAPTER 6

Cofacial Structures of 1:1 Complexes of Perfluorophenazine with Polynuclear Aromatic Compounds

Part of this chapter is in process as a manuscript to be submitted by Prabin Rai, Scott

Bunge, Brett Ellman, Robert J. Twieg

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CHAPTER 6 Cofacial Structures of 1:1 Complexes of Perfluorophenazine with Polynuclear Aromatic Compounds

6.1 Introduction

The co-crystallization of a mono/polynuclear aromatic unit with a perfluoromono

/polynuclear aromatic unit has been known for decades. Patrick and Posser first reported the simplest and first co-crystallization of the aromatic mononuclear unit benzene and the perfluoroaromatic mononuclear hexafluorobenzene in 1960. In this case an equimolar mixture of both liquid monomers were mixed to afford a white solid274. There exists a strong electrostatic interaction between benzene and hexafluorobenzene in this co-crystal, which is composed of infinite stacks of the two alternating parallel aromatic molecules.

Notably, in their pure state, both benzene and hexafluorobenzene crystallize in a herringbone fashion274,275. The herringbone pattern is a molecular conformation that maximizes the electrostatic interaction energy amongst molecular electric quadrupole moments for an assemblage of like-quadrupolar molecules. In terms of orbitals, the herringbone pattern minimizes electrostatic π-orbital repulsion by edge-to-face arrangement and maximizes the electrostatic attraction forming two dimensional layers276. Recognizing the possibility of co-crystallization, Marder et. al. prepared a series of co-crystals such as acenaphthalene with hexafluorobenzene and octafluoronaphthalene277, octafluoronaphthalene with biphenyl, biphenylene278; 9,10 dihydrophenanthrene279, trans-stilbene and trans-azobenzene280, and polyfluorinated tolans281 to observe the arene-perfluoroarene interactions. There is a cofacial pattern of

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co-crystallization with 1:1 stoichiometric ratio in all of these cases possessing mixed stacks of alternating, nearly parallel molecules of the two components whereas independent parent arene and perfluoroarenes generally possess the herringbone (edge to face) arrangement.

Beside these studies there are many other reports on arene-perfluoroarene interactions utilized for different purposes, for example pentafluorophenyl-copper- pyridine p-chloride (applied in solid state luminescence)282, 5,5’-bis(perfluorophenyl)—

2,2’-bithiophene (theoretical studies on the electro optical properties)283, (E)-N,N- dimethyl-4-(perfluorostyryl)aniline (applied in solid state photodimerization)284, (E)-4- azido-N-(4-ethynylbenzylidene)aniline (applied in Copper free cycloaddition reaction)285,

Hexafluorobenzene,5-phenylpyrimidin-2-amine (applied in self-assembly of arenes in Ag

(I) complex formation)286, 1,2,3,4,5-pentafluoro-6-((1E,3E,E)-6-phenylhexa-1,3,5- trienyl)benzene (applied in emission shift due arene-perfluoroarene interactions)287, and perfluoro β-diketone and β-diketone complexes of Cu (applied in magnetic and conductive nanometer size materials)288

There are some explanations for the forces that bind the closely matched or moderately different shaped aromatic units and perfluorinated polynuclear aromatic units in face-to-face assemblies. No single force is usually involved, but rather it may best be described by a combination of van der Waals and multipolar interactions289 such as interaction between C – F and H – C. The C – F∙∙∙H – C interaction also plays a significant role in the mode of crystal packing and the stability of the crystals290.

Although arene-perfluoroarene co-crystallization is largely of theoretical interest to

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understand the nature of intermolecular interactions in these complexes and the mode of crystal packing, there are reports of arene-perfluoroarene interactions in co-crystalline state which may be useful in photodimerization and polymerization291, cycloaddition in the crystalline state292, luminescence from arene–perfluoroarene interactions in co- crystals293,294, self- assembling systems of simple organic compounds295-297, liquid crystals298,299, solution phase macrocycles synthesis300 and metal complexes301.

We chose here to examine perfluorophenazine as a component in these co-crystals wherein it would mimic the behavior of perfluoroanthracene. Phenazine itself is somewhat electron deficient relative to anthracene and the addition of the eight fluorine atoms further activates the heterocycle. Perfluorophenazine is readily prepared in a single step from pentafluoroaniline while perfluoroanthracene is prepared with significantly more difficulty302. The co-crystals of perfluorophenazine with polynuclear aromatic molecules may provide valuable information about the packing characteristics of organic molecules.

6.2.0 Results and discussion

6.2.1 Synthesis of perfluorophenazine

Perfluorophenazine (1) was synthesized by oxidation of perfluoroaniline. This reaction was attempted by a procedure reported by Leyva at. el.,303 carried out using perfluoroaniline, potassium ferricyanide as an oxidizing agent in ethanolic water solution.

The yield was not good (3.0%) and the compound was not isolated pure.

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6.1 6.2 6.3

Scheme 6.1 The synthesis of perfluorophenazine 6.2 with side product perfluoroazobenzene 6.3

In an alternative preparation, the oxidation was performed with lead tetraacetate in toluene as solvent. The desired compound PFP (6.2) (20.0%) and the byproduct decafluoroazobenzene 6.3 (37.0%) were obtained in satisfactory yield (but less than the yields reported) Scheme 6.1304. The melting point of 6.2 is 215ºC while the reported melting points range from 230ºC305 to 260ºC306. The separation of 6.2 and 6.3 was performed with pure hexane in a very slow pace monitoring with UV lamp. An attempt to increase the rate of elution by increase in polarity of the eluent by adding 1% ethyl acetate in hexane failed since all the black materials eluted along with the desired product. The byproduct 6.3 also forms a co-crystal with trans-stilbene307 and with azomesitylene308.

6.2.2 X-ray crystallographic discussion of PFPpolynuclear aromatic compounds

In a single component system one influence promoting non-cofacial packing is the strong electrostatic attraction between the instantaneous negatively charged center of

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one molecule and the instantaneous positively charged perimeter of an adjacent molecule

(or vis-à-vis, the mutual repulsion of molecular centers and the mutual repulsion of molecular peripheries)274,309-311 which is a typical case of herringbone motif during crystal formation. In a typical herringbone motif generally there is an edge to face contact of molecules. Pure perfluorophenazine in its crystal state occupies a herringbone motif in which there is edge to edge contact between adjacent molecules but there is no edge to face contact as expected for pure single component for most of the compounds unless otherwise modified some functional moieties [Figure 6.1].

Figure 6.1 Molecular packing of perfluorophenazine in its pure state adapting a herringbone motif along the c-axis, green-F, blue-N, grey-C.

PFP has been synthesized on several occasions but its crystal structure has not been reported so far in the Cambridge structural database (2013 release). However, parent phenazine has 149 co-crystals reported with different aromatic moieties and salts. Since there is ample evidence of co-crystallization amongst different shapes and different sizes of fluorinated and nonfluorinated aromatic molecules, an attempt to co-crystallize PFP

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with a number of different polynuclear aromatic moieties was anticipated to afford some interesting co-crystals.

Not all attempts to grow co-crystals were successful. During attempted co- crystallization, there exists a risk of crystallization of only one component or both components separately from the mixture of two in the solution. For example several attempts to co-crystallize PFP with a variety of aromatic moieties such as benzene, triphenylmethane, [2,2]paracyclophane, azulene, pentacene, and tetrathiafulvalene were unsuccessful. In these cases either crystals of each component of the pair grew separately or only one component crystallized out and the other remained in solution. Herein, we report some of the co-crystal structures of PFP and some polynuclear aromatic compounds and their detailed X-ray crystallographic descriptions in the experimental section.

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6.2.3 X-ray crystallography of Perfluorophenazine

Capped stick plot Packing along a axis

Packing along b-axis Packing along c axis

Figure 6.2 Capped stick plots of the perfluorophenazine showing herringbone packing along different axes (hydrogen atoms have been omitted for clarity)

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6.2.4 X-ray crystallography of Perfluorophenazine∙Naphthalene

The slow evaporation of dichloromethane from a solution of equimolar PFP (mp

215°C) and naphthalene (mp 80.26°C) afforded fine needle shaped crystals with continuous alternating stacks of PFP and naphthalene in the co-crystal of

PFP∙naphthalene (mp 173°C). The molecular structure of this co-crystal is shown in

Figure 6.3 and crystal packing is shown in Figure 6.5 adapting monoclinic system with space group P21/n with four molecules per unit cell.

Figure 6.3 The thermal ellipsoid plot of co-crystal PFPnaphthalene (50% probability)

The co-crystal did not grow in parallel and face to face geometry as we expected but rather the alternate molecules are packed parallel in one column facing opposite direction with the alternating stack of the adjacent column. This packing motif is very different from typical herringbone (edge to face) packing as in pure naphthalene312 or pure PFP but rather it possesses edge to edge contacts. The PFP∙naphthalene structure is similar to the co-crystal between octafluoronaphthalene and biphenyl278. The PFP molecules are perfectly parallel in a given stack and the naphthalene molecules are perfectly parallel but the PFP and naphthalene are twisted by a small angle in a given

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stack which can be expressed by the inter-component inter-planar angle between their mean molecular planes as explained by Marder et. al.313 and is measured as 1.88°. The two adjacent molecules in a stack are separated by a distance which is referred to as inter- planar distance and is measured by the space between the two centroids of adjacent molecules in a stack. The PFP molecules in one column and the PFP molecules in the next column intersect by an angle 50.91°(as shown in Figure 6.4) whereas naphthalene molecules in one column intersect the naphthalene molecules in another intersect by

51.17°.

Figure 6.4 PFP molecules in one column and the PFP molecules in the next column intersect by an angle 50.91°

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Capped stick plot Packing along a axis

Figure 6.5 Capped stick plots of the co-crystal PFP.naphthalene showing herringbone packing along different axes (hydrogen atoms have been omitted for clarity)

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Here the inter-centroid distance between adjacent PFP and naphthalene molecules is

3.790Å. Since the two adjacent molecules are not in a perfect parallel plane for this co- crystal, the inter-planar distance can be measured in a different way which is a mean distance between the centroid of one molecule and the plane of the adjacent molecule in a stack. The inter-planar distance of PFP∙naphthalene is 3.382Å

Figure 6.6 The inter-planar distance between PFP and naphthalene.

The stacking of alternate PFP and naphthalene is columnar but there exists a slip between the adjacent molecules in the stack. This extent of slip can be measured by slip angle which is the angle created between the stacking axis and the normal to the mean molecular planes (Figure 6.7) The slip angle for PFP is 26.874° and for naphthalene is

27.377° in PFP∙naphthalene crystal. The slip of molecule away from the axis with respect to similar molecule in a given column can also be expressed by slip distance. The slip distance of PFP with respect to the other PFP in the same column is 3.487Ǻ and the slip distance between naphthalene with respect to other naphthalene is 3.4232 Ǻ.

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Figure 6.7 A Slip distance of a molecule with respect to the similar molecule. The similar molecules in most co-crystals are perfectly parallel to each other in a given column. When the centroid of one molecule is determined and the distance between the two molecules is obtained by drawing a perpendicular from the centroid of the molecule to the plane of the other molecule (6.730Ǻ). The centroids distance can be calculated in a straight forward way which is (7.660Ǻ). The third distance is now obtained by simple calculation.

Although PFP molecules run perfectly parallel to each other in a given column, they make certain angle with PFP molecules in the adjacent column which is 50.91° and similarly 51.17° for naphthalene. All these physical parameters are summarized in Table

6.1. A combination of fluorinated aromatic molecules and non-fluorinated aromatic molecules known as arene-perfluoroarene effect has some influence in the crystal packing stability by the C – H···F – C bond interaction. If the intermolecular distance between H·and·F is in the order of 2.3-2.8Å, then it is more likely that C – H···F – C interaction can occur since the sum of the van der Waal radii of hydrogen and fluorine has been reported in a range from 2.3 - 2.5 Å but the interaction can occur up to 2.8 Å290.

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For the co-crystal between PFP and naphthalene, there are 8 comparable H and F contacts which range from 2.546 Å x 2, 2.583 Å x 2, 2.619 Å x 6 to 2.633 Å x 2 which suggest a significant number of C – H···F – C interactions considering PFP molecule (Table 6.2).

There is one C∙∙∙C close contact of PFP with adjacent naphthalene on the upper face and one C∙∙∙C close contact on the bottom face which are separated by a distance of 3.357 Å.

Figure 6.8 PFP∙naphthalene co-crystal showing close contacts

6.2.5 X-ray crystallography of perfluorophenazine and anthracene

Anthracene easily co-crystalizes with PFP by slow evaporation of dichloromethane from the equimolar solution of both components to afford fine needle shaped orange crystals adapting a triclinic system with space group P-1 with one formula unit per unit cell (Z = 1).

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Figure 6.9 The thermal ellipsoid plot of co-crystal PFP∙anthracene (50% probability)

The molecular structure of co-crystal is shown in Figure 6.9 and the crystal packing is shown in Figure 6.10. The interaction between the polynuclear aromatic molecule and the highly fluorinated PFP molecules leads to an infinite alternating PFP and anthracene face to face stacks (π-stacking) running parallel in the c direction (1D lamellar). Note that, once again, PFP and anthracene 314 individually adapt herringbone motifs.

Perfluorophenazine and anthracene both possess three aromatic rings of almost the same size. In this co-crystal both PFP and anthracene molecules lie at the crystallographic mirror plane symmetry (Figure 6.9). The PFP and anthracene molecules stack almost perfectly to each other in a unit cell (Figure 6.10). There exists a small tilt between PFP and adjacent anthracene molecule in a given stack therefore the inter- component inter-planar angle is 1.6°.

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Capped stick plot packing along a axis

Packing along b axis packing along c axis

Figure 6.10 Capped stick plots of the co-crystal PFP∙anthracene showing almost perfect parallel packing along different axis and face to face stacking to adjacent molecules (hydrogen atoms have been omitted for clarity)

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There are three C∙∙∙C close contacts of PFP with an adjacent anthracene on the upper face and three identical C∙∙∙C contacts on the bottom face. The closest contact distance between C∙∙∙C is 3.326 Å. The average closest distance between C∙∙∙C of two planes is

3.35 Å. In addition, there are six C – H∙∙∙F – C contacts with PFP surrounding. The distance of H∙∙∙F ranges from 2.480 Å to 2.616 Å which are more or less under the influence of van der Waal radii (interaction can occur up to 2.8 Å290).

Figure 6.11 PFP∙anthracene co-crystal showing close contacts

The inter-centroid distance between two adjacent PFP and anthracene molecules is 3.639 Å. The PFP and anthracene molecules are not in perfectly parallel planes.

However, the inter-planar distance can be described by a mean distance between the centroid of one molecule and the plane of the adjacent molecule in a stack, which is

3.365Å. The stacking of alternate PFP and anthracene molecules are not a perfectly

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columnar but rather there exists a certain slip between the molecules adjacent to each other in the stack. The slip angle for PFP is 20.88° and the slip angle for anthracene is

22.569° in PFP∙anthracene crystal. The slip distance of anthracene with respect to another anthracene in a given column is 2.594 Å whereas the slip distance of PFP with respect to another PFP is in a given column is 2.782 Å.

6.2.6 X-ray crystallography of perfluorophenazine and phenanthrene

The slow evaporation of dichloromethane in equimolar mixture of PFP and phenanthrene affords fine cube shaped yellow co-crystal adapting monoclinic system with space group C2/c with four formula units per unit cell (Z = 4).

Figure 6.12 The thermal ellipsoid plot of co-crystal PFP∙phenanthrene (50% probability)

There are infinite alternating PFP and phenanthrene stacks running in the c direction. The molecular structure of the co-crystal is shown in Figure 6.12 and the

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molecular packing is shown in Figure 6.13. In the pure phenanthrene crystal structure, all the phenanthrene molecules are facing in the same direction in herringbone motif315.

Capped stick plot Packing along a axis

Packing along b axis Packing along c axis

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Figure 6.13 Capped stick plots of the co-crystal PFP∙phenanthrene showing almost perfect parallel packing along different axes (hydrogen atoms have been omitted for clarity)

There is a complex orientation of phenanthrene in the columnar stacks. Two phenanthrene molecules occupy an exact opposite orientation to each other in different columns. The co-crystal between PFP and phenanthrene does not possess a crystallographic center of symmetry (Figure 6.12). The PFP and adjacent phenanthrene in a given stack have the inter-component inter-planar angle of 4.92° suggesting a significant decrease in parallelism compared to PFP∙anthracene co-crystal which has an interplaner angle 1.6 Å. There are three C···C close contacts of PFP with adjacent phenanthrene on the upper face and three C···C contacts on the bottom face. PFP and anthracene overlapped almost perfectly in the PFP∙anthracene co-crystal but in the

PFP∙phenanthrene co-crystal, there is only partial overlap of the PFP and phenanthrene molecules. The closest C···C contact is 3.322 Å. The average closest C···C distance between of two planes is 3.351 Å. There are also eight close H···F contacts which ranges from the shortest 2.486 Å to the longest 2.653 Å which is comparable to the sum of the van der Waal radii of hydrogen and fluorine. The inter-centroid distance between PFP and phenanthrene molecules is 3.655 Å and inter-planar distance is 3.383Å. Compared to the PFP∙anthracene co-crystal, the PFP∙phenanthrene occupies a large volume due to their larger inter-planar spacing which may be affected by twisted orientation of the molecules in the crystal (Table 6.1). The slip angle of PFP and phenanthrene in the

PFP∙phenanthrene co-crystal are 23.18° and 22.34° respectively. The slip distance of phenanthrene with respect to next phenanthrene in a given column is 2.782 Å.

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6.2.7 X-ray crystallography of perfluorophenazine and tetracene

The co-crystal of PFP and tetracene was successfully grown from 1-octanol (after screening numerous other solvents for the slow evaporation method). After several attempts the co-crystal grew as fine dark brown needles. The PFP∙tetracene adapts triclinic system with space group P-1 with one formula unit of either component per unit cell (Z = 1).

Figure 6.14 The thermal ellipsoid plot of co-crystal PFP∙tetracene (50% probability)

The molecular structure of the co-crystal is shown in Figure 6.14 and the crystal packing is shown in Figure 6.15. The aromatic hydrocarbon unit and highly fluorinated heteroaromatic unit leads to an infinite alternating face to face stacking running parallel along the c axis. As in previous cases, tetracene alone exists in herringbone motif in its crystal form316. Both PFP and tetracene molecules lie at the crystallographic mirror plane symmetry (Figure 6.14). The PFP and tetracene stack face to face almost perfectly with each other in a unit cell (Figure 6.15) and all the molecules run almost perfectly parallel along c axis. The inter-component inter-planar angle is 0.85Å which is smaller than that is found in PFP∙anthracene crystal.

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Capped stick plot Packing along a axis

Packing along b axis Packing along c axis

Figure 6.15 Capped stick plots of the co-crystal PFP∙tetracene showing almost perfect parallel packing along different axes and face to face stacking to adjacent molecules (hydrogen atoms have been omitted for clarity)

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The crystal packing is constructed with six C···C contacts on the upper face and six

C···C contacts on the bottom face of each PFP molecule. The average closest contact between C···C is 3.345 Å which ranges from 3.320 Å to 3.362 Å. There are ten H···F contacts ranges from 2.497 Å to 2.652 Å which are comparable to van der Waal radii.

These ten H···F contacts are formed with the surrounding six tetracene molecule in three different planes. The inter-centroid distance between two adjacent PFP and tetracene molecules is 3.338 Å. The measure of inter-planar distance from the mean distance between the centroid of one molecule and the plane of the adjacent molecule in a stack is

3.341Å. This is a slight decrease of inter-planar distance compared to PFP∙anthracene is due to the fitting of one stack into the inter-planar gaps of the adjacent molecule. The slip angle for PFP is 12.838° and for tetracene is 13.83° in PFP∙tetracene crystal. The slip distance of PFP with respect to another PFP in a given column is 1.623 Å whereas the slip distance of tetracene with respect to another tetracene is in a given column is 1.525

Å.

6.2.8 X-ray crystallography of PFP∙DTT (dithieno[3,2-b’:2’,3’-d]thiophene)

Apart from the co-crystal investigation of PFP with polynuclear aromatic compounds, we were also interested in co-crystallization of PFP with polynuclear heteroaromatic compounds. A co-crystal of PFP and dithieno[3,2-b’:2’,3’-d]thiophene

(DTT) grew by slow evaporation of DCM solvent in 3:2 ratio of PFP and DTT (DDT used in this study was provided by Yulia Getmanenko). The co-crystal of PFP∙DTT was

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mechanically separated from the excess DTT under a microscope. The PFP∙DTT adapts monoclinic system with space group P2(1)/n with two formula unit with 3:2 ratio per unit cell (Z=2).

Figure 6.16 Thermal ellipsoid plot of co-crystal PFP∙DTT (50% probability)

The molecular structure of co-crystal is shown in Figure 6.16 and the crystal packing is shown in Figure 6.17. The crystal grew in alternate molecules packed parallel in one column facing opposite direction with the alternating stack of the adjacent column with herringbone motif. Although the molecules are packed in alternate fashion as in 1:1 ratio of molecules, the total number of molecules counts in a ratio of PFP:DTT (3:2) in a unit cell. The packing motif of PFP∙DTT is similar with PFP∙naphthalene when a column is compared but the spatial arrangement of individual molecule in a given column is different as two DTT molecules alternately resides on either side of tetrafluorobenzene part of PFP which are in face to face orientation with PFP in a given column. The two

DTT molecules are in the same plane in a given column. The PFP molecules are perfectly parallel in a given stack and the DTT molecules are also perfectly parallel to each other

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but the PFP and DTT in a given column are twisted by a small angle (inter-component inter-planar angle) which is 1.59°. The intercentroid distances between PFP and two DTT molecules are 5.362 Ǻ and 4.358 Ǻ. The differences in the intercentroid distance between

PFP and DTT are due to the certain slip of the molecules away from the axis. The slip distance between two PFP molecules in a given column is 2.48 Ǻ. The interplanar distance between PFP and DTT is 3.419Ǻ. The PFP molecules in one column and the

PFP molecules in the adjacent column intersect by an angle 34.19° whereas DTT in one column intersect the naphthalene molecules in another column intersect by 33.45°.

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Stick plot Packing along a axis

Packing along b axis Packing along c axis

Figure 6.17 Capped stick plots of the co-crystal PFP∙DTT showing herringbone pattern packing along different axes (hydrogen atoms have been omitted for clarity)

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There is one S∙∙∙C close contact between DTT in one side and PFP of distance 3.481Ǻ where as there is no close contact of DTT with the same PFP molecule but rather the second DTT molecule has close contact with PFP molecules in the other layer with same distance 3.481Ǻ. There is one C∙∙∙C close contact between PFP and DTT 3.349 Ǻ at the upper layer of DTT and similar C∙∙∙C (3.349 Ǻ) close contact extends from the next DTT molecule to the PFP at the bottom. There is F∙∙∙H∙∙∙N close contact between DTT and PFP molecules (F∙∙∙H, 2.489 Ǻ and H∙∙∙N, 2.678 Ǻ). There is another continuous

F∙∙∙S∙∙∙S∙∙∙S∙∙∙S close contact between PFP, DTT and DTT molecules (F∙∙∙S 3.141 Ǻ, S∙∙∙S

3.339 Ǻ, 2 x S∙∙∙S 3.329 Ǻ). There is one H∙∙∙F (2.635 Ǻ) bond between one DTT with the

PFP in adjacent column.

Figure: 6.18 PFP∙DTT co-crystal showing close contacts

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There are two H··· F contacts at each DTT and two H··· F contacts at each PFP ranges from 2.489 Å to 2.635 Å that are comparable to the sum of van der Waal radii of hydrogen and fluorine.

Figure 6.19 Schematic representation of the intermolecular parameter

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Table 6.1 Intermolecular parameters

Complex PFP. PFP PFP PFP PFP naphthalene anthracene phenanthrene tetracene DTT

Inter-planar angle/° 1.88 1.6 4.92 0.85 1.59

Inter-centroid distance/Å 3.790 3.639 3.655 3.338 5.362, 4.358

Inter-planar distance/Å 3.365 3.362 3.383 3.341 3.419

PFP slip angle/° 26.874 20.88 23.18 12.838 ---

Arene slip angle/° 27.377 22.569 22.343 13.83 ---

Slip distance/ Å 3.423a 2.594a 2.782a 1.525a --- 3.487b 2.782b ---- 1.623b 2.48b a Polynuclear aromatic compounds slip distance a column bPFP slip distance in a column

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Table 6.2 Intermolecular close contacts in co-crystals per PFP

PFP∙Naphthalene Intermolecular close contacts Quantity Average distance (Å) Closest distance (Å) H – F 8 2.595 2.546 F – F 8 2.934 2.930 C – F 4 2.979 2.979 C – C 2 3.357 3.357 PFP∙Anthracene Intermolecular close contacts Quantity Average distance (Å) Closest distance (Å) H – F 6 2.554 2.480 F – F 2 2.812 2.812 C – C 6 3.35 3.322 PFP∙Phenanthrene Intermolecular close contacts Quantity Average distance (Å) Closest distance (Å) H – F 8 2.588 2.578 F – F 2 2.926 2.716 C – F 4 2.716 3.072 C – C 6 3.351 3.322 PFP∙Tetracene Intermolecular close contacts Quantity Average distance (Å) Closest distance (Å) H – F 10 2.578 2.497 C – C 12 3.345 3.320 PFP∙DTT Intermolecular close contacts Quantity Average distance (Å) Closest distance (Å) H – F 1 2.489 2.489 C – C 1 3.349 3.349 S – C 1 3.481 3.481 H – F 1 2.635 2.635 N – H 1 2.678 2.678 S – F 1 3.141 3.141 S – S 2 3.334 3.329

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6.2.9 Summary

In most cases whenever there is a possibility of occurrence of arene-perfluoroarene interaction, a co-crystal grown with aromatic and perfluoroaromatic compounds adopts a cofacial stacking of the molecules. This study has suggested that perfluoroheteroaromatic compounds such as PFP behave in a similar way with perfluoroarenes in terms of crystal packing in the solid state. The crystal complexes possess infinite stacks of alternating PFP and arene molecules. The molecular packing in

PFP∙naphthalene is completely different from the packing of PFP∙anthracene,

PFP∙phenanthrene and PFP∙tetracene in which PFP∙naphthalene adapts a herringbone motif with no edge to face contacts. Although PFP∙phenanthrene consists of similar infinite alternate packing of the molecules in an almost parallel fashion, the spatial orientation of molecules in the crystal is very different from PFP∙anthracene and

PFP∙tetracene. There is only a subtle difference in the crystal structure of PFP∙anthracene and PFP∙tetracene. The co-crystal of PFP∙anthracene and PFP∙tetracene are tightly packed cofacial molecules with significant arene-perfluoroarene interactions. The co-crystals we have prepared may contribute some valuable information about the packing characteristics of organic molecules and may be used in some organic reactions and materials.

CHAPTER 7

Synthesis of partially fluorinated heteroaromatic compounds for possible use in Organic Semiconductors

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CHAPTER 7. Synthesis of partially fluorinated heteroaromatic compounds for possible use in Organic Semiconductors

7.1 Introduction

Over the last 60 years, silicon based inorganic semiconductors have dominated the semiconductor field. During the last two decades, however, highly conjugated organic materials as organic semiconductors have attracted significant attention as complimentary materials relative to silicon based semiconductors317-320. The major reasons for this attraction, basically, is due to their potential for low manufacturing cost, easy processibility and fabrication as well as mechanical flexibility321. The organic semiconducting materials have a tremendous diversity of structural and functional group modifications so that they can be optimized for low cost solution based processing technologies, whereas silicon based semiconductors have fewer options and often involving vacuum deposition processing techniques319.

The operating mechanisms of organic semiconductors are different from their inorganic counterparts. The inorganic semiconductors are essentially covalently bonded highly crystalline three dimensional solid structures. In the inorganic materials strong interactions of the overlapping atomic orbitals are responsible for the charge transport whereas the organic semiconductors are molecular crystals. Here molecules are bound together by weak van der Waals interactions, hydrogen bonding, and π - π interactions.

Their charge transport properties depend on number of factors. Amongst them is an

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efficient π-orbital overlap between the constituent molecules. Basically, any organic crystal which possesses an extended π-system and the appropriate supramolecular arrangement in the solid state may exhibit some charge transport322. The state-of-the-art organic conjugated molecular crystals such as pentacene exhibit charge carrier mobilities

(µ)-reported to be as high as 35 cm2/Vs at room temperature in a single crystal at room temperature. This value exceeds the charge mobility of amorphous silicon320 for holes in excess of 1 cm2/Vs. A derivative of pentacene, 6,13-dichloropentacene (DCP),323 single crystalline ribbon transistors displayed charge mobility up to 9.0 cm2/Vs. In the same way, single crystal rubrene is reported to have a charge carrier mobility as high as 20 cm2/Vs at room temperature324,325. In a recently report, single crystal field-effect transistors based on DPC8-DTT (2,6-dioctylphenyldithieno[3,2-b:2',3'-d]thiophene)326 microribbons exhibits a high mobility of 1.1 cm2/Vs.

For technological applications, although the single crystal state possesses the highest charge carrier mobility, thin films have more processing options and are considered to be more useful. As such, thin films with a well-defined supramolecular organization are of real interest. Many promising compounds (e.g., the acenes, including even pentacenes, and often even disc shaped aromatic molecules) crystallize in a herringbone motif, which diminishes the π-overlap. However, there are some examples of high mobility systems known, which have behaviors contrary to this common design tenet, e.g., 1,4-diiodobenzene, and no co-facial structures whatsoever but still has a high

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hole charge mobility of µ ≈ 13 cm2V-1s-1 for a small molecule crystalline system327.

Beside herringbone motif there are some other molecular arrangement patterns as described in Figure7.1318.

Figure 7.1. Packing motifs of organic molecule in crystal (A) Herringbone packing (edge to face) without π- π overlap (face to face) between adjacent molecules (eg: pentacene); (B) herringbone packing with π- π overlap between adjacent moleculres (eg: rubrene); (C) lameral motif, 1-D π-stacking (eg: hexylsubstitutednaphthalene diimide); (D) lamellar motif, 2-D π-stacking (eg: TIPS- PEN) [Adapted from Chem. Rev. 2012, 112 Wang, C.]

The charge mobility in 1,4-diiodobenzene cannot be explained by π-π interaction.

Rather, there is a significant orbital overlapping of iodine atoms between the adjacent molecules328. Similarly, sexithiophene329, a very promising organic semiconductor candidate, has a charge mobility of µ = 2 x 10 cm2V-1s-1. The sexithiophene molecules

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crystallize in an edge-to-face (herringbone packing motif) motif, which can be explained by the strong electrostatic interaction between the negatively charged center and the positively charged perimeters of the ring systems of the adjacent molecules (or, vis-a-vis, the repulsion of molecular centers and the repulsion of molecular peripheries)317. The adoption of such a structure minimizes the electrostatic repulsion between the faces.

Besides these examples, a herringbone motif still may not be considered as the most suitable structure for optimal charge carrier mobility. Theoretical studies showed that charge carrier mobility can be improved by π-π stacking of organic molecule (face to face arrangement of molecules)330,331. One possibility to create such co-facial packing is through the co-crystallization between a highly electron deficient molecule (acceptor) and a complimentary electron rich molecule (donor) as in classical charge transfer complexes such as picric acid (acceptor) and methyl substituted aromatic hydrocarbons (donors) forms complexes in solution332. In this context, the study of charge transfer in co-crystals possessing face-to-face arrangements of highly electron deficient molecules such as perfluorinated aromatic compounds and their parent hydrocarbons is appropriate333 for possible improvement of the charge carrier mobility. However, another unavoidable problem exists when dealing with co-crystals/multicomponent crystals, which puts a potential fundamental limit to the charge mobility of the semiconductors. An electron or a hole moving from molecule to molecule faces an energy barrier imposed by different ionization potential of the molecules. In this situation, there will be no charge mobility or very small mobility will be observed.

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7.2 Option for avoiding ionization potential issue to improve charge mobility

In order to overcome (or at least mitigate) the problem generated by the difference in the ionization potential of different molecules, such as in the case of a co-crystal of a perfluorinated aromatic compound and its unsubstituted parent aromatic compound, it is possible to synthesize and grow a crystal of a polycyclic aromatic compound possessing unsubstituted aromatic rings condensed with perfluorinated aromatic rings in the same intramolecular framework. There are a number of examples of such molecules that adopt a co-facial orientation in packing, for example, as in 1,2,3,4 tetrafluoronapthalene and

1,2,3,4-tetrafluoroanthracene 334. The individual parent hydrocarbons naphthalene, anthracene and phenanthrene adopt herringbone crystal packing motifs whereas the

1,2,3,4-tetrafluoro derivatives of each of them adopts layered face-to-face stacking (π- stacking). The face-to-face assembly of the 1,2,3,4-tetrafluoronaphthalene, anthracene and phenanthrene (Figure 7.2) can be explained by a well-established fact of arene- perfluoroarene interactions which involves the minimization of the significant dipole moment possessed by these molecules.

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Figure 7.2 (A) A face to face (cofacial) packing of 1,2,3,4-tetrafluoronaphthalene molecules (B) A cofacial molecular packing of 1,2,3,4-tetrafluoroanthracene

In the co-facial structure and alternating orientations of the molecules, the perfluoroarene ring of one molecule overlaps with the arene ring of the other molecule.

This alternate parallel orientation extends across the whole crystal structure. However, the crystal packing motifs of monofluorinated (2-fluoronaphathelene 335), difluorinated naphthalene (1,5 and 1,8 difluoronaphthalene 318) and perfluoronaphthalene are all herringbone which indicates that there must be a well-balanced arene-perfluoroarene interaction in perfluoroaromatic rings and the unsubstituted ring in the same compound which results in the parallel orientation of adjacent molecules in layers. Such well- balanced interaction is not present in these mono and difluoroaromatic compounds.

We are also interested in synthesizing and studying crystals incorporating not only fluorine substituents but also nitrogen built in the aromatic systems. The substitution of one or more carbon atoms with nitrogen in extended π-system containing C=N double bonds have electron accepting properties which lowers the LUMO energy level resulting increase in orbital interaction and favorable for electron injection in n-type organic field

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effect transistors336. The valence band theory, described by conduction band and valence band, is developed for the crystalline inorganic semiconductor which is comparable to the organic semiconductor materials described by HOMO and LUMO energy levels, and they often gave the correct predictions 337. The HOMO and LUMO energy level are the intrinsic properties of individual molecules, however this energy level gap is extremely dependent on the packing mode of the organic molecules330 because of the interaction of

π-orbitals of the adjacent molecules318,330. The packing of unsubstituted polycyclic hydrocarbons is influenced by a delicate balance of π-π stacking and C – H∙∙∙∙ π interactions. In the quest of modification of the organic semiconductor, we sought nitrogen containing heterocyclic aromatic compounds.

We have synthesized partially fluorinated molecules in the acridine and phenazine classes and have attempted to grow their respective crystals. A number of related acridine class compounds have been synthesized previously such as 5,6,7,8-tetrafluoro-N,N dimethylacridin-2-amine, 1,2,3,4-tetrafluoro-7-methoxyacridine and 1,2,3,4,7- pentafluoroacridine338. The 1,2,3,4-tetrafluoroacridine and its derivatives have a strong potential for application as n-type semiconductors. All these molecules show their arrangements in perfect parallel fashion with face-to-face orientation of the molecules.

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Figure 7.3 Molecular packing of 1,2,3,4-tetrafluoro-7-methoxyacridine in a perfect parallel fashion.

Therefore, we further attempted to synthesize some larger examples with extended π- conjugation over a four-ring system.

7.3 Results and discussion

There are only 5 hits for 1,2,3,4-tetrafluoroacridine derivatives found in the

Cambridge Structural Database System (CSDS) 2012, v 5.33 and no crystal structure of

1,2,3,4-tetrafluoroacridine has been reported. Therefore, we first attempted to see the molecular packing in the 1,2,3,4-tetrafluoroacridine itself.

7.4 Synthesis of partially fluorinated acridine and its derivatives

The reaction between 1,2,3,4,5-pentafluorobenzaldehyde (7.1)and 2- aminonaphthalene (7.2) afforded 1,2,3,4-tetrafluorobenzo[b] acridine (7.3)(Scheme 7.1).

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The result was a mixture of two distinct products; possibly one is its isomer. The mixture was separated in a painful column chromatography due to a solubility issue. The desired product (7.3) was obtained in the first fraction as a yellow solid (25.0%). The compound was recrystallized from DMSO, which afforded a fluffy type shining yellow solid.

7.1 7.2 7.3 7.4 Scheme 7.1 Synthesis of 1,2,3,4-tetrafluorobenzo[b] acridine (7.3)

In an alternate route for the synthesis of 7.3, we used 2,6-dimethylaniline to make a Schiff base (Scheme 7.2). The idea behind making the Schiff’s base is to minimize the possible isomer formation as well as to save the possible loss expensive amines. The

Schiff’s base was prepared simply by stirring 1,2,3,4,5-pentafluorobenzaldehyde (7.1) and 2,6-dimethyl aniline with no solvent at room temperature. The desired product was obtained by refluxing the Schiff’s base with 2-naphthylamine in xylene until all the starting materials were consumed (36 hours). The desired product (7.3) precipitated and was filtered off (48%). Application of the method in Scheme 7.2 helped in improving the yield and no column chromatography was required.

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7.1 7.5 7.3 7.4

We tried several different solvent systems to get a crystal of 7.3 that is suitable for X-ray crystallography but none were successful. We also tried high vacuum deposition but still a suitable crystal was not obtained.

7.5 Synthesis of dibenzo[a,c]phenazine and partially fluorinated dibenzophenazine derivatives

We synthesized perfluorophenazine and successfully grew co-crystals of PFP and other aromatic hydrocarbons as described in Chapter 6. We wanted to further examine the crystal packing of phenazine derivatives that possess extended π-conjugation such as is found in dibenzophenazine. There are only 3 hits for dibenzophenazine derivatives in

CSDS 2013, v 5.33 2012 and there are no reports of X-ray crystallographic studies on any partially fluorinated dibenzo[a,c]phenazines. Therefore, we first synthesized the unsubstituted dibenzo[a,c]phenazine which is a known compound with its known crystal packing ID: MIVRUE following the literature procedure 339,340. We synthesized 7.8 by

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refluxing perfluorinated phenylene-1,2-diamine and phenanthrene-9,10-dione. The crystals structure of 7.8 is known (CSDS, MIVRUE).

7.8 (R1 = R2 = R3 = R4 = H) 7.9 (R1 = R3 = R4 = H; R2 = F) 7.10 (R1 = R2 = R3 = R4 = F) Scheme 7.3 Synthesis of partially fluorinated dibenzophenazine derivatives.

We synthesized the tetrafluoro derivative 7.9 by a similar procedure. We tried several solvents for the recrystallization of 7.9 and 7.10 that might give suitable crystals for the X-ray crystallographic studies but unfortunately no solvents were found which provided adequate crystals. Our objective was to study the effect of fluorine atoms on the molecular packing pattern. We used vacuum sublimation method to recrystallize 7.8 and

7.9 (25µ at 120°C and 25µ at 150°C respectively), which afforded only fine dust adhered on the condensation tube. Prior to this synthesis, we synthesized the starting material perfluoro-1,2-aminobenzene which is commercially available (but costs a fortune,1g/$476, lowest price).

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7.6 Synthesis of 6-nitroperfluoroaniline

7.11 7.12 7.13

Scheme 7.4 Synthesis of 6-nitroperfluoroaniline (7.12).

According to the literature procedure, the synthesis of 6-nitroperfluoroaniline

(7.11) can be carried out by passing dry ammonia gas through a solution of pentafluoronitrobenzene in diethyl ether at room temperature for 3h 114. However, we did not use ammonia gas but rather we used 9 equivalents of ammonium hydroxide stirring at room temperature in diethyl ether. This reaction was attempted several times with different solvent systems and different amounts of ammonium hydroxide. The use of 9 equivalent of ammonium hydroxide in ether afforded predominantly the ortho isomer

(7.12) (56%).

7.7 Synthesis of perfluoroaniline-1,2-phenylenediamine following literature procedure

7.12 7.7

Scheme: 7.5 Synthesis of perfluoroaniline-1,2-difluorobenzene (7.7)

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The reduction of 6-nitroperfluoroaniline using a mixture of tin (II) chloride and concentrated HCl in dry ethanol at room temperature 114 afforded the diamine in excellent yield (91%).

7.8 Synthesis of partially fluorinated phenanthrophenazine derivatives

Once the perfluorophenylenediamine was synthesized, we synthesized some additional extended π-conjugated molecules with a framework of phenanthrophenazine such as 7.12 (Scheme 7.6).

7.9 Synthesis of 10,11,12,13-tetrafluorphenathro[4,5-abc]phenazine

The intermediate, pyrene-4,5-dione (7.15 )required in this reaction was synthesized by following a literature procedure 341 using sodium periodate and ruthenium(III) chloride as a catalyst. The yield of dione (7.15) was lower than reported.

The yield did not improve even though several attempts were made. The dione (7.15) so obtained was condensed with perfluoro-1,2-phenylenediamine to afford the desired product 339,340 in excellent yield 7.14 (98%). The crystal suitable for X-ray crystallographic discussion was grown in m-xylene which afforded tiny orange needles.

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7.14 7.15 7.14

Scheme 7.6 Synthesis of 10,11,12,13-tetrafluorophenathro[4,5-abc]phenazine (7.14) (a) RuCl3.2H2O (10 mol%), 4 equiv NaIO4, DCM, MeCN, H2O, rt (b) glacial acetic acid, 120°C

7.10 X-ray crystallographic discussion of 10,11,12,13-tetrafluorphenathro[4,5- abc]phenazine (12)

We wanted to see the effect of fluorine atoms on the molecular packing in the crystal and wanted to carry out a comparative study of all the molecules but crystal structures of the molecules were not available although they were synthesized. However, we were able to grow crystals of 7.16. The compound 7.16 has a similar crystal packing pattern to the co-crystal between PFP and naphthalene which we grew previously and it is also similar to the co-crystal of octafluoronaphthalene and biphenyl 278 in terms of their molecular packing.

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Capped stick packing view along a-axis

view along b-axis view along c-axis

Figure 7.4 Capped stick plots of 10,11,12,13-tetrafluorphenanthro[4,5- abc]phenazine showing along different crystallographic axis (H atoms have been omitted for clarity).

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We expected that the molecule would pack in parallel motif due to the presence of partial fluorination in the molecule but they adopted a herringbone type packing motif.

The packing of 10,11,12,13-tetrafluorophenathro[4,5-abc]phenazine molecules is perfectly parallel in a given column i.e. the inter-component inter-planar angle between two molecules in the same column is 0° as explained by Marder et al 313 but molecules in a column make an angle 70.78° with molecules of the adjacent column. The inter-planar distance of two molecules packed in parallel in a given column is 3.195 Å with their inter-centroid distance of 4.339 Å. This suggests that the molecules in a given column possess certain slip. The extent of slip is measured by slip angle, which is 28.2°. With inter-planar distance 3.195 Å, we can say that molecules in a column are tightly packed, and there exists strong arene-perfluoroarene interaction as the perfluoro arene part of the molecule stacks over the arene part of the adjacent molecule in a given column. The crystal packing in partially fluorinated molecules is believed to be governed partially by

C – H∙∙∙F – C bonds interactions (Table 7.1). There are four H∙∙∙F/F∙∙∙H bonds from each molecule, which can interact with the neighboring molecules. Since all the H∙∙∙F/F∙∙∙H bonds are separated by a distance that is on the order of 2.545-2.734Ǻ, then it is more likely C – H∙∙∙F – C interaction can occur which are within the sum of van der Waal radii of hydrogen and fluorine atom. There are 8 C∙∙∙C close contacts of one molecule with the adjacent molecule, 4 at the upper face and 4 at the lower face. The upper face 4 C∙∙∙C ranges from 3.364 Ǻ to 3.398 Ǻ. The closest C∙∙∙C contact in upper face is 3.364 Ǻ and the average C∙∙∙C is 3.381 Ǻ. There are 4 C∙∙∙C contacts in the lower face that ranges from

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3.323 Ǻ to 3.348 Ǻ. The closest C∙∙∙C contact in lower face is 3.323 Ǻ and the average

C∙∙∙C is 3.381 Ǻ.

Figure 7.5 The close contact of atoms between 10,11,12,13-tetrafluorophenathro[4,5- abc]phenazine molecules

Table 7.1 Intermolecular close contacts in 10,11,12,13-tetrafluorphenathro[4,5- abc]phenazine molecule

Intermolecular close Closest distance Quantity Average Distance Ǻ contancts Ǻ H∙∙∙F 1 2.545 2.545

H∙∙∙F 1 2.627 2.627

F∙∙∙H 1 2.692 2.692

F∙∙∙H 1 2.734 2.734

C∙∙∙C 4 3.381 3.364

C∙∙∙C 4 3.335 3.323

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7.11 Crystal parameters of 10,11,12,13-tetrafluorophenathro[4,5-abc]phenazine

(7.16)

-1 Crystal data for 12: C22 H8 F4N2, MW = 376.30 g mol , crystal dimensions 0.35 x 0.10 x

0.05 mm, monoclinic, space group P21/n, a = 11.44 (3) Å, b = 8.139 (2) Å, c = 16.711 (5)

3 -3 Å, β = 105.019(6)º, V = 1502.7(8) Å , Z = 4, ρcalc = 1.663 mg cm , Bruker SMART

APEX II diffractometer, 1.95º < θ < 25.04º, Mo(Kα) radiation (λ = 0.71073 Å), ω scans,

T = 160 (2) K; of 11623 measured reflections 2663 were independent with I > 2σ (I), -13

< = h < = 13, - 9 < = k < = 9, - 19 < = l < = 19; R1 = 0.0514, wR2 = 0.1187, GOF = 0.870

-3 for 254 parameters, Δρmax = -0.251 e. Å . The structure was solved by direct methods

(SHELXS-97) and refined by full matrix least-squares procedures (SHELXL-97),

Lorentzian and polarization corrections and absorption correction SADABS were applied, µ = 0.134 mm-1.

7.12 10,11,12,13-tetrafluorophenanthro[4,5-abc]phenazine-4,5-dione

We attempted to synthesize another highly conjugated phenanthrophenazine derivative 7.19 by a condensation reaction between perfluoro-1,2-phenylenediamine and

7.17.

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7.14 7.17 7.18 7.19

Scheme 7.7 Synthesis of 10,11,12,13-tetrafluorophenanthro[4,5-abc]phenazine-4,5-dione (a) RuCl3.2H2O (20 mol%), 8 equiv NaIO4, DCM, MeCN, H2O, rt (b) glacial acetic acid, 120°C

The intermediate required in this reaction can be prepared in the same way as we prepared pyrene-4,5-dione. We used RuCl3.2H2O ~ 20 mol% and 8 equivalents of NaIO4.

A reaction of 7.17 with perfluorophenylene-1,2-diamine afforded 10,11,12,13- tetrafluorophenanthro[4,5-abc]phenazine-4,5-dione (7.18) in good yield but there was a problem as two products were produced and their separation was very difficult due to solubility. Without purification we decided to make 7.18 that we also faced a severe problem as the crude intermediate 7.18 is very insoluble in all the common polar solvents. So, we could not perform the next step to 7.19.

7.13 Concluding remarks

Synthesis of the desired compounds such as 7.3, 7.8, 7.9, 7.10, 7.16, and 7.18 were successful. We wanted to see the effect of fluorine substitution on the crystal packing in molecules where nitrogen is also present in place of one or more carbon atoms. The presence of C=N in the aromatic system makes the conjugated system even

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more electron deficient which is reinforced by the presence of fluorine atoms. We expected the overall net effect of this kind of system in the close packing of molecules

(hopefully in co-facial packing), but unfortunately we could not get the X-ray crystallographic structure of several molecules due to the difficulties with solvent based recrystallization. The vacuum transport crystallization was also tried in some instances but it also did not work. We were successful in getting a crystal structure of tribenzo[a,b,c]-2,3,4,5-tetrafluorophenazine (7.16). In the case of crystal 7.16, we expected the molecules would pack in parallel face to face orientation but it packed in similar to herringbone pattern. Coplanar orientation was achieved withing a given column, but in the bulk the molecules are packed in two sets of columns.

CHAPTER 8

Experimental

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CHAPTER 8. Experimental

The characterization of the all the synthesized materials was carried out in 1H (400 MHz),

13C (100 MHz), 19F (376.5 MHz), and 39P (162 MHz) NMR (Bruker Avance 400 MHz spectrometer using Topspin version 2.1 software) in CDCl3, CD3OH, D2O or DMSO-d6

19 with tetramethylsilane as internal standard. CFCl3 was used as internal standard for F

NMR. EI-MS was obtained at 70 eV using a Finnigan Polaris ion trap MS coupled with a

Trace GC instrument. Differential scanning calorimetry (DSC) measurements were performed using a TA Instruments Differential Scanning Calorimeter 2920 at heating and cooling rates of 5-10°C per minute (unless otherwise stated) with indium as the internal standard. Thin Layer Chromatography (TLC) was carried out using Silicycle brand plastic backed plates (250 μm thick layer of 60 Å silica gel with UV 254 and 354 nm fluorescence indicator). Column chromatography (flash) was carried out using Silicycle brand 60 Å, 40-63 μm particle size silica. THF, Et2O, benzene and toluene were dried by distilling over sodium benzophenone. Anhydrous DMF and DMSO were purchased from

Sigma Aldrich. Recrytallization was performed in the solvents available in the lab. All chemicals were used as received. UV Vis (Ultra Violet and Visible) spectra were recorded on Agilent/HP8453 diode array spectrometer. Samples were prepared by dissolving in an appropriate solvent and measurements were done in standard quartz cuvettes of 1.0 cm path-length. Melting point was obtained from Nikon eclipse E600

POL with temperature controller (Metter FP90). In many occasion microwave reactor

CEM (Discover) was used for better yield and short reaction time. In some occasion, the

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purity and molecular mass of the sample was checked by using GC-MS (Finnegan Trace

GC Ultra equipped with mass detector (Finnegan Polaris Q).

Experimental of Chapter 2

1H-isoindolo<1,2-a>benzimidazole-1-one (2.6)

A mixture of phthalic anhydride 1.48 g (10.0 mmol) and 1,2-phenylenediamine dissolved in NMP (4.0 mL) was placed in a long neck round bottom flask fitted with condenser.

The microwave was set to 300W of power, 100°C temperature, and the holding time was set to 30 minutes. During this set up the cooling was turned on simultaneously. The mode of the microwave setting was chosen to “open vessel” which applies for open vessel reaction with no application of pressure during reaction. After 30 minutes the reaction was stopped and cooled it to room temperature. Yellow needle shaped crystals were precipitated out from the solution at room temperature which was filtered. Yield: 1.48 g

1 (67%); mp 214.5°C (sharp); H NMR (400 MHz, CDCl3) δ 7.85 – 7.82 (m, 2H), 7.78 (d,

J = 7.36 Hz, 1H), 7.69 (d, J = 7.88 Hz, 1H), 7.64 (dt, J = 7.58, 0.96 Hz, 1H), 7.52 (dt, J =

7.62, 0.8 Hz, 1H), 7.33 (dt, J = 7.68, 1.24 Hz, 1H), 7.30 - 7.266 (m, 1H). 13C NMR (100

MHz, CDCl3) δ 160.96, 156.68, 149.16, 135.05, 134.77, 132.33, 131.65, 129.81, 126.47,

125.90, 125.11, 122.34, 121.29, 112.72.

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Tetra-N-ethyl-spiro[benzo[4,5]imidazo[2,1-a]isoindole-11,9'-xanthene]-3',6'-diamine (P-71)

A mixture of 1H-isoindolo<1,2-a>benzimidazole-1-one (P-71) (0.1 g, 0.45 mmol) and 3-

3-diethylamino phenol in 1,2-dichlorobenzene (3.0 mL) and boric acid (0.056 g, 0.9 mmol) was placed in microwave vial with a magnetic stirrer bar. The microwave reactor was set to 300W of power, 200°C temperature, pressure 250 psi and the holding time was set to 30 minutes. During this set up, the cooling was turned on simultaneously. The mode of the microwave setting was chosen to “discover” which applies for closed vessel reaction. After 30 minutes the reaction was stopped and cooled it to room temperature.

The reaction mixture was checked by TLC which indicated the presence of a halochromic compound with a single major spot with some other spots for other compounds. The mixture was separated by column chromatography using ethyl acetate:hexane (2:3);

1 0.075 g (32 %); mp 217°C (sharp); H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 7.56 Hz,

1H), 7.72 (d, J = 8.16 Hz, 1H), 7.41 (dt, J = 7.52, 0.94 Hz, 1H), 7.31 (dt, J = 7.54, 1.08

Hz, 1H), 7.15 (d, J = 7.72 Hz, 1H), 7.08 (dt, J = 8.04, 1.2 Hz, 1H), 6.94 (dt, J = 8.04, 3.24

Hz, 1H), 6.86 (d, J = 7.88 Hz, 1H), 6.41 (d, J = 2.56 Hz, 2H), 6.22 (d, J = 8.88 Hz, 2H),

6.08 (d, J = 8.92 Hz, 1H), 6.07 (d, J = 8.92 Hz, 1H), 3.23 (q, J = 7.08 Hz, 8H), 1.06 (t, J

13 = 7.06 Hz, 12H). C NMR (100 MHz, CDCl3) δ 153.05, 148.92, 134.32, 130.4, 128.60,

128,28, 124.84, 123.89, 122.51, 121.90, 121.35, 120.10, 110.20, 108.10, 106.25, 97.82,

44.32, 12.59.

266

8-methoxy-1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinoline or 8- methoxy julolidine (2.11)

A mixture of 3-methoxyaniline (6.0 mL, 6.72 g, 54.54 mmol), 1-bromo-3-chloropropane

(87.2 mL 128.1 g, 817.7 mmol) and anhydrous sodium carbonate (23.0 g, 218.8 mmol) was heated in a 100 mL flask at 145ºC and maintained the same temperature overnight.

The reaction mixture turned to pink after heating for 3h. The mixture was cooled to room temperature. Concentrated HCl (81.6 mL) and water (27.5 mL) was added slowly to the cooled solution. The entire solid did not dissolve so the solution was further treated with aqueous HCl (10%). The organic phase was separated and washed with aqueous HCl

(10%) to remove the product. This washing was added to the aqueous phase which was washed with ether to remove excess 1-bromo-3-chloropropane. The aqueous phase was made basic with 50% aqueous NaOH and extracted with ether until the organic phase was no longer colored. The ethereal solution was dried over MgSO4 and the solvent was removed under reduced pressure which afforded a dark orange liquid which was purified further by column chromatography using hexane:ethylacetate (19:1 ratio) (7.8 g, 65.2%);

1 H NMR (400 MHz, CDCl3,) δ 6.79-6.77 (m, 1H), 6.15 (d, J = 8.4 Hz, 1H), 3.79 (s, 1H),

3.14-3.09 (m, 4H), 2.74 (dd, J = 6.8 Hz, 6.4 Hz, 2H), 2.69 (dd, J = 6.8 Hz, 6.8 Hz, 2H),

2.02-1.97 (m, 4H), 7.26-7.22 (m 1H), 7.05 (d, J = 16.0 Hz, 1H), 1.45 (s, 6H); 13C NMR

(400 MHz, CDCl3) δ 155.8, 143.7, 126.3, 114.7, 109.7, 98.5, 27.2, 27.2, 22.4, 21.7, 21.2,

21.1, 21.1

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8-hydroxy-2,3,6,7-tetrahydro-1H,5H-benzo[ij]quinolizine or 8- hydroxy julolidine (2.12)

To a solution containing HI (55%, 28.4 mL), concentrated HCl (52.7 mL) and water

(132.0 mL) was added 8-methoxy julolidine (6.6 g, 33.0 mmol). The solution was heated at reflux (155-165ºC). After 15h more concentrated HCl (28.4 mL) was added. After 38h, the solution was cooled to room temperature and in an ice bath. The solution was made slightly basic by slow addition of saturated NaHCO3 solution. The organic compound was extracted in DCM. The organic phase was then dried over MgSO4 and the excess solvent was removed under reduced pressure. The desired product was obtained as creamy solid (3.352 g, 92.0%); mp 132.5 ºC (reported 132 ºC); 1H NMR (400 MHz,

CDCl3,) δ 6.65 (d, J = 8.0 Hz, 1H), 6.05 (d, J = 8.0 Hz, 1H), 3.11-3.07 (m, 4H), 2.72 (dd,

J = 8.0 Hz, 8.0 Hz, 2H), 2.68 (dd, J = 7.2 Hz, 6.8 Hz, 2H), 2.07-1.92 (m, 4H); 13C NMR

(400 MHz, CDCl3) δ 151.7, 143.9, 136.7, 114.3, 107.7, 102.8, 50.2, 49.5, 27.2, 22.3,

21.6, 20.9

Benzo[de]benzo[4,5]imidazo[2,1-α]isoquinolin-7-one (2.17)

A mixture of naphthalic anhydride 1.0 g (5.05 mmol) and 1,2-phenylenediamine (0.55g,

5.05) dissolved in NMP (5.0 mL) was placed in a long neck round bottom flask. The

268

microwave was set to 150W of power, 200°C temperature, pressure 250 psi, and the holding time was set to 30 minutes. During this set up the cooling was turned on simultaneously. The mode of the microwave setting was chosen to “discover” which applies for closed vessel reaction maintaining pressure during reaction. After 30 minutes the reaction was stopped and cooled it to room temperature. Yellow needle shaped crystals were precipitated out from the solution at room temperature which was filtered.

1 Yield: Quantitative; mp 214.5°C (sharp); H NMR (CDCl3, 400MHz) δ 8.87(d, J = 7.24

Hz 1H,), 8.82 (d, J = 6.84 Hz 1H), 8.61-8.55 (m, 1H), 8.30 (d, J=8.12 Hz 1H), 8.16(d,

1H, J=8.36Hz), 7.93-7.87(m, 1H), 7.38(dd, J=7.56Hz, 8.12Hz, 2H), 7.52-7.46(m, 2H).

13 C NMR (CDCl3, 100 MHz) δ 158.3, 149.43, 143.91, 137.03, 135.36, 132.29, 131.90,

131.71, 127.40, 127.16, 126.93, 125.81, 125.44, 123.23, 120.79, 119.98, 115.92, 109.67.

12H-isoindolo[2,1-a]perimidin-12-one (2.20)

A mixture of 1,8-naphthalenediamine (1.0 g, 6.33 mmol) and phthalic anhydride (0.94 g,

6.33 mmol) dissolved in NMP (10.0 mL) was placed in a long neck round bottom flask fitted with condenser. The microwave was set to 150W of power, 200°C temperature, and the holding time was set to 30 minutes. During this set up the cooling was turned on simultaneously. The mode of the microwave setting was chosen to “open vessel” which

269

applies a reaction without maintaining a pressure during reaction. After 30 minutes the reaction was stopped and cooled it to room temperature. A dark red solid was precipitated out. The chunk of red solid was crushed into fine power and dissolved in ethyl acetate, washed with water and dried under reduced pressure to afford brick red solid. Yield:

1 Quantitative; mp 223.0°C (sharp); H NMR (CDCl3, 400MHz): δ 8.52 (d, J = 7.24 Hz,

1H), 8.18 (d, J = 6.4Hz, 1H), 8.04 (d, J = 6.4Hz, 1H), 7.83-7.74 (m, 2H), 7.58 (dd, J =

13 7.2, 2.4Hz, 1H), 7.56-7.52 (m, 1H), 7.51-7.45 (m, 3H). ). C NMR (CDCl3, 100MHz) δ

164.63, 156.15, 143.43, 139.27, 134.10, 133.10, 132.60, 130.47, 128.06, 127.78, 125.65,

123.73, 122.97, 122.36, 122.05, 118.95, 109.66

General Procedure for synthesizing tetralkyl Rhodamine salts (2.22, 2.23)

R = CH3 (2.22) and CH2CH3 (2.23)

A mixture of 3-Dialkylaminophenol (11.24 mmol) and phthalic anhydride (1g, 7.29 mmol) were heated in a 50 mL round bottom flask with reflux condenser in an oil bath at

160ºC for six hours. The initial melt was mobile and was thoroughly mixed by hand- rotating the flask, and slowly become viscous. To the cooled mixture was added 3- dialkylaminophenol (11.24mmol) and 2.85mL of 85% phosphoric acid. The contents

270

were heated under reflux in an oil bath at 170ºC for three hours, and the flask was intermittently agitated for the first fifteen minutes to ensure even dispersal. To the still warm mixture were added a methanol solution of perchloric acid (2.85mL of 50% aqueous perchloric acid in 12 mL of methanol), and the flask was refluxed for 10 minutes and cooled to room temperature. The flask was stored overnight at 0ºC, and filtration gave 1.55g (25%) of green crystals. Recrystallization in methanol gave shining green crystals. Melting point >250oC for both rhodamine salts. );

9-(2-carboxyphenyl)-3,6-bis(dimethylamino)xanthylium perchlorate;

1 H NMR (CDCl3, 400MHz): δ 8.00-7.98 (m, 1H), 7.87 (t, J = 15.2, 6.8Hz, 1H), 7.8 (t, J =

15.2, 8.4Hz, 1H), 7.75 (d, J = 6 Hz, 1H), 7.09 (d, J = 2.4Hz, 2H), 7.04 (d, J = 9.6Hz, 2H),

13 6.96 (d, J = 2.4Hz, 2H), 3.27 (s, 12H); C NMR (100 MHz, CDCl3) δ 166.7, 157.98,

156.63, 154.52, 132.72, 131.99, 130.95, 130.32, 130.10, 130.04, 129.97, 114.10, 113.59,

99.32, 42.

9-(2-carboxyphenyl)-3,6-bis(diethylamino)xanthylium perchlorate

1 H NMR (CDCl3, 400MHz): δ 8.02-7.98 (m, 1H), 7.51-7.47 (m, 2H), 7.08-7.07 (m, 1H),

6.64 (d, J = 8.8Hz, 2H), 6.32 (dd, J = 8.8, 2.8 Hz, 2H),6.25 (d, J = 2.8 Hz, 2H), 3.31 (q, J

13 = 7.0 Hz, 8H), 1.16 (t, J = 6.8 Hz, 12H); C NMR (100 MHz, CDCl3) δ 167.7, 158.97,

157.83, 155.51, 133.88, 133.0, 131.95, 131.32, 130.32, 130.04, 129.97, 114.10, 113.59,

99.32, 46.03, 12.60.

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A representative procedure following Scheme 2.16 for the rhodamine lactam synthesis [preparation of 3',6'-bis(diethylamino)-2-(4-iodophenyl)spiro[isoindoline- 1,9'-xanthen]-3-one] P-91 as an example

To a stirred solution of rhodamine B base (3.0 g, 6.78 mmol) in dichloromethane (36 mL) were added toluenesulfonylchloride (2.21 g, 11.64 mmol), and DMAP (1.82 g, 15.83 mmol) at room temperature. After stirring for 30 min, a solution of 4-iodoaniline (1.35 g,

6.17 mmol) in dichloromethane (36 mL) was added. The reaction mixture was stirred for three hours. The reaction mixture was monitored by TLC and a strong halochromic spot was appeared at the top of the TLC plate. The mixture was quenched with saturated aqueous sodium bicarbonate and the organic phase was extracted with ethylacetate. The organic solution was washed with saturated aqueous sodium bicarbonate, dried over anhydrous magnesium sulfate and evaporated. The crude reaction mixture was purified by column chromatography using a mixture of hexane:ethyl acetate (1:1) as eluent. The desired compound was obtained from the first fraction as a creamy creamy white solid

(2.76 g, 63%); mp 173-176ºC; FTIR 3016, 2975, 2935, 1700.20, 1615.33; 1120.31,

1 786.46; H NMR (400 MHz, CDCl3,) δ 8.02-7.97 (m, 1H), 7.53-7.46 (m, 2H), 7.45-7.41

(m, 2H), 7.17-7.12 (m, 1H), 6.66-6.57 (m, 4H), 6.32-6.27 (m, 4H), 3.29 (q, J = 7.2 Hz,

13 8H), 1.18 (t, J = 7.2 Hz, 12H); C NMR (100 MHz, CDCl3) δ 167.7, 153.3, 153.0, 148.8,

137.6, 136.7, 133.0, 130.4, 128.65, 128.6, 128.1, 123.9, 123.3, 108.1, 106.05, 97.8, 91.7,

67.3, 44.3, 12.5.

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3',6'-bis(diethylamino)-2-phenylspiro[isoindoline-1,9'-xanthen]-3-one (P-80)

Yield (198 mg, 49%): mp 207ºC; FTIR 3010, 2972, 2930, 2250, 1697, 1614, 1546, 1511,

-1 1 1429, 1401, 1329 cm ; H NMR (400 MHz, CDCl3) δ 8.0-7.97 (m, 1H), 7.52-7.44 (m,

2H), 7.43-7.39 (m, 2H ), 7.26-7.23 (m, 2H), 7.12-7.09 (m, 1H), 6.57 (s, 1H), 6.55 (s, 1H),

6.32 (s, 1H), 6.31 (s, 1H), 6.27 (d, J = 2.8 Hz, 1H), 6.25 (d, J = 2.8 Hz, 1H), 3.31 (q, J =

13 7.2 Hz, 8H), 1.15 (t, J = 6.8 Hz, 12H); C NMR (100 MHz, CDCl3) δ 168.3, 153.7,

148.9, 141.6, 133.7, 132.5, 129.3, 128.24, 125.2, 123.8, 123.5, 118.8, 108.3, 105.8, 97.8,

45.7, 44.3, 12.5, 8.6; UV-Vis (DCM) λmax (lg ε) 240 (1.63), 280 (1.22), 320 (0.45) and cut off at 420 nm

3',6'-bis(diethylamino)-2-phenylspiro[isoindoline-1,9'-xanthen]-3-one (P-81)

Yield (127 mg, 68%): mp 197.5ºC; FTIR 3020.7, 2970.9, 1689.91, 1616.79 cm-1, 1H

NMR (400 MHz, CDCl3,) δ 8.07-8.04 (m, 1H), 7.72-7.68 (m, 1H), 7.61 (d, J = 8.88 Hz,

1H ), 7.57-7.50 (m, 3H), 7.40-7.33 (m, 2H), 7.26 (d, J = 2.0 Hz, 1H), 7.25-7.19(m, 1H),

273

7.04 (dd, J = 8.88, 2.0 Hz, 1H), 6.74 (s, 1H), 6.72 (s, 1H), 6.36 (d, J = 2.4 Hz, 1H), 6.34

(d, J = 2.8 Hz, 1H), 6.25 (s, 1H), 6.24 (s, 1H), 3.33 (qd, J = 8.72, 1.92 Hz, 8H), 1.16 (t, J

13 = 7.0 Hz, 12H); C NMR (100 MHz, CDCl3) δ 167.8, 153.4, 153.1, 148.7, 134.4, 133.4,

132.9, 131.9, 131.0, 128.8, 128.1, 127.3, 125.6, 125.5, 124.0, 123.3, 108.2, 106.4, 97.8,

67.6, 44.3, 12.5; UV-Vis (DCM) λmax (lg ε) 241 (1.78), 278 (1.13), 320 (0.43) and cut off at 350 nm

3',6'-bis(diethylamino)-2-phenylspiro[isoindoline-1,9'-xanthen]-3-one (P-82)

Yield 326 mg, 57%: mp 230ºC; FTIR 3012.7, 2975.8, 1691.62, 1613.18 cm-1; 1H NMR

(400 MHz, CDCl3,) δ 8.02-7.98 (m, 1H), 7.51-7.48 (m, 2H), 7.17-7.08 (m, 4H ), 6.80 (d,

J = 2.0 Hz, 1H), 6.7 (d, J = 1.2 Hz, 1H), 6.64 (s, 1H), 6.62 (s, 1H), 6.32 (d, J = 2.8 Hz,

1H), 6.29 (d, J = 2.8 Hz, 1H), 6.25 (s, 1H), 6.24 (s, 1H), 3.31 (qd, J = 7.0 Hz, J = 1.6 Hz,

13 8H), 1.14 (t, J = 7.2 Hz, 12H); C NMR (400 MHz, CDCl3) δ 167.7, 153.2, 153.1, 148.7,

136.6, 132.8, 131.0, 128.8, 128.5, 128.1, 127.3, 126.6, 124.0, 123.3, 108.1, 106.4, 97.7,

44.3, 12.5. UV-Vis (DCM) λmax (lg ε) 241 (1.78), 278 (1.13), 320 (0.43) and cut off at

350 nm

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3',6'-bis(diethylamino)-2-(4-((4-nitrophenyl)ethynyl)phenyl)spiro[isoindoline-1,9'- xanthen]-3-one (P-84)

Yield (408 mg, 55%): mp 238ºC; FTIR 3021.7, 2974.14, 2212.21, 1692.78, 1634.2,

1510.47 cm-1; 1H NMR (400 MHz, DMSO) δ 8.26-8.22 (m, 2H), 7.92-7.89 (m, 1H),

7.75-7.72 (m, 2H), 7.6-7.52 (m, 2H), 7.43-7.39 (m, 2H), 7.08-7.04 (m, 3H), 6.56 (s, 1H),

6.54 (s, 1H), 6.37 (d, J = 2.0 Hz, 1H), 6.35 (d, J = 2.8 Hz, 1H), 6.33 (s, 1H), 6.32 (s, 1H),

13 3.29 (q, J = 7.2 Hz, 8H); 1.06 (t, J = 6.8 Hz, 12H), C NMR (100 MHz, CDCl3) δ 167.5,

153.8, 152.7, 149.2, 147.4, 138.8, 134.0, 132.8, 132.2, 129.7, 129.6, 128.9, 128.5, 125.6,

124.2, 124.1, 123.4, 119.0, 109.0, 106.4, 98.2, 94.4, 88.4, 67.2, 44.1, 12.8; UV-Vis

(DCM) λmax (lg ε) 240 (1.83), 279 (1.36), 350 (0.86) and cut off at 430 nm

2-(4-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2- yl)benzylidene)malononitrile (P-85)

Yield 27.3 mg, (4%): mp 123-130ºC; FTIR: 3023.5, 2986.7, 2945.9, 2240.1, 1725.5,

-1 1 1670, 1501.7, 1182.2 cm ; H NMR (400 MHz, CDCl3,) δ 7.99-7.96 (m, 1H), 7.72-7.68

275

(m, 2H), 7.55-7.43 (m, 5H), 7.09-7.06 (m, 1H), 6.57(s, 1H), 6.55 (s, 1H), 6.36 (s, 1H),

6.35 (s, 1H), 6.26 (s, 1H), 6.25 (s, 1H), 6.25 (d, J = 2.6 Hz, 1H), 6.23 (d, J = 2.84 Hz,

1H), 3.34 (q, J = 6.8 Hz, 8H); 1.17 (t, J = 7.2 Hz, 12H); UV-Vis (DCM) λmax (lg ε) 240

(1.5), 279 (0.84), 326 (0.6), 370 (0.63) and cut off at 450 nm

2-(4-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2- yl)benzylidene)malononitrile (P-86)

Yield (0.4 g, 58 %): mp 168ºC (sharp); FTIR 3012, 2975, 2935, 1687.31, 1613.09;

-1 1 1116.61, 784.45 cm ; H NMR (400 MHz, CDCl3,) δ 8.1-7.97 (m, 1H), 7.53-7.46 (m,

2H), 7.27-7.22 (m, 2H), 7.17-7.12 (m, 1H), 6.76-6.71 (m, 2H ), 6.60 (s, 1H), 6.58 (s, 1H),

6.31-6.26 (m, 4H), 3.31 (q, J = 7.2Hz, 8H), 1.15 (t, J = 8Hz, 12H); 13C NMR (400 MHz,

CDCl3) δ 167.9, 153.2, 153.2, 148.9, 135.1, 133.0, 131.9, 130.5, 128.7, 128.6, 128.2,

124.1, 123.3, 120.02, 108.1, 106.0, 97.2, 67.54, 45.6, 12.5; UV-Vis (DCM) λmax (lg ε)

240 (1.78), 278 (1.13), 320 (0.43) and cut off at 345 nm

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2-butyl-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one (P-88)

Yield (0.32 g, 57%): mp 168ºC (sharp); FTIR 3012, 2975, 2935, 1687.31, 1613.09;

-1 1 1116.61, 784.45 cm ; H NMR (400 MHz, CDCl3,) δ 7.93-7.89 (m, 1H), 7.47-7.42 (m,

2H), 7.1-7.09 (m, 1H), 6.46 (s, 1H), 6.44 (s, 1H ), 6.4 (s, 1H), 6.4 (s, 1H),6.3 (d, J = 2.4

Hz, 1H), 6.3-6.27 (d, J = 2.4Hz, 1H), 3.31 (q, J = 7.2Hz, 8H), 1.15 (t, J = 7.2 Hz, 12H);

13 C NMR (100 MHz, CDCl3) δ 168.1, 153.5, 153.0, 148.9, 132.1, 131.9, 129.0, 128.0,

124.9, 123.9, 108.0, 106.0, 98.0, 66.0, 44.5, 40.1, 30.1, 20.5, 13.5, 12.5; UV-Vis (DCM)

λmax (lg ε) 240 (1.9), 275 (1.1), 315 (0.43) and cut off at 345 nm

trans-4-Amino--stilbazole (2.40)

In a 100 mL round bottom flask, equipped with a stir bar stirrer, condenser, oil bath, nitrogen was placed 4-iodoaniline (2.19 g, 10 mmol), vinyl pyridine (1.07 mL, 10 mmol),

DMF (50 mL), TDA-1 (0.3 mL) and potassium carbonate (1.4 g). The reaction mixture was heated to 80°C and palladium acetate (22.5 mg) was added all at once. The reaction was then heated to 110°C and monitored by TLC. The reaction was completed in 1 hour

277

and to make sure the reaction was further heated half an hour more. When water was added to the mixture, a dense light greenish precipitate was appeared. It was filtered to obtain a product of aminostilbazole (1.6 g, 82%): mp >250°C. 1H NMR (400 MHz,

DMSO) δ 8.47 (dd, J = 4.6 Hz, 1.6 Hz, 2H), 7.44 (dd, J = 4.6 Hz, 1.2 Hz, 2H), 7.35 (d, J

= 16.48 Hz, 1H), 7.33 (d, J = 8.44 Hz, 2H), 6.86 (d, J = 16.4 Hz, 1H), 6.59 (s, 1H), 6.56

(s, 1H), 5.5 (s, 2H). 13C NMR (100 MHz, DMSO) δ 150.2, 145.7, 134.3, 128.9, 124.0,

120.6, 120.2, 114.

(E)-3',6'-bis(diethylamino)-2-(4-(2-(pyridin-4-yl)vinyl)phenyl)spiro[isoindoline-1,9'- xanthen]-3-one (P-89) by Jeffrey Heck reaction:

In a 100 mL round bottom flask, equipped with stir bar stirrer, condenser, oil bath, nitrogen was placed rhodamine B lactam of 4-iodoaniline P-91 (0.2527 g, 0.39 mmol), vinyl pyridine (0.042 mL, 0.39 mmol), DMF (16 mL), TDA-1 (0.01 mL) and potassium carbonate (0.05 g, 0.39 mmol). The reaction mixture was heated to 80ºC and palladium acetate (0.88 mg) was added all at once. The reaction was then heated to 110ºC and monitored by TLC. The reaction was completed in 1 hour and to make sure the reaction was complete it was heated half an hour more. The reaction mixture was cooled to room temperature. Water was added to this cooled mixture and a dense creamy white precipitate appeared. It was filtered and dissolved in ethyl acetate and dried over

278

magnesium sulfate. The solvent was removed by rotary evaporation. The product was stuck on the wall of flask. It was scraped off and collected by using hexane and filtered to

º 1 obtain a creamy white mass (0.21 g, 86%). mp 153-158 C; H NMR (400 MHz, CDCl3,) δ

8.54 (d, J = 6.0 Hz, 2H), 8.03-8.01 (m, 1H), 7.52-7.50 (m, 2H), 7.31-7.265 (m, 4H), 7.17-

7.13 (m, 2H), 6.98 (s, 1H), 6.96 (s, 1H), 6.86 (d, J = 16.4, 1H), 6.66 (s, 1H); 6.64 (s, 1H),

13 6.33 (d, J = 2.4, 1H), 6.32-6.31 (m, 3H). C NMR (100 MHz, CDCl3) δ 167.8, 153.5,

152.9, 150.0, 148.8, 144.6, 137.5, 133.8, 133.0, 1332.7, 130.4, 128.7, 128.1, 127.2, 126.6,

125.6, 123.9, 123.3, 120.7, 108.1, 106.3, 97.8, 67.4, 44.3, 12.5.

3',6'-bis(diethylamino)-2-(4'-iodobiphenyl-4-yl)spiro[isoindoline-1,9'-xanthen]-3-one (P-92)

Yield: Creamy white solid (0.35g, 43%): mp 144-148oC (wide); 1H NMR (400 MHz,

CDCl3,) δ 8.05-8.02 (m, 1H), 7.74-7.67 (m, 2H), 7.55-7.48 (m, 2H), 7.38-7.34 (m, 1H),

7.32-7.29 (m, 1H ), 7.27-7.25 (m, 1H), 7.23-7.20 (m, 1H), 7.18-7.55(m, 1H), 6.97-6.658

(m, 2H), 6.69 (s, 1H), 6.65 (s, 1H), 6.34 (d, J = 2.8 Hz, 1H), 6.32 (d, J = 2.8 Hz, 1H), 6.3

(s, 1H), 6.29 (s, 1H), 3.34 (q, J = 7.2 Hz, 8H), 1.16 (t, J = 7.2 Hz, 12H); 13C NMR (400

MHz, CDCl3) δ 167.9, 153.5, 153.0,148.7, 140.0, 137.6, 136.9, 132.9, 130.6, 128.8,

128.7, 128.1, 127.1, 126.9, 123.9, 123.3, 108.1, 106.2, 97.7,92.8, 44.3, 12.2.

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(E)-4-(4-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2-yl)styryl)- 1-methylpyridinium iodide (P-93)

Rhodamine B lactam of trans-4-amino-stilbazol (0.1g, 0.16 mmol) was dissolved in acetonitrile (2 mL). Methyl iodide (0.02 mL, 0.32 mmol) dissolved in acetonitrile (2 mL) was added to the rhodamine B lactam solution and refluxed under nitrogen for two hours.

The reaction was monitored by TLC. When TLC of the sample was taken, it showed the consumption of all starting material. The solvent and excess methyl iodide was removed under reduced pressure. A yellowish compound was strongly adhered on the wall of the flask. A little amount of dichloromethane was used to dissolve the compound and excess hexane was used to crash out the desired yellow compound (115 mg, 94%); mp >260°C.

1 H NMR (400 MHz, DMSO,) δ 8.81 (d, J = 6.88 Hz, 2H), 8.10 (d, J = 6.88 Hz, 2H),

7.91-7.81 (m, 2H), 7.62-7.53 (m, 4H), 7.41-7.39 (d, J = 16.4 Hz, 1H), 7.09-7.06 (m, 3H),

6.57 (s, 1H), 6.55 (s, 1H), 6.39 (d, J = 2.4, 2H); 6.39 (d, J = 2.4, 2H, 6.36 (d, J = 2.4, 2H),

6.32 (d, J = 2.8, 2H), 4.23 (s, 3H), 3.31 (q, J = 6.8, 8H), 1.07 (t, J = 6.8, 12H); 13C NMR

(100 MHz, DMSO) δ 167.4, 154.0, 152.7, 152.5, 148.8, 145.5, 140.1, 139.4, 134.1,

132.9, 129.5, 129.0, 128.7, 125.6, 124.2, 123.8, 123.7, 123.4, 108.7, 105.9, 97.6, 66.9,

47.3, 44.0, 12.8, UV-Vis (DCM) λmax (lg ε) 245 (1.7), 280 (0.89), 325 (0.4), 390 (0.7) and cut off at 495 nm

280

2-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2-yl)acetic acid (P- 100) [Following Scheme 2.16]

Yield: Creamy white (16.4 mg, 3%): mp 168ºC; FTIR: 3102, 2985, 2945, 1720.5, 1670,

-1 1 1501.7, 1182.2 cm , H NMR (400 MHz, CDCl3,) δ 7.88-8.87 (m, 1H), 7.42-7.41 (m,

2H), 7.06-7.04 (m, 1H), 6.73 s, 1H), 6.71 (s, 1H), 6.40-6.38 (m, 3H), 6.32 (d, J = 2.4 Hz,

1), 6.30 (d, J = 2.4 Hz, 1H), 3.33 (q, J = 7.2 Hz, 8H), 1.16 (t, J = 7.2 Hz, 12H); 13C NMR

(100 MHz, CDCl3) δ 169.8, 155.6, 152.3, 148, 132.9, 129.6, 127.9, 123.7, 123.1, 107.9,

107.1, 97.9, 59.9, 44.39, 12.6; UV-Vis (DCM) λmax (lg ε) 240 (1.36), 275 (0.73), 320

(0.28) and cut off at 430 nm

2-(4-(biphenyl-4-ylethynyl)phenyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'- xanthen]-3-one (P-101) by Sonogashira cross coupling reaction (P-101)

A mixture of 4-ethynylbiphenyl (0.29 mmol, 5.7 mg), rhodamine B lactam of 4- iodoaniline (31.6 mg, 0.24 mmol), Pd(OAc)2 (5.6 mg, 0.024 mmol), PPh3 (12.8 mg,

281

0.060 mmol), CuI (9.2 mg, 0.028 mmol) and anhydrous diethylamine (4 mL) were stirred under nitrogen for 6h. All the materials were dissolved in the solution to give a clear solution but after one and half hours later, light yellowish precipitate was observed. A small portion of ppt was dissolved in methanol and took its TLC, which showed two spots, a very light blue fluorescence of unreacted ethynylbiphenyl was observed and a big spot of lactam was observed which was halochromic. The reaction was stopped, cooled and filtered. The residue was dissolved in excess of ethylacetate and washed with water.

The organic phase was dried by magnesium sulphate. The two compounds were separated in column using hexane:ethylacetate (1:1) mixture. A yellowish solid was obtained as second fraction (136 mg, 79%); mp >250°C; FTIR: 3050, 2960, 2940,

-1 1 1692.6, 1614.5, 1511.3, 1371.7, 1217.8, 1118.1, 813.9 cm ; H NMR (400 MHz, CDCl3,)

δ 7.91-7.89 (m, 1H), 7.73-7.694 (m, 4H), 7.58-7.55 (m, 4H), 7.50-7.46 (m, 2H), 7.40-

7.37 (m, 3H ), 7.07 (d, J = 7.2 Hz, 1H), 7.01 (s, 1H), 6.99 (s, 1H), 6.57 (s, 1H), 6.55(s,

1H), 6.39 (d, J = 2.4 Hz, 1), 6.37 (d, J = 2.4 Hz, 1H ), 6.33 (s, 1H), 6.32 (s, 1H), 3.32 (q,

13 J = 7.6 Hz, 8H), 1.07 (t, J = 7.2 Hz, 12H); C NMR (100 MHz, CDCl3) δ 167.4, 154.0,

152.6, 148.8, 140.6, 139.5, 137.9, 134.1, 133.5, 132.3, 132.0, 129.5, 129.4, 129.0, 128.7,

128.4, 127.5, 127.3, 127.2, 127.1, 125.7, 124.2, 123.4, 120.0, 108.6, 105.8, 97.6, 90.0,

44.0, 12.8; UV-Vis (DCM) λmax (lg ε) 231 (1.2), 275 (0.88), 320 (0.81) and cut off at 375 nm

282

4-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2-yl)benzoic acid (P-102) [Following Scheme 2.16]

Yield: Light yellow solid (0.22g, 31%): mp 152-158ºC (wide); FTIR 3025, 2966, 2945,

1 1694.67, 1599.77; 1510.54, 1111.1, 824.42; H NMR (400 MHz, CDCl3,) δ 8.53-8.52 (d,

J = 2.4 Hz, 1H), 8.51-8.15 (d, J = 1.2 Hz 1H), 8.03-7.81 (m, 1H), 7.5-7.478 (m, 2H ), 7.3-

7.26.5 (m, 4H), 7.16-7.12 (m, 2H), 6.95 (s, 1H), 6.93 (s, 1H), 6.85 ( d, J = 16.4 Hz, 1H),

6.64 (s, 1H), 6.62 (s, 1H), 6.3-6.28 (m, 4H), 3.32 (q, J = 7.2 Hz, 8H), 1.17 (t, J = 7.2 Hz,

13 12H); C NMR (400 MHz, CDCl3) δ 189.1, 167.2, 153.5, 153.0, 150, 148.8, 148.5,

144.1, 137.6, 134.8, 133.1, 132.7, 130.5, 128.3, 127.1, 126.8, 125.6, 124.2, 123.2, 108,

105.3, 96, 44.5, 12.5.

4-Iodomethyl benzoate (2.43)

The 4-iodobenzoic acid (4.2 g, 17 mmol) was treated with a solution of concentrated sulfuric acid (0.51 mL, 30L/mmol) in freshly distilled anhydrous methanol (34 mL, 2 mL/mmol) and refluxed for one hour. The solution was cooled to room temperature and concentrated in vacuo. Diethyl ether was added and the organic layer was washed with

283

5% aqueous solution of NaHCO3 followed by brine, dried over MgSO4 and concentrated in vacuo to afford white crystals of methyl-4-iodobenzoate (3.62 g, 82%): mp 114.5°C;

1 H NMR (400 MHz, CDCl3,) δ 7.80 (d. J = 8.40 Hz, 2H), 7.73 (d. J = 8.48 Hz, 2H), 3.91

13 (s, 3H); C NMR (100 MHz, CDCl3) δ 166.5, 137.7, 131.0, 129.5, 100.7, 52.3.

Methyl 4-((trimethylsilyl)ethynyl)benzoate (2.44)

A solution of methyl 4-iodobenzoate (2.0 g, 7.63 mmol), triphenylphosphine (50 mg, 0.2 mmol) in diisopropylamine (7.0 mL) was heated at 30 oC under nitrogen. To the clear solution were added catalyst PdCl2 (7.04 mg, 0.04 mmol), Cu(OAc)2•H2O (7.03 mg, 0.04 mmol). The solution was degassed and then to this solution was added TMS acetylene

(0.88 g, 8.96 mmol) slowly by a syringe. The reaction mixture was gradually heated with an oil bath (80°C) for 2 h. filtrate was evaporated to dryness and treated with concentrated

HCl (0.3 mL) and crushed ice. Organic material was extracted with hexane (2 x 50 mL) and the organic phase was washed with water (100 mL) dried over MgSO4, filtered and evaporated. The mixture was separated by column chromatography using hexane: ethylacetate (4:1). The desired compound was obtained in first fraction as a yellowish

1 solid (1.38 g, 78%); mp 50 - 58°C; H NMR (100 MHz, CDCl3,) δ 7.96 (d, J = 8.8 Hz,

284

13 2H), 7.51 (d, J = 8.8 Hz, 2H), 3.90 (s, 3H), 0.25 (s, 9H); C NMR (400 MHz, CDCl3) δ

166.5, 137.7, 131.8, 131.0, 129.6, 129.3, 127.7, 104.0, 97.6, 52.2, -0.16

Methyl 4-ethynylbenzoate (2.45)

To a solution of methyl 4-((trimethylsilyl)ethynyl)benzoate (0.96g, 4.13 mmol) in anhydrous THF (6 mL) was added a 1.0 M solution of n-Bu4NF in THF (4.2 mL, 4.2 mmol) with stirring at -20°C. The solution was stirred for 30 min at -20°C and then it was diluted with water (100 mL) and extracted with ether (2 x 50 mL). The combined extracts were washed with brine (2 x 30 mL), dried over anhydrous MgSO4 and evaporated. The residue was purified by chromatography with hexane:ether (10:1) as eluent to afford

° 1 yellowish solid (0.52 g, 74%); mp 89 C (sharp); H NMR (400 MHz, CDCl3,) δ 8.00-7.98

13 (m, 2H), 7.56-7.53 (m, 2H), 3.92 (s, 3H), 3.22 (s, 1H); C NMR (100 MHz, CDCl3) δ

166.4, 137.7, 132.0, 131.0, 129.4, 82.8, 80.0, 52.3.

285

Methyl 4-((4-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2- yl)phenyl)ethynyl)benzoate (P-109)

A solution of rhodamin B lactam of 4-iodoaniline (1.72 g, 2.67 mmol), triphenylphosphine (17.7 mg, 0.07 mmol) in diisopropylamine (3.0 mL) was heated at

° 30 C under nitrogen. To the clear solution were added catalyst PdCl2 (2.5 mg, 0.01 mmol), Cu(OAc)2·H2O (2.5 mg, 0.01 mmol). The mixture was further heated to 60°C for half an hour. The solution was degassed and then 2-(4-(methoxycarbonyl)phenyl)ethyn-

1-ylium (0.5 g, 3.07 mmol) was added to the solution which was gradually heated with an oil bath (80°C) for 2 h. A grayish salt was formed and after 2 h, the solution was cooled to room temperature, filtered off and washed well with hexane. The filtrate was evaporated to dryness and treated with concentrated HCl (0.3 mL) and crushed ice (5 g).

The organic material was extracted with hexane (2 x 50 mL) and the organic phase was washed with water (100 mL) dried over MgSO4, filtered and evaporated. The mixture was separated by column chromatography using hexane: ethylacetate (4:1). The desired compound was obtained in first fraction as a yellowish solid (0.4 g, 22%); mp 244°-

247°C; FTIR :2986.2, 2796.2, 1689.17, 1613.3, 1513.5, 1488.3, 1329.1, 1305.7, 1269.8,

-1 1 1116.57 cm , H NMR (400 MHz, CDCl3,) δ 8.00-7.96 (m, 3H), 7.51-7.47 (m, 4H), 7.31-

7.29 (m, 2H), 7.14-7.12 (m, 1H), 6.95-6.93 (m, 2H), 6.62-6.60 (m, 2H), 6.30-6.28 (m,

4H), 3.9 (s, 3H), 3.31 (q, J = 7.2 Hz, 8H), 1.15 (t, J = 7.2 Hz, 12H); 13C NMR (100 MHz,

286

CDCl3) δ 167.4, 166.5, 156.2, 155.4, 152.7, 150.2, 147.2, 138.9, 134.1, 132.5, 132.2,

129.9, 128.8, 129.6, 129.5, 128.6, 125.4, 124.2, 124.0, 123.4, 119.0, 109.1, 106.4, 94.4,

88.4, 54.5, 44.1, 12.8; UV-Vis (DCM) λmax (lg ε) 234 (1.12), 279 (0.83), 326 (0.83), and cut off at 385 nm

3’,6’-bis(diethylamino)-2-(4-((trimethylsilyl)ethynyl)phenyl)spiro[isoindoline -1,9’- xanthen]-3-one (P-114) by Sonogashira cross coupling reaction (P-114)

To a mixture of rhodamine B lactam of 4-iodoaniline [3’,6’-bis(diethylamino)-2-(4- iodophenyl)spiro[isoindoline-1,9’-xanthen] -3-one] (1.32 g, 2.06 mmol), Pd(PPh3)2Cl2

(36.73 mg, 0.52 mg) and CuI (32.14 mg, 0.16 mmol) in THF (15.0 mL) was added a mixture of N,N-diisopropylethylamine (2.05 mL) and trimethylsilylacetylene (0.63 mL,

4.36 mmol) and the mixture was stirred overnight at room temperature. The reaction was checked by TLC which indicated the absence of starting materials and presence of single spot suggested that there were no other side products. The mixture was filtered and the filtrate was evaporated to dryness. The residue was dissolved in ethyl acetate. It was washed with water and the organic phase was then washed with brine and dried over

MgSO4 and concentrated in rotavap. The compound was crashed out in hexane and filtered. The filtrate was yellowish liquid and the desired product was obtained as a

287

grayish white solid: (1.24 g, 98%), FTIR: 2970.97, 2158.14, 1694.49, 1615.07, 1549.14,

-1 1 1015.33, 865.02, 820.00 cm ; H NMR (400MHz, CDCl3,) δ 7.99-7.97 (m, 1H), 7.49-

7.46 (m, 2H), 7.22-7.20 (m, 2H), 7.13-7.11 (m, 1H), 6.84-6.82 (m, 2H), 6.59 (s, 1H), 6.57

(s, 1H), 6.28 (d, J = 2.8 Hz, 1H), 6.26-6.25 (m, 3H), 3.31 (q, J = 7.2 Hz, 8H), 1.14 (t, J =

13 6.8 Hz, 12H), 0.191 (s, 9H); C NMR (100MHz, CDCl3) δ 167.7, 153.4, 152.9, 148.7,

137.2, 133.0, 132.2, 130.5, 128.6, 128.1, 126.2, 123.9, 123.3, 120.6, 108.1, 106.1, 104.9,

97.7, 94.1, 44.3, 12.5, 0.07.

3',6'-bis(diethylamino)-2-(4-ethynylphenyl)spiro[isoindoline-1,9'-xanthen]-3-one (P- 121)

To a solution of Rhodamine B lactam of 4-((trimethylsilyl)ethynyl)aniline (0.85 g, 1.38 mmol) in anhydrous THF (2.0 mL) was added a 1.0 M solution of n-Bu4NF in THF (1.4 mL, 1.4 mmol) with stirring at 0°C. The solution was stirred for 1 h at 0°C and then it was diluted with water (20 mL) and extracted with ethyl acetate (2 x 50 mL). The combined extracts were washed with brine (2 x 30 mL), dried over anhydrous MgSO4 and evaporated. The residue was purified by chromatography with ethylacetate:hexane (1:1) as eluent to afford light gray solid (0.52 g, 69 %); mp 181.7 oC (sharp); FTIR: 3249.52,

2967.07, 1693.84, 1633.85, 1616.93, 1548.93, 1346.78, 1015.77, 787.63 cm-1; 1H NMR

(400 MHz, CDCl3,) δ 8.00-7.97 (m, 1H), 7.49-7.47 (m, 2H), 7.24-7.23 (m, 2H), 7.14-7.11

288

(m,1H), 6.89-6.86 (m, 2H), 6.61-6.58 (m, 2H), 6.29-6.29 (m, 1H), 6.26-6.27 (m, 3H),

3.31(q, J = 7.2 Hz, 8H), 2.98 (s, 1H), 1.49 (t, J = 6.8 Hz, 12H); 13C NMR (100 MHz,

CDCl3) δ 167.8, 153.4, 152.9, 148.8, 137.5, 133.0, 132.4, 130.4, 128.6, 128.1, 126.2,

123.9, 123.4, 119.6, 108.1, 106.1, 97.7, 83.5, 67.4, 44.3, 12.5

3',6'-bis(diethylamino)-2-(pyridin-4-yl)spiro[isoindoline-1,9'-xanthen]-3-one (P-135) [Following Scheme 2.16]

Yield: Creamy white solid; (0.81 mg, 72%): mp 222 ºC (sharp); FTIR 3025, 2970.9,

2885.0, 2745.1, 1702.7, 1633.8, 1511.98, 1317.69 1115.47, 788.11 cm-1; 1H NMR (400

MHz, CDCl3,) δ 8.33 (d, J = 1.6 Hz, 2H), 8.31 (d, J = 1.6 Hz 1H), 7.48-7.44 (m, 2H), 7.5-

7.42 (m, 2H), 7.35 (d, J = 1.56 Hz, 1H), 6.54 (d, J = 8.84 Hz, 2H), 6.37 (d, J = 2.6 Hz,

2H), 6.23 (dd, J = 8.88, 2.64 Hz, 2H), 3.31 (q, J = 7.08 Hz, 8H), 1.15 (t, J = 7.04 Hz,

13 12H; C NMR (400 MHz, CDCl3) δ 168.7, 154.2, 152.3, 150.1, 148.9, 144.9, 133.8,

128.4, 128.2, 127.9, 123.7, 123.5, 116.8, 108.3, 106.0, 97.9,67.5, 44.3, 12.5. 23

4-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2-yl)-1- methylpyridinium iodide (P-137)

289

Rhodamine B lactam of 4-aminopyridine (0.1g, 0.19 mmol) was dissolved in acetonitrile

(2 mL). Methyl iodide (0.02 mL, 0.32 mmol) dissolved in acetonitrile (2 mL) was added to the rhodamine B lactam solution and refluxed under nitrogen overnight. The reaction was monitored by TLC. In ethyl acetate the pink spot did not move at all indicating there was no starting material left. The solvent and excess methyl iodide was removed under reduced pressure. A yellowish adduct was strongly adhered on the wall of the flask. It was dissolved in a little amount of dichloromethane and excess hexane was used to precipitate it. The brick red adduct was filtered and dried under suction (0.12 g, 99%): mp

198-200 ºC; FTIR: 3426.8, 2968.7, 2929.5, 2868.9, 1720.6, 1610.5, 1511.1, 1320.7,

-1 1 1213.6, 1113.8 cm ; H NMR (400 MHz, CDCl3,) δ 8.70 (s, 1H), 8.68 (s, 1H), 8.26 (s,

1H ), 8.25 (s, 1H), 7.99 (d, J = 7.48 Hz, 1H), 7.56 (t, J = 6.52 Hz 1H), 7.48 (t, J = 8.6 Hz

, 1H), 7.07 (d, J = 7.68 Hz, 1H), 6.46 (s, 1H), 6.45 (s, 1H), 6.43 (s, 1H), 6.41 (s, 1H), 6.22

(d, J = 2.8 Hz, 1H); 6.2 (d, J = 2.8 Hz, 1H), 4.5 (s, 3H), 3.32 (q, J = 7.2 Hz, 8H) 1.16 (t, J

13 = 7.2 Hz, 12H); C NMR (400 MHz, CDCl3) δ 166.5, 152.0, 149.5, 145.4, 144.2, 141.9,

135.9, 133.58, 128.9, 126.9, 125.6, 124.2, 115.7, 108.7, 103.6, 98.2, 44.3, 12.5.

6-Nitro-2-phenyl-benzo[de]isoquinoline-1,3-dione (2.47)

To a solution of 4-nitro-1,8-naphthalic anhydride (0.5 g, 2.05 mmol) in 15 mL of glacial acetic acid was added aniline 0.19 mL (2.05 mmol). The solution was refluxed for 8 h.

290

and then cooled to room temperature. The product formed was filtered off washed with water and dried under vacuum overnight (0.45 g, 65%): mp 250ºC; 1H NMR (400 MHz,

CDCl3,) δ 9.27 (d, J = 2.4 Hz, 1H), 9.2 (d, J = 2.4 Hz, 1H), 8.26 (dd, J = 7.2 Hz, 1.2 Hz,

1H), 8.41 (dd, J = 8.4 Hz, 0.8 Hz, 1H), 7.91 (dd, J = 7.6 Hz, 7.2 Hz, 1H), 7.53-7.46 (m,

13 1H), 7.45-7.43 (m, 2H), 7.26-7.24 (m, 1H), 7.19 (s, 1H); C NMR (400 MHz, CDCl3) δ

160.1, 148.0, 141.1, 135.8, 134.8, 131.5, 131.2, 129.5, 129.2, 129.2, 129.1, 128.4, 124.8,

124.6.

6-amino-2-phenyl-benzo[de]isoquinoline-1,3-dione (2.48)

A solution of 6-nitro-2-phenyl-benzo[de]isoquinoline-1,3-dione (0.45 g, 1.42 mmol) was added in 10 mL glacial acetic acid and was dissolved by heating. A solution of stannous chloride (2.4 g, 12.77 mmol) was prepared by dissolving 4.5 mL of HCl (12N) in a separate beaker. The stannous chloride solution was added to the acetic acid solution of

6-nitro-2-phenyl-benzo[de]isoquinoline-1,3-dione and stirred vigorously at 118 ºC for overnight. The mixture was monitored with TLC. A big bright fluorescent spot was observed along with other two non-fluorescent spots. The mixture was neutralized with saturated NaHCO3. The organic phase was extracted with dichloromethane and dried over MgSO4. The excess solvent was removed under reduced pressure and the desired compound was separated by column chromatography. In the second fraction, a yellow

291

1 compound was obtained (0.21g, 52 %): mp >270 ºC; H NMR (400 MHz, CDCl3,) δ 8.35

(dd, J = 7.4, 1.2 Hz 1H), 8.08 (d, J = 2.0 Hz, 1H), 7.98 (dd, J = 8.4, 0.8 Hz, 1H), 7.64

(dd, J = 8.4, 7.2 Hz, 1H), 7.54 (m, 2H), 7.47 (m,1H), 7.34 (d, J = 2.4 Hz, 1H), 7.31 (m,

13 2H), 4.21 (s, 2H); C NMR (100 MHz, CDCl3) δ 145.4, 135.5, 133.6, 132.0, 129.4,

129.3, 128.8, 128.6, 127.8, 127.3, 123.7, 122.5, 114.3.

6-bromo-2-phenyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (2.50)

To a solution of 4-bromo-1,8-naphthalic anhydride (0.57 g, 2.06 mmol) in 20 mL of glacial acetic acid was added aniline 0.2 mL (2.06 mmol). The solution was refluxed for

8 h and then cooled to room temperature. The product was precipitated out at room temperature which was filtered off, washed with water and dried under vacuum overnight

1 (0.42 g, 59%): mp 231.5 ºC; H NMR (400 MHz, CDCl3,) δ 8.65 (d, J = 7.2 Hz, 1H), 8.64

(d, J = 8.4 Hz, 1H), 8.46 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 7.6 Hz, 1H), 7.89 (dd, J = 8.2,

7.46 Hz, 1H), 7.58-7.53 (m, 2H), 7.51-7.47 (m, 2H), 7.32-7.29 (m, 2H); 13C NMR (100

MHz, CDCl3) δ 163.7, 135.1, 133.6, 132.4, 131.6, 131.2, 130.8, 130.6, 129.4, 128.8,

128.5, 128.2, 123.2, 122.4.

292

3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one (P-142)

To a stirred solution of rhodamine B base (0.5 g, 1.13 mmol) in DCM (6 mL) were added toluenesulfonylchloride (0.23 g, 1.13 mmol), and DMAP (0.29 g, 1.23 mmol) at room temperature. After having been stirred for 30 min, a controlled supply of ammonia was arranged with the facility of trapping excess ammonia. The reaction mixture was stirred for four hours. The reaction mixture was monitored by TLC. A strong halochromic spot- typical to rhodamines was appeared at the top of the TLC plate and unreacted starting material rhodamine B was at the bottom. The mixture was quenched with saturated aqueous sodium bicarbonate and the organic phase was extracted with ethyl acetate. The organic solution was washed with saturated aqueous sodium bicarbonate and dried over anhydrous magnesium sulfate and the excess solvent was removed under reduced pressure. The crude reaction mixture was purified by a column chromatography by using a polar gradient from hexane:ethyl acetate (2:3 to pure ethyl acetate) as an eluent. The desired compound was obtained in third fraction as a creamy white solid (0.31 g, 65%): mp 225°C (sharp); FTIR 3208.48, 2971.22, 2933.46, 1692.85, 1583.8, 1116.12, 814.5 cm-

1 1 ; H NMR (400 MHz, CDCl3,) δ 8.53-8.52 (m, 1H), 8.51-8.15 (m, 2H), 8.03-7.81 (m,

1H), 6.72 (d, J = 7.2 Hz, 2H ), 6.40 (s, 1H), 6.38 (d, J = 2.6 Hz, 2H), 6.31 (dd, J = 8.86,

2.62 Hz, 2H), 3.33 (q, J = 7.02 Hz, 8H), 1.16 (t, J = 7.2 Hz, 12H); 13C NMR (100 MHz,

293

CDCl3) δ 169.7, 155.6, 152.3, 148.6, 132.9, 129.6, 128.3, 127.9, 123.7, 123.1, 108.0,

107.2, 97.9, 60.01, 44.3, 12.5.

6-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2-yl)-2-phenyl-1H- benzo[de]isoquinoline-1,3(2H)-dione (P-145) by Buchwald Hartwig reaction

A Schlenk tube was charged with CuI (9.6 mg, 0.05 mmol, 5 mol%) and K2CO3 (0.28 g,

1 2.03 mmol), evacuated and backfilled with N2. N -ethylethane-1,2-diamine (0.1 mmol,

11μL, 10 mol%), 6-bromo-2-phenyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (0.35 g,

1.01 mmol), 3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one (0.55 g, 1.24 mmol) and toluene (4 mL) were added under N2. The reaction mixture was heated at

80ºC for 24 h. The reaction was monitored by TLC. After 24 h, the mixture was checked again by TLC. It indicated the presence of starting material Rhodamine B P-142 so additional CuI (9.6 mg) and N1-ethylethane-1,2-diamine (11μL) was added and ran for another 12 h. The reaction did not show any change even after 12 h so it was stopped.

The solvent was removed under reduced pressure and the compound was separated in a column chromatography using DCM:EtOAc (1:1) as eluent to obtained desired yellow compound (0.243 g, 24%); mp >250 ºC; FTIR 2966.53, 1706.12, 1691.66, 1191.86 cm-1;

1 H NMR (400 MHz, CDCl3,) δ 8.46 (d, J = 7.2 Hz, 1H), 8.33 (d, J = 7.6 Hz, 1H), 8.10

294

(dd, J = 6.8 Hz, 1.2 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.69-7.65 (m, 2H), 7.55-7.51 (m,

2H), 7.47-7.38 (m, 3H), 7.28-7.27 (m, 2H), 6.75 (d, J = 9.2 Hz, 1H), 6.72 (d, J = 8.8 Hz,

1H), 6.62 (d, J = 7.6 Hz, 1H), 6.45 (dd, J = 8.8, 7.6 Hz, 2.4 Hz, 1H), 6.26 (d, J = 2.4 Hz,

1H), 6.23 (d, J = 2.4 Hz, 1H), 5.91 (d, J = 2.4 Hz, 1H), 3.37 (dq, J = 7.08, 3.72 Hz, 4H),

3.18 (q, J = 7.2 Hz, 4H), 1.21 (t, J = 6.8 Hz, 6H), 1.03 (t, J = 7.2 Hz, 6H); 13C NMR (100

MHz, CDCl3) δ 167.0, 164.3, 164.0, 154.4, 153.7, 151.2, 149.3, 148.9, 140.0, 135.4,

133.2, 132.1, 131.4, 129.9, 129.3, 129.1, 128.8, 128.6, 127.7, 126.1, 124.7, 123.8, 122.6,

122.2, 108.4, 107.7, 106.6, 105.9, 97.7, 97.5, 44.48, 44.35, 12.52, 12.35

2,5-dioxopyrrolidin-1-yl 3-iodopropanoate (2.51)

A solution of NHS hydroxide (0.6 g, 5.3 mmol), 3-iodopropanoic acid (1.9 g, 9.54 mmol), DCC (2.0 g, 9.7 mmol), DMAP (90 mg, cat. amount) in acetonitrile (90.0 mL) was stirred overnight at room temperature. The reaction mixture was monitored by TLC.

A distinct non-polar compound compared to the starting material was observed. The reaction mixture was filtered and the filtrate was concentrated under vacuum. The resulting yellow oil was subjected to column chromatography. The column was first washed down with pure hexane to remove non-polar impurities. The desired compound was obtained in the first fraction as a white crystalline solid (0.61 g, 22%); mp 95.3°C

295

1 (sharp). H NMR (400 MHz, CDCl3,) δ 3.38-3.34 (m, 2H), 3.29-3.25 (m, 2H), 2.84 (s,

13 4H); C NMR (400 MHz, CDCl3) δ 168.7, 166.5, 35.5, 25.5.

(E)-4-(4-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2-yl)styryl)- 1-(3-(2,5-dioxopyrrolidin-1-yloxy)-3-oxopropyl)pyridinium iodide (P-146)

Rhodamine B lactam of trans-4-amino-γ-stilbazole (0.2 g, 0.32 mmol) was dissolved in 6 mL of warm acetonitrile and 2,5-dioxopyrrolidin-1-yl 3-iodopropanoate (0.1 g, 0.34 mmol) was added in the solution of acetonitrile and refluxed overnight. The TLC of the reaction indicated the presence of starting material rhodamine lactam. The reaction was refluxed for another 24 hours until complete consumption of starting material. The excess acetonitrile was removed under reduced pressure. A brown solid was obtained which was crushed into power and stirred in ethyl acetate overnight and filtered to afford a brown solid (0.44 g, 71%); FTIR: 2955.25, 2925.50, 1736.3, 1698.20, 1614.14, 1513.57,

1 1327.36, 1116.18, 784.66; H NMR (400 MHz, CDCl3,) 8.90 (m, 2H), 8.00-7.99 (m, 1H),

7.93 (s, 1H), 7.92 (d, J = 6.8 Hz, 2H), 7.58 (d, J = 16.4 Hz, 1H), 7.54-7.48 (m, 2H), 7.40

(d, J = 8.64 Hz, 2H), 7.15-7.13 (m, 1H), 7.04-6.99 (m, 2H), 6.61 (d, J = 9.48 Hz, 2H),

6.31-6.29 (m, 4H), 5.07 (t, J = 5.6 Hz, 2H), 3.47 (t, J = 6 Hz, 2H), 3.30 (q, J = 6.4 Hz,

13 8H), 2.80 (s, 4H), 1.13 (t, J = 7.2 Hz, 12H); C NMR (100 MHz, CDCl3) δ 168.6, 168.0,

165.8, 154.8, 152.8, 144.1, 142.0, 139.9, 136.4, 133.4, 132.1, 130.0, 129.3, 129.1, 128.5,

296

126.6, 124.1, 123.4, 121.8, 108.5, 106.3, 97.9, 96.3, 92.1, 89.9, 55.3, 44.3, 33.2, 25.6,

12.5; UV-Vis (DCM) λmax (lg ε) 242 (1.63), 279 (0.86), 325 (0.45), 395 (0.7), and cut off at 490 nm

2-(2-(2-ethoxyethoxy)ethoxy)ethanol 4-methylbenzenesulfonate (2.52)

A three neck 100 mL reaction flask was fitted with a dropping funnel. A solution of

NaOH (2.86 g, 72 mmol) in distilled water (10 mL) and 2-(2-(2- ethoxyethoxy)ethoxy)ethanol (40.2 mmol, 6.63 g) in THF (10 mL) were introduced into the reaction flask and cooled to 0 ºC with rapid stirring. A solution of p-toluene sulfonyl chloride (7.65 g, 40.2 mmol) in THF (12 mL) was transferred to the dropping funnel and added over a 15 minutes period while maintaining the temperature 0 ºC. The reaction was then allowed to slowly warm to room temperature while stirring for 12 h. The reaction was monitored by TLC. A distinct new spot was observed with no starting material. The reaction mixture was extracted with ethyl acetate and washed with water and dried over magnesium sulfate. The excess solvent was removed under reduced pressure to obtain the desired product as a clear liquid (quantitative); HNMR liquid (11.086 g, 90%); 1H NMR

(400 MHz, CDCl3,) δ 7.80-7.78 (m, 2H), 7.34-7.32 (m, 2H), 4.16-4.14 (m, 2H), 3.69-3.67

(m, 2H), 3.60-3.56 (m, 8H), 3.51 (q, J = 7.2, 2H), 2.44 (s, 3H), 1.19 (t, J = 6.8, 3H); 13C

NMR (100 MHz, CDCl3) δ 144.7, 133.0, 129.8, 127.9, 70.7, 70.6, 70.5, 69.7, 69.2, 68.6,

66.5, 21.6, 15.1.

297

(E)-4-(4-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2-yl)styryl)- 1-(2-(2-(2-ethoxyethoxy)ethoxy)ethyl)pyridinium 4-methylbenzenesulfonate (P-140)

Rhodamine B lactam of trans-4-amino-γ-stilbazol (0.2 g, 0.32 mmol) was dissolved in 6 mL of warm acetonitrile. 2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate

(0.5 mL, 0.58 g, 1.76 mmol) was added in the solution of acetonitrile and refluxed overnight. The TLC of the reaction indicated the presence of starting material-rhodamine lactam so additional 0.1 mL of 2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4- methylbenzenesulfonate was added. The reaction was run for 6 h with the complete consumption of starting material rhodamine lactam P-83. The reaction was stopped and the solvent was removed under reduced pressure and the excess of the 2-(2-(2- ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate was also removed under high vacuum (25µ) at 150°C. A brown sticky solid compound was obtained. The HNMR indicated that the compound was still not pure. The sticky mass was cooled in a refrigerator overnight. The brown mass was broken by spatula and further crushed into powder by stirring overnight in hexane. A yellow compound was obtained (0.41 g, 88%); mp 75-78 ºC; FTIR 3046.4, 2971.0, 2927.5, 2869.1, 1696.6, 1614.6, 1346.1, 1175.4,

-1 1 1010-785 cm , H NMR (400 MHz, CDCl3,) δ 9.11-9.09 (m, 2H), 8.0-7.98 (m, 1H),

7.82-7.79 (m, 4H), 7.51-7.44 (m, 3H), 7.38-7.36 (m, 2H), 7.15-7.12 (m, 5H), 6.93 (d, J =

16.20 Hz, 1H), 6.61 (d, J = 8.7 Hz, 2H), 6.31-6.27 (m, 4H), 4.92-4.90 (m, 2H), 4.01-3.99

298

(m, 2H), 3.63-3.54 (m, 8H), 3.50 (q, J = 7.2 Hz, 2H), 3.31 (q, J = 7.2 Hz, 8H), 2.31 (s,

13 3H), 1.17 (t, J = 7.2 Hz, 3H), 1.15 (t, J = 4 Hz, 12H); C NMR (100 MHz, CDCl3) δ

172.2, 158.8, 158.1, 157.3, 153.6, 151.0, 149.9, 145.2, 144.2, 142.7, 138.9, 137.6, 135.3,

134.2, 133.8, 133.5, 133.4, 133.2, 132.8, 130.7, 130.3, 128.9, 128.5, 128.4, 128.2, 113.4,

110.7, 102.3, 74.9, 74.8, 74.7, 74.3, 73.8, 70.7, 48.8, 25.9, 20.2, 17.6; UV-Vis (DCM)

λmax (lg ε) 240 (1.4), 277 (0.8), 325 (0.4), 390 (0.67), and cut off at 480 nm

3',6'-bis(diethylamino)-2-(4-(hydroxymethyl)phenyl)spiro[isoindoline-1,9'-xanthen]- 3-one (P148) [Following Scheme 2.16]

Yield: Yellowish white solid (0.12 g, 20%); mp 124-125 oC; FTIR, 3447.1, 2971.9,

-1 1 2925.5, 2849.6, 1677.8, 1633.7, 1117.4 cm ; H NMR (400 MHz, CDCl3,) δ 8.00-7.98

(m, 1H), 7.50-7.48 (m, 2H), 7.18-7.16 (m, 1H), 7.13 (d, J = 8.24 Hz, 2H), 6.8 (d, J = 8.32

Hz, 2H), 6.66 (d, J = 8.80 Hz, 2H), 6.33 (dd, J = 8.86 Hz, 2.42 Hz, 2H), 6.28-6.27 (m,

2H), 4.56 (s, 2H), 3.31 (q, J = 7.2 Hz, 8H), 1.63 (s, 1H), 1.14 (t, J = 7.2 Hz, 12H); 13C

NMR (400 MHz, CDCl3) δ 167.8, 153.3, 153.0, 148.7, 139.0, 136.0, 132.8, 130.8, 128.8,

128.1, 127.3, 127.1, 124.0, 123.3, 108.1, 106.3, 97.7, 64.9, 44.3, 26.9, 12.5

299

3',6'-bis(diethylamino)-2-(4-(4-((E)-2-(pyridin-4- yl)vinyl)styryl)phenyl)spiro[isoindoline-1,9'-xanthen]-3-one (P-149)

In a 100 mL round bottom flask, equipped with stir bar stirrer, condenser, oil bath, nitrogen was placed the rhodamine B lactam of 4-iodoaniline (0.25 g, 0.4 mmol), vinyl pyridine (0.081 g, 0.4 mmol), DMF (2 mL), TDA-1 (0.012 mL) and potassium carbonate

(0.24 g, 0.42 mmol). The reaction mixture was heated to 80 oC and palladium acetate (8.8 mg) was added all at once. The reaction was then heated to 110 oC and monitored by

TLC. The reaction was completed in 1 hour and to make sure the reaction was further heated half an hour more. When water was added to the mixture, a dense creamy white precipitate was appeared. It was filtered off, washed with water, air dried and then dissolved in ethyl acetate and dried with magnesium sulfate. The solvent was removed by rotary evaporation. The product was collected using hexane and filtered to obtain a creamy white mass (0.235 g, 82%). mp 145-165 oC; FTIR 3018.2, 2968.29, 1694.32,

-1 1 1613.46, 1512.17 cm ; H NMR (400 MHz, CDCl3,) δ 8.57 (d, J = 6.0 Hz, 2H), 8.01-

7.99 (m, 1H), 7.51-7.47 (m, 4H), 7.46-7.43 (m, 2H), 7.37-7.35 (m, 2H), 7.30-7.27 (m,

3H), 7.15-7.13 (m, 1H), 7.02-6.97 (m, 3H), 6.91-6.89 (m, 2H); 6.65 (s, 1H), 6.33 (s, 1H),

6.31 (d, J = 2.8 Hz, 1H), 6.30-6.28 (m, 3H), 3.31 (q, J = 7.2 Hz, 8H), 1.15 (t, J = 7.2 Hz,

13 12H); C NMR (100 MHz, CDCl3) δ 167.5, 152.3, 149.5, 147.9, 144.8, 139.2, ,137.3,

300

137.1, 134.5, 133.6, 132.5, 128.5, 127.9, 127.5, 126.8, 124.5, 121.0, 110.3, 107.8, 106.5,

98.7, 83.7, 44.2, 12.5

2-(acridin-9-yl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one (P-154)

To a stirred solution of rhodamine B base (0.5 g, 1.13 mmol) in DCM (6 mL) were added toluenesulfonylchloride (0.23 g, 1.13 mmol), and DMAP (0.29 g, 1.23 mmol) at room temperature and stirring was continued for 30 min. To this solution was added a solution of acridin-9-amine hydrochloride (0.18 g, 1.028 mmol) and triethylamine (0.33 mL,

1.028 mmol) in DCM (6 mL). The mixture was stirred for 2 hours. The reaction mixture was monitored by TLC. A distinct halochromic spot was observed. The mixture was quenched with saturated aqueous sodium bicarbonate and the organic phase was extracted with ethyl acetate. The organic solution was washed with saturated aqueous sodium bicarbonate and dried over anhydrous magnesium sulfate and evaporated. The crude reaction mixture was purified by a column chromatography by using hexane:ethyl acetate (1:9) as an eluent. The desired compound was obtained in the first fraction as a yellow solid (0.2 g, 29 %); mp 280°C; FTIR, 2971.9, 2968.0, 2849.6, 1689.1, 1608.6,

-1 1 1509.7 cm ; H NMR (400 MHz, CDCl3,) δ 8.22-8.20 (m, 1H), 8.07 (s, 1H), 8.22-8.21

(m, 1H), 8.07 (s, 1H), 8.05 (s, 1H), 7.80-7.75 (m, 2H), 7.57-7.56 (m, 1H ), 7.49-7.44 (m,

301

2H), 7.15-7.13 (m, 2H), 6.93-6.89 (m, 2H), 6.73 (s, 1H), 6.72 (s, 1H), 6.31 (d, J = 2.4 Hz,

1H), 6.28 (d, J = 2.4 Hz, 1H), 5.53 (s, 1H), 5.52 (s, 1H), 3.20-3.13 (m, 8H); 1.00n (t, J =

13 8.4 Hz, 12H); C NMR (100 MHz, CDCl3) δ 166.7, 154.8, 149.3, 149.1, 148.3, 138.5,

133.4, 132.8, 129.1, 128.7, 128.4, 128.2, 125.3, 125.2, 124.7, 124.47, 124.4, 107.8, 107.4,

97.9, 97.6, 44.5, 12.369.7; UV-Vis (DCM) λmax (lg ε) 250 (2.2), 277 (1.3), 320 (0.6), 360

(0.38), and cut off at 420 nm

(E)-2-(4-bromostyryl)-3,3-dimethyl-3H-indole (2.53)

A mixture of 4-bromobenzaldehyde (0.92 g, 5.0 mmol) and 2,3,3-trimethyl-3H-indole

(0.81 g, 5.0 mmol) in 15 mL of ethanol was boiled for 2 h. The reaction mixture was checked by TLC, which indicated the presence of starting materials with a small new spot. The mixture was further refluxed for additional 1 h and then refluxed further overnight. The reaction never went to completion. Then solvent was removed under reduced pressure and the residue was separated by column chromatography using dichloromethane as eluent. The 4-bromobenzaldehyde was separated in small quantity in the first fraction as yellow solid and then the desired product was obtained in the second fraction as yellow solid (0.95g, 58%); mp-124.5 ºC (sharp); FTIR 3049.5, 2957.9,

-1 1 2925.6, 2860.2, 1625.2, 1515.4, 1066.6 cm , H NMR (400 MHz, CDCl3,) δ 7.65 (d, J =

302

16.4 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.54-7.51 (m, 2H), 7.48-7.45 (m, 2H), 7.36-7.31

(m, 2H), 7.26-7.22 (m 1H), 7.05 (d, J = 16.0 Hz, 1H), 1.45 (s, 6H); 13C NMR (100 MHz,

CDCl3) δ 182.9, 153.8, 146.5, 136.5, 135.0, 132.0, 128.8, 127.9, 125.8, 123.3, 121.1,

120.7, 120.3, 52.7, 23.

9-nitro-5H-benzo[a]phenoxazin-5-one (Nitro nile red) (P-153)

To a suspension of 2-hydroxy-1,4-naphthoquinone (11.85 g, 0.07 mol) in 80% acetic acid

(100 mL) was added 2-hydroxy-4-nitroaniline (10.35 g, 0.07 mol). The mixture was stirred for 12 h at 100ºC. The mixture was checked by TLC. A faint yellow fluorescent, a red fluorescence and a yellow spot of starting material were observed. The mixture was diluted with water and a heavy dark brown precipitate was obtained. The mixture was then neutralized by adding Na2CO3. The precipitate was extracted with 2x100 mL of chloroform. The first 200 mL extract was filtered through a silica pad to obtain the desired product. The precipitate was further extracted several times with chloroform and the excess solvent was removed under reduced pressure to obtain a yellow solid mass

(1.57 g, 8.0%, reported-8.33%); mp-264.5 ºC (sharp); FTIR 3062.02, 1631.08, 1590.54,

-1 1 1517.63, 1340.40, 1117.90 cm , H NMR (400 MHz, CDCl3,) δ 8.75 (dd, J = 3.6 Hz, 2.4

Hz, 1H), 8.32 (dd, J = 3.2 Hz, 2.4 Hz, 1H), 8.23 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 8.19 (d, J =

303

2.4 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.86-7.84 (m, 2H), 6.54 (s, 1H); 13C NMR (100

MHz, CDCl3) δ 183.2, 151.1, 150.9, 148.4, 144.2, 137.2, 133.5, 133.1, 132.1, 131.1,

130.7, 125.9, 125.4, 120.5, 111.9, 108.3

9-amino-5H-benzo[a]phenoxazin-5-one (P-156)

A suspension of 9-nitro-5H-benzo[a]phenoxazin-5-one was made in MeOH (20 mL) followed by addition of Pd/C (0.11 g). The air inside the flask was evacuated under vacuum and backfilled with hydrogen. The mixture was stirred until all the starting material was consumed which was monitored by TLC. It took around 3 hrs to complete the reaction. The yellow precipitate inside the flask turned into dark red as it came in contact with the air. The reaction mixture was dissolved in pyridine and filtered through

Celite. The filtrate was diluted with water to form precipitate. The precipitate was partially soluble with dichloromethane and ethylacetate mixture so it was extracted several times with dichloromethane:ethyl acetate (1:1) followed by several silica pad filtration. The excess solvent was removed under reduced pressure to afford a dark red solid (0.4 g, 40%, reported-45.7%); mp->280ºC; FTIR 3411.6, 3330.0, 3218.9, 1646.4,

-1 1 1574.0, 1114.6 cm , H NMR (400 MHz, CDCl3,) δ 8.57 (d, J = 8.0 Hz, 1H), 8.13 (d, J =

8.0 Hz, 1H), 7.82 (dd, J = 8.0 Hz, 1H), 7.73 (dd, J = 8.0 Hz, 1H), 7.56 (d, J = 8.8 Hz,

1H), 6.72 (s, 2H), 6.71 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 6.32 (s,

304

13 1H); C NMR (100 MHz, CDCl3) δ 182.4, 154.5, 152.2, 146.8, 138.6, 132.0, 131.7,

131.5, 130.4, 125.5, 125.0, 123.8, 113.2, 105.0, 98.0.

3',6'-bis(diethylamino)-2-(4-nitrophenyl)spiro[isoindoline-1,9'-xanthen]-3-one (P- 166) [Following Scheme 2.16]

Yield: Yellow solid (2.1 g, 85.0 %): mp 199ºC (sharp); FTIR 3016.2, 2972.5, 2931.2,

1 2868.7, 1714.4, 1545.3, 1346.5, 788.4; H NMR (400 MHz, CDCl3,) δ 8.01-7.98, (m,

3H), 7.51-7.47 (m, 2H), 7.39-7.36 (m, 2H), 7.12-7.10 (m, 1H), 6.58 (s, 1H), 6.56 (s, 1H),

6.33 (s, 1H), 6.33 (s, 1H), 6.27 (d, J = 2.8 Hz, 1H), 6.25 (d, J = 2.4 Hz, 1H), 3.31 (q, J =

13 6.8 Hz, 8H), 1.15 (t, J = 7.2 Hz, 12H); C NMR (100 MHz, CDCl3) δ 168.4, 153.7,

152.6, 148.9, 144.4, 143.6, 133.7, 129.0, 128.3, 128.1, 124.4, 124.0, 123.8, 123.5, 108.3,

105.8, 97.8, 67.61, 44.3, 12.5

2-(4-aminophenyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one (P- 167)

305

To a stirred solution of 3',6'-bis(diethylamino)-2-(4-nitrophenyl)spiro[isoindoline-1,9'- xanthen]-3-one (1.0 g, 1.78 mmol) in methanol (7.0 mL) was added palladium on charcoal catalyst (84.0 mg, 10% w/w). The atmosphere was then evacuated under vacuum and hydrogen gas was filled back with a balloon. The yellow color of the starting material started to fade away to a grayish solid mass. The reaction mixture was monitored by TLC until the reaction was complete in around 2 h. The mixture was filtered through celite pad, concentrated and then purified by column chromatography. The desired compound was obtained as a grayish white solid (0.7 g, 74%); mp 197-199°C, FTIR

-1 1 3330.6, 2967.5, 2928.9, 1666.4, 1332.4 cm ; H NMR (400 MHz, CDCl3,) δ 7.99-7.97

(m, 1H), 7.49-7.47 (m, 2H), 7.16-7.14 (m, 1H), 6.62 (s, 1H), 6.59 (s, 1H), 6.45-6.39 (m,

4H), 6.31 (d, J = 2.8 Hz, 1H), 6.29 (d, J = 2.8 Hz, 1H), 6.24 (s, 1H), 6.23 (s, 1H), 3.31

13 (dq, J = 7.2, 2.8 Hz, 8H), 1.14 (t, J = 6.8 Hz, 12H); C NMR (100 MHz, CDCl3) δ 167.6,

153.3, 152.9, 148.6, 145.2, 132.5, 131.6, 129.0, 129.0, 128.0, 127.1, 124.0, 123.3, 115.2,

108.0, 106.5, 97.8, 67.3, 44.3, 12.5

2-(4-nitrophenyl)-1,3-dithiane (2.34)

A reaction mixture of 4-nitrobenzaldehyde (2.0 mL, 2.35 g, 18.93 mmol), 1,3- propanedithiol (1.99 mL, 2.15 g, 19.88 mmol), and silica- sulfuric acid (946.86 mg) in acetonitrile (40 mL) was stirred at room temperature. The reaction was monitored by

306

1HNMR indicating the disappearance of CHO proton for the completion of reaction.

After completion of reaction in 1 hour, the excess acetonitrile was removed under reduced pressure and residue solid mass was combined with DCM and filtered. The catalyst was washed with DCM. The organic layer was washed with NaHCO3 (200 mL,

10%), followed by water (200 mL) and brine (100 mL). The organic layer was dried over

MgSO4 and filtered. The excess solvent was removed under reduced pressure and the pure product (white solid) was obtained quantitatively (4.3 g, 92.33%); mp 105.5ºC;

1 HNMR (400 MHz, CDCl3) δ 8.21-8.19 (m, 2H), 7.66-7.64 (m, 2H), 5.24 (s, 1H), 3.12-

3.048 (m, 2H), 2.97-2.92 (m, 2H), 2.24-2.18 (m, 1H), 1.92-1.89 (m, 1H); 12CNMR (100

MHz, CDCl3) δ 147.82, 146.15, 128.94, 124.00, 50.42, 31.76, 24.82.

4-(1,3-dithian-2-yl)aniline (2.35)

A solution of 2-(4-nitrophenyl-1,3-dithiane (1.0 g, 4.14 mmol), and SnCl2.2H2O (3.93 g

(20.71 mmol) in THF/EtOH (3/1, 83 mL) was prepared and heated to 60°C. To this mixture, NaBH4 (0.078 g, 2.07 mmol) in EtOH (20.0 mL) was added over a period of 30 minutes with stirring. After stirring for further 30 min, the reaction mixture was cooled to

5-10C and chilled water (83 mL) was added followed by neutralization with 3.5 NaOH to pH7. The THF and ethanol was evaporated off and the aqueous solution was continuously extracted with ether. The ethereal solution was dried over MgSO4 and filtered through a thin silica pad. The unreacted and other non polar compounds washed

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away with ether and the desired product remained in the silica pad which was then washed with EtOAc and collected in a separate flask. The excess solvent was removed under reduced pressure to give a grayish solid (0.32 g, 37%); mp 138ºC (sharp); 1HNMR

(400 MHz, CDCl3) δ 7.26-7.23 (m, 2H), 6.62-6.60 (m, 2H), 5.08 (s, 1H), 3.06-2.99 (m,

2H), 2.9-2.84 (m, 2H), 2.16-2.11 (m, 1H), 1.91-1.87 (m, 1H); 12CNMR (100 MHz,

CDCl3) δ 146.59, 135.02, 128.77, 121.27,115.08, 50.95, 32.25, 25.12.

4-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2-yl)benzaldehyde (P–422)

To a stirred solution of rhodamine B base (0.5 g, 1.043 mmol) in DCM (5.0 mL) was added toluenesulfonylchloride (0.216 g, 1.137 mmol), and DMAP (0.278 g, 2.277 mmol) at room temperature. After having been stirred for 45 min, a solution of 4-(1,3-dithian-2- yl)aniline (0.34 g, 1.61 mmol) in DCM (5.0 mL) was added. The reaction mixture was stirred for 3 h. The reaction mixture was checked by TLC. A new halochromic spot was appeared. The reaction mixture was quenched with saturated aqueous sodium bicarbonate and the organic phase was extracted with ethyl acetate. The organic phase was washed with saturated aqueous sodium bicarbonate, water, brine, and dried over anhydrous magnesium sulfate. The excess solvent was removed under reduced pressure. The desired

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product was separated from crude mixture by a flash column chromatography using a mixture of hexane:ethyl acetate (2:3) as an eluent. In the second fraction, a creamy white compound was obtained: (0.425 g, 64.0%): mp 220ºC (sharp); 1H NMR (400 MHz,

CDCl3,) δ 7.99-7.96 (m, 1H), 7.47-7.45 (m 2H), 7.26-7.20 (m, 2H), 7.11-7.709 (m, 1H),

6.90-6.88 (m, 2H), 6.63-6.59 (m, 2H), 6.29-6.27 (m, 4H), 5.03 (s, 1H), 3.31 (q, J = 7.09

Hz, 8H), 3.01-2.94 (m, 2H), 2.87-2.82 (m, 2H), 2.14-2.08 (m, 1H), 1.91-1.84 (m, 1H)

1.14 (t, J = 7.04 Hz, 12H)

4-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2-yl)benzaldehyde (P–424)

To a stirred solution of 2-(4-(1,3-dithian-2-yl)phenyl)-3',6'-bis(diethylamino)spiro

[isoindoline-1,9'-xanthen]-3-one (0.16 g, 0.236 mmol), pyridine (28 µL) in DCM (1 mL) and water (0.17 mL) was added pyridine tribromide (76 mg, 0.236 mmol) followed by tetraethyl ammonium bromide (3.36 mg, 0.016 mmol). The resulting mixture stirred for

24 hours at room temperature. The orange slowly faded away. The reaction was monitored by TLC. The reaction mixture was poured into water (70.0 mL) and extracted with DCM (3x50 mL). The combined organic fractions were washed with water (250 mL), brine (250 mL), and dried over MgSO4, filtered and evaporated to give yellow oil

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which was further separated in column chromatography (hexane:ethyl acetate/4:1). Yield

1 (quantitative); mp 216; H NMR (400 MHz, CDCl3,) δ 9.86 (s, 1H), 8.03-8.01 (m 1H),

7.68-7.66 (m, 2H ), 7.53-7.49 (m, 2H), 7.32-7.28 (m, 2H), 7.15-7.13 (m, 1H), 6.63 (s,

1H), 6.61 (s, 1H), 6.34-6.29 (m, 4H), 3.33 (q, J = 7.10 Hz, 8H), 1.72 (t, J = 7.04 Hz, 12H)

2-(6-(pyrrolidin-1-yl)-3-(pyrrolidinium-1-ylidene)-3H-xanthen-9-yl)benzoate or (3’,6’-pyrrolidinorhodamine) [P-168]

To a finely ground mixture of 3’,6’-dichloroflouran (1.0 g, 2.71 mmol) and anhydrous zinc chloride (1.84 g, 13.5 mmol) was added pyrrolidine (2.25 mL, 27.0 mmol) and the resulting mixture was heated in an oil bath at 140 ºC for 4 h. The dark purple residue was dissolved in concentrated HCl (42.3 mL), and the resulting dark red solution was filtered under a suction and then diluted with 200 mL of water, allowed stand for 2 h at room temperature and filtered to afford the desired HCl salt (1.21 g 100%); mp >250 ºC; 1H

NMR (400 MHz, CDCl3,) δ 8.23 (dd, J = 7.6, 1.2 Hz, 1H), 7.87 (dt, J = 7.4, 1.6 Hz, 1H),

7.81 (dt, J = 7.6, 1.2 Hz, 1H), 7.03 (s, 1H), 7.01 (s, 1H), 6.95 (d, J = 2.0 Hz, 1H), 6.93 (d,

J = 2.4 Hz, 1H), 6.82 (s, 1H), 6.82 (s. 1H), 3.58 (s, 8H), 2.04 (s, 8H); 13C NMR (400

MHz, CDCl3) δ 166.7, 159.4, 157.0, 154.5, 133.7, 133.1, 131.4, 131.3, 131.2, 130.7,

130.6, 115.9, 113.4, 96.9, 49.2, 25.2

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2-(4-iodophenyl)-3',6'-di(pyrrolidin-1-yl)spiro[isoindoline-1,9'-xanthen]-3-one, (P- 169) [Following Scheme 2.16]

Yield: Creamy white solid (0.25 g, 30%); mp 186-188 ºC (wide); FTIR 3012.2, 2973.1,

1 2955.6, 1703.20, 1625.3; 1121.1, 787.4; H NMR (400 MHz, CDCl3,) δ 8.01-7.97 (m,

1H), 7.53-7.46 (m, 2H), 7.45-7.41 (m, 2H), 7.17-7.12 (m, 1H), 6.66-6.57 (m, 4H ), 6.32-

13 6.27 (m, 4H), 3.28 (s, 8H), 2.01 (s, 8H); C NMR (400 MHz, CDCl3) δ 168.3, 155.2,

151.5, 150.0, 139.1, 137.6, 134.1, 131.1, 129.5, 128.8, 123.5, 123.5, 108.0, 106.1, 98.0,

91.5, 67.0, 43.5, 12.8.

Rhodamine 101 hydrochloride (RhD 101)

A mixture of 8-hydroxyjulolidine (0.5 g, 2.64 mmol) and phthalic anhydride (0.61 g, 4.7 mmol) was heated in a 50 mL of round bottom flask with reflux condenser in an oil bath at 160 ºC for 2 hours. The initial melt was mobile and was thoroughly mixed by hand- rotating the flask, and slowly became viscous. The mixture was cooled to room

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temperature. To this cooled mixture were added 8-hydroxyjulolidine (0.5 g, 2.64 mmol) and 1.16 mL of 85.0% phosphoric acid. The contents were heated under reflux in an oil bath at 170ºC for 4 hours, and the flask was intermittently agitated for the first fifteen minutes to ensure even dispersal of the contents. The cooled solid mixture was dissolved in conc. HCl (75.0 mL) and water (150.0 mL) was added and left for 2h. A thick sticky layer was collected at the bottom of the flask. It was dissolved in DCM and dried over

MgSO4. The sticky nature of the substance could not be removed. It was again dissolved in DCM and toluene was added and rotavapped. The desired product was obtained as a

1 dark purple solid (1.35 g, 62%); H NMR (400 MHz, CDCl3,) δ 8.21-8.19 (m, 1H), 7.83-

7.81 (m, 1H), 7.79-7.77 (m, 1H), 7.36-7.34 (m, 1H), 6.5 (s, 2H), 3.53 (t, J = 5.2, 4H),

3.48 (t, J = 5.6, 4H m), 3.12 (t, J = 6.0, 4H), 2.63 (t, J = 6.0, 4H), 2.01 (m, 4H), 1.84(m,

13 4H); C NMR (100 MHz, CDCl3) δ 166.7, 156.2, 151.7, 150.9, 134.2, 133.0, 131.3,

131.3, 130.8, 130.4, 128.8, 125.8, 123.9, 112.6, 105.1, 50.6, 50.2, 27.3, 20.5, 19.8, 19.6

N-(4-Hydroxy-cyclohexyl)-4-methyl-benzenesulfonamide (2.62)

A two neck 100 ml reaction flask was fitted with a dropping funnel, a solution of NaOH

(1.04 g, 26 mmol, 1.5 equiv) in distilled water (5.0 mL) and then trans-4-aminocyclohexanol-(1) (2.0 g, 17.4 mmol) in THF (5.0 mL) was introduced to the reaction flask at room temperature with rapid stirring. A solution of p-TsCl (3.3 g, 17.4 mmol) in

THF (12.0 mL) was transferred to the dropping funnel and added over a 30 min period.

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The reaction was allowed to run for 3 h monitoring with TLC. A creamy white solid was observed in the reaction mixture, which disappeared as soon as dilute HCl (1N) was added and stirred. The organic phase was extracted with ethyl acetate and dried with

MgSO4. A colorless liquid was obtained which upon cooling turned to a faint brown viscous solid. It was dissolved in ethyl acetate and impregnated with silica gel and ran a column chromatography using DCM:ethyl acetate (4:1) as eluent. The desired product was obtained in the first fraction as white crystals (4.02 g. 86%); mp 98-100.2 ºC;1H

NMR (400 MHz, CDCl3) δ 7.78-7.7 (m, 2H), 7.33-7.28 (m, 2H), 4.97 (d, J = 7.6 Hz, 1H),

3.59-3.55 (m, 1H), 3.12-3.08 (m, 1H), 3.03 (s, 3H), 1.92-1.83 (m, 4H), 1.32-1.18 (m,

13 4H); C NMR (100 MHz, CDCl3) δ 143.3, 138.1, 129.7, 126.9, 69.1, 51.8, 33.5, 31.3,

21.5

7-(Toluene-4-sulfonyl)-7-aza-bicyclo[2.2.1]heptane (2.63)

To a stirred solution of N-(4-hydroxy-cyclohexyl)-4-methyl-benzenesulfonamide (3.42 g,

19.9 mmol, 1 equiv) in anhydrous THF (100.0 mL) under a nitrogen atmosphere at -10

ºC, was added PPh3 (7.43 g, 28.33 mmol). After ~ 5 min, a solution of DIAD (3.92 mL,

19.9 mmol) in anhydrous THF (24 mL) was added drop wise by syringe pump over 1 h period. The reaction was stirred at -10 ºC for 1 h and then ran overnight at room

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temperature. There were three distinct spot was observed with a great difference in their

Rf values. A column chromatography was run with dichloromethane as eluent. The desired compound was obtained in the first fraction as a white solid (2.5 g, 65%); mp

1 121.5-122.0 ºC; H NMR (400 MHz, CDCl3,) δ 7.80-7.78 (m, 2H), 7.28-7.28 (m, 2H),

4.19-4.16 (m, 2H), 2.42 (s, 3H), 1.79-1.75 (m, 4H), 1.40-1.35 (m, 4H); 13C NMR (400

MHz, CDCl3) δ 121.5, 116.5, 116.4, 116.2, 109.6, 69.9, 52.2, 31.7, 29.2, 29.1, 29.0, 25.8,

22.6, 14.0

7-aza-bicyclo[2.2.1]heptane.hydrochloride (2.64)

A solution of naphthalenide in THF was prepared by adding THF (10.0 mL) to a mixture of sodium (0.3 g, 13.0 mmol) and naphthalene (2.1 g, 16.0 mmol) and stirring the resulting mixture at room temperature for 2 h. A dark green solution was obtained. It was cooled in a dry-ice/isopropanol bath. A solution of 7-(toluene-4-sulfonyl)-7-aza- bicyclo[2.2.1]heptane (0.34 g, 2.0 mmol) in THF (35.0 mL) was added dropwise and stirred for half an hour. The reaction was quenched by addition of saturated NaHCO3 (1.0 mL) followed by addition of anhydrous K2CO3 (5.0 g). The green color turned to colorless mixture, which was then stirred overnight. The white solid mass was filtered

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and washed with diethyl ether. The filtrate then dried over MgSO4. To this dry solution, freshly prepared HCl gas was bubbled. A light white precipitate was obtained which was

1 filtered and dried under vacuum (0.116 g, 65%); mp 261°C; H NMR (400 MHz, CDCl3,)

13 δ 4.09 (m, 2H), 1.84-1.82 (m, 4H), 1.82-1.57 (m, 4H); C NMR (100 MHz, CDCl3) δ

57.7, 27.2

trans-4-tert-butoxycarbonylaminocyclohexanol (2.68)

To a solution of trans 4-aminocyclohexanol (1.5 g, 13.02 mmol) in anhydrous ethanol

(26.0 mL) at 0 ºC was added di-tert-butyl-dicarbonate (2.86 g, 13.02 mmol). After stirring at room temperature for 2 h, the reaction mixture was diluted with DCM, washed with brine and organic layer was dried over MgSO4. The solvent was removed to get the

1 desired product-solid white (2.55 g, 92%); mp 163.5°C; H NMR (400 MHz, CDCl3,) δ

4.37 (s, 1H), 3.63-3.55 (m, 1H), 3.42-3.41 (m, 1H), 2.03-1.94 (m, 4H), 1.43 (s, 12H),

13 1.41-1.32 (m, 2H), 1.18-1.14 (m, 2H); C NMR (100 MHz, CDCl3) δ 155.2, 69.8, 48.8,

33.9, 31.1, 28.4, 27.4

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trans-4-(tert-butoxycarbonylamino)cyclohexyl methanesulfonate (2.69)

To a cooled (ice bath 0º-4ºC) stirred solution of trans-4-tert- butoxycarbonylaminocyclohexanol (8.0 g, 37.12 mmol) and Et3N (8.0 mL, 55.7 mmol) in

DCM (320 mL) was added methanesulfonylchloride (4.3 mL, 55.7 mmol). The reaction mixture was stirred at this temperature for half an hour then allowed to warm to room temperature and stirring was continued for 3 hrs. To this solution was added saturated

NaHCO3 (320 mL) followed by vigorous stirring for another half an hour. The reaction mixture was extracted with DCM. The organic phase was washed with water and dried

1 over MgSO4 to afford a white solid (9.3 g, 85%); mp 147.4°C; H NMR (400 MHz,

CDCl3,) δ 4.65-4.58 (m, 1H), 4.43-4.41 (m, 1H), 3.47-3.46 (m, 1H), 3.02 (s, 3H), 2.17-

2.05 (m, 1H), 1.73-1.52 (m, 2H), 1.42 (s, 9H), 1.41 (m, 2H); 13C NMR (100 MHz,

CDCl3) δ 155.1, 79.7, 47.9, 45.9, 38.8, 31.0, 30.5, 28.3.

trans-4-aminocyclohexyl methanesulfonate.trifluoroacetic acid (2.70)

A solution of trans-4-(tert-butoxycarbonylamino)cylohexylmethanesulfonate (9.0 g, 30.6 mmol) was prepared in DCM (20 mL) under nitrogen. To this solution was added trifluoroacetic acid (17.5 mL) in two batches in half an hour interval of time. The mixture

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was stirred overnight at room temperature resulting a clear solution. To this reaction mixture, dry diethyl ether was added. The trifluoroacetate salt was precipitated out as

1 white solid (8.24 g, 92.5%); H NMR (400 MHz, CDCl3,) δ 8.04 (s, 4H), 4.56-4.51 (m,

1H), 3.49 (s,1H), 3.19 (s, 3H), 3.04 (s, 1H), 2.09-2.05 (m, 2H), 1.98-1.95 (m, 2H), 1.61-

13 1.50 (m, 4H); C NMR (100 MHz, CDCl3) δ 159.8 (q, J = 30.43 Hz), 123.3 (dd, J =

1161.27, 274.48 Hz), 79.4, 38.1, 30.1, 28.2

7-azabicyclo[2.2.1]heptane hydrochloride (Another method)

A solution of trans-4-aminocyclohexylmethanesulfonate-TFA salt (6.5 g, 21.12 mmol) was prepared by dissolution in water (71.5 mL, 11 vol). To this aqueous solution was slowly added sodium hydroxide (2.5 g, 63.4 mmol, 3 equiv) keeping the reaction temperature below 25ºC and the mixture was stirred overnight. The reaction mixture was checked by HNMR for its completion. The absorption peaks of the cyclohexane protons no longer appeared. DCM (50 mL, 7 vol) was then added and the mixture was stirred, and the organic layer was separated. The aqueous layer was then extracted second time with DCM (50 mL, 7 vol), and the DCM layers were combined. To this organic layer was added concentrated HCl (3.53 mL, 12M, 42.5 mmol, 2 equiv) and the mixture was stirred for 1 hr. It was then concentrated in a rotary evaporator to dryness. To this residue was added acetonitrile (10 vol) and concentrated by rotavap. This process was continued for several times until all the trace of water was azeotropically removed to dryness to provide

317

bicyclic salt. The salt was further purified by recrystallization in acetonitrile (2.36 g,

1 86%); H NMR (400 MHz, CDCl3,) δ 9.08 (s, 2H), 4.08 (m, 2H), 1.85-1.83 (m, 4H),

13 1.58-1.56 (m, 4H); C NMR (100 MHz, CDCl3) δ 57.7, 27.2

Experimental of Chapter 4

Diethyl bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylate (4.12)

Diethyl fumarate (5.0 g, 4.76 mL, 29.0 mmol) was dissolved in dry DCM (9 mL) under nitrogen at 0ºC and to this solution was added cyclopentadiene (5.75 g, 7.31 mL, 87 mmol) dropwise by a syringe. The colorless solution was stirred at room temperature for

12 hrs under nitrogen. The mixture was monitored by TLC. Three well separated spots were observed. The solution was concentrated under reduced pressure and purified by column chromatography (hexane:EtOAc/4:1). The compound was obtained as a colorless

1 liquid (6.90 g, quantitative); H NMR (400 MHz, CDCl3,) δ 6.3 (dd, J = 5.64 Hz, 5.6 Hz,

1H), 6.08 (dd, J = 5.64 Hz, 5.64, 1H), 4.18 (q, J = 7.21 Hz, 2H), 4.11 (dq, J = 7.16 Hz,

2.04 Hz, 2H), 3.39 (dd, J = 3.88 Hz, 4.36 Hz, 1H), 3.29-3.27 (m, 1H), 3.14-3.12 (m, 1H),

2.70-2.68 (m, 1H), 1.65-1.62 (m, 1H). 1.46 (dq, J = 8.76 Hz, 1.76 Hz, 1H) 1.29 (t, J =

13 7.12 Hz, 3H), 1.25 (t, J = 7.12 Hz, 3H); C NMR (100 MHz, CDCl3) δ 174.4, 173.5,

137.5, 135.0, 60.8, 60.5, 47.8, 47.7, 47.2, 47.2, 45.6, 14.2

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Dimethyl bicyclo[2.2.1]hepta-2,5-diene-2,3-dicarboxylate (4.16)

To a freshly distilled neat cyclopentadiene (1.47 g, 22.35 mmol) was added dimethyl acetylenedicarboxylate (2.75 mL, 22.35 mmol) dropwise. The reaction mixture was cooled by water bath since the mixing was exothermic and exceed. The mixture was

1 stirred for 3 hrs to afford a clear liquid (5 g, quantitative); HNMR (400 MHz, CDCl3) δ

6.92 (dd, J = 2.0 Hz, 2H), 3.93 (dq, J = 1.64 Hz, 0.52, 2H), 3.78 (s, 6H), 2.27 (dt, J =

13 6.76, 1.64 Hz, 1H), 2.10 (dt, J = 6.8, 1.52 Hz, 1H); C NMR (100 MHz, CDCl3) δ 165.4,

152.4, 142.3, 72.9, 53.4, 52.0

Benzonorbornene (4.24)

A solution of 1,2-dibromobenzene (4.0 g, 8.58 mmol) and cyclopentadiene (1.14 g, 8.58 mmol) in toluene (25.0 mL) was stirred at 0°C under nitrogen. To this solution was added, n-BuLi in hexane (3.43 mL, 2.5M in hexane, 8.58 mmol) dropwise over 10 min during which the reaction solution became faint yellow. After an additional 10 min at

0°C, the mixture was allowed to warm to room temperature, stirred overnight and treated with water (50 mL) and extracted with hexane (2x10 mL). The organic layer was washed with brine, dried over MgSO4, filtered and concentrated to oil that was purified by

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column chromatography on silica gel eluting with hexane to provide clear colorless oil

1 (1.02 g, 42%); H NMR (400 MHz, CDCl3,) δ 7.21 (dd, J = 5.12, 3.08 Hz, 2H), 6.91 (dd,

J = 5.16, 3.08 Hz, 2H), 6.78 (dd, J = 1.88, 1.84 Hz, 2H), 3.87 (m, 2H), 2.31 (dt, J = 7.09,

13 1.56 Hz, 1H), 2.23 (m, 1H); C NMR (100 MHz, CDCl3) δ 151.8, 143.0, 124.2, 121.5,

70.1, 50.3.

Bicyclo[2.2.1]heptane-2-carboxylic acid [mixture of exo and endo] (4.52)

To a solution of (1R,2R,4R)-bicyclo [2.2.1]hept-5-ene-2-carboxylic acid (3.0 g, 21.71 mmol) in freshly distilled AcOEt (30.0 mL) was added 10% Pd/C (0.105 g). The air inside the flask was removed by vacuum and back filled with hydrogen. The mixture was stirred for 4 hours under hydrogen atmosphere. The reaction was checked with HNMR, the disappearance of the alkene H confirmed that the reaction was complete. The reaction catalyst was removed by filtration through Celite and the filtrate was concentrated in a vacuo to give quantitative the titled compound as colorless oil which was partially crystallized on standing at room temperature and turned into white solid upon

1 refrigeration (quantitative); H NMR (400 MHz, CDCl3) δ 11.38 (s, 1H), 2.79-2.78 (m,

1H), 2.59-2.56 (m, 1H), 2.28-2.28 (m, 1H), 1.53-1.17 (m, 8H); The 13C NMR showed a distinct mixture of two isomer endo and exo. Endo isomer is predominant; [exo-isomer

13 C NMR (100 MHz, CDCl3) δ 180.95, 45.87, 40.52, 40.24, 37.00, 31.72, 29.07, 24.83]

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13 [endo isomer, C NMR (100 MHz, CDCl3) δ 181.98, 46.30, 40, 36.533, 36.01, 34.07,

29.44, 28.57]

2-Bromobicyclo[2.2.1]heptane-1-carboxylic acid (4.54)

A mixture of (1S,2R,4R)-bicyclo[2.2.1]heptane-2-carboxylic acid (1.70 g, 11.98 mmol), bromine (0.7 mL, 13.56 mmol) and red phosphorus (9.46 mg) was stirred at 80ºC for 10 hours. Bromine (0.38 mL, 7.24 mmol) was added and the stirring was continued overnight. Heptane (3 mL) was added and the precipitated product was filtered to afford a creamy white solid (1.6 g, 60.2% yield based on acid); mp 148.7ºC (sharp); 1HNMR (400

MHz, CDCl3) δ 4.21 (ddd, J = 7.28 Hz, 3.12 Hz, 1.84 Hz, 1H), 2.37 (t, J = 3.85, 1H),

13 2.34-2.05 (m, 4H), 1.76-1.63 (m, 3H), 1.35-1.29 (m, 1H); C NMR (100 MHz, CDCl3) δ

178.3, 58.9, 52.9, 43.8, 36.9, 36.6, 32.5, and 29.2; 13C APT 178.25 C, 58.92 C, 52.9 CH,

43.84 CH2, 36.90 CH2, 36.6 CH, 32.5 CH2, and 29.2 CH2

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Methyl 2-bromobicyclo[2.2.1]heptane-1-carboxylate (4.55)

To a solution of (1S,2R,4S)-2-bromobicyclo[2.2.1]heptane-1-carboxylic acid (1.0 g, 4.56 mmol) in DMF (65.0 mL) and MeI (13.0 mL) at room temperature was added K2CO3 (3.3 g, 23.7 mmol) in one portion under nitrogen. After 3 hrs of stirring, the mixture was poured into water and extracted with ether. The organic extract was washed with water, brine and dried over MgSO4 and concentrated to afford the desired product as light yellow liquid. The crude was further purified by Kugelrohr distillation (40°C and 75µ

1 vacuum) to afford clean product (0.78 g, 74%); H NMR (400 MHz, CDCl3) δ 4.19-4.16

(m, 1H), 3.73 (s, 3H), 2.33-2.32 (m, 1H), 2.30-2.10 (m, 3H), 1.99-1.92 (m, 1H), 1.72-

13 1.62 (m, 3H), 1.27-1.24 (m, 1); C NMR (100 MHz, CDCl3) δ 173.49, 59.11, 53.80,

51.78, 43.86, 37.07, 36.62, 32.24, and 29.16

Ethyl 2-aminoacetate hydrochloride (4.62)

A solution of SOCl2 (2.6 mL, 22 mmol, freshly distilled) in absolute ethanol 200 proof

(20 mL) was prepared at -10°C. To this solution was added glycine (1.5 g, 20 mmol) and so formed suspension was subjected to ultrasonication at RT until completion of the reaction. The mixture turned into a clear solution after half an hour. The reaction mixture

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was monitored by IR until the disappearance of typical carboxylic peak (1725-1700 cm-

1). When a single peak of carbonyl at 1743.09 cm-1 (typical peak of aliphatic ester) was observed, the reaction was stopped. The excess solvent was removed under reduced pressure and the white solid mass was washed with hexane and vacuum dried to afford

1 clean white solid (98%); mp 146.7°C; H NMR (400 MHz, CDCl3,) δ 7.21 (dd, J = 5.12,

3.08 Hz, 2H), 6.91 (dd, J = 5.16, 3.08 Hz, 2H), 6.78 (dd, J = 1.88, 1.84 Hz, 2H), 3.87 (m,

13 2H), 2.31 (dt, J = 7.09, 1.56 Hz, 1H), 2.23 (m, 1H); C NMR (100 MHz, CDCl3) δ

166.2, 62.7, 13.8.

Ethyl 2-aminoacetate hydrochloride (4.63)

To a solution of 2.5 g (17.86 mmol) of ethyl 2-aminoacetate hydrochloride in water (5.0 mL) was added DCM (12.0 mL) in a three neck round bottom flask and cooled to -5ºC.

To this cold solution, an ice cold solution of NaNO2 (1.5 g, 21.4 mmol) in water (4.5 mL) was added with stirring. The temperature of the mixture then lowered to 10º-15ºC, and

H2SO4 (1.7 g, 5% by Wt) was added from the dropping funnel during a period of about 3 minutes. The temperature of the mixture was further lowered to -20ºC. The reaction was monitored by HNMR. The reaction was stopped after an hour. The reaction mixture was then transferred to a separatory funnel containing a solution of ice cold Na2S2O3 (5%,

20.0 mL). The yellow green DCM layer was separated. The aqueous phase was once again washed with DCM (20.0 mL). The combined golden yellow organic phase was

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dried over MgSO4. The excess solvent was removed under reduced pressure at room temperature. The desired product was obtained as a yellow liquid (1.51 g, 65%); 1H NMR

(400 MHz, CDCl3,) δ 4.74 (s, 1H), 6.91 (q, J = 6.49 Hz, 2H), 1.29 (t, J = 6.50 Hz, 3H);

13 C NMR (100 MHz, CDCl3) δ 159.2, 138.9, 60.8, 14.4

endo (syn, anti)and exo(syn, anti) 3-(ethoxycarbonyl) tricycle[3.2.1.02,4]oct-6-ene (4.27a/4.27b)

In an oven dried two neck 100 mL round bottom flask with a magnetic stirrer bar were placed norbornadiene (2.76 g, 3.05 mL, 30.0 mmol), palladium acetate (2.24 mg, 1 mo1

%) and ethyl diazoacetate (0.65 mL, 0.7 g, 6.198 mmol) and stirred 24 hrs at room temperature. The mixture was filtered through a silica pad. The silica pad was washed with ethyl acetate. The excess solvent and reactants were removed in a reduced pressure.

A wine red thick liquid was obtained. The crude was further separated in a column chromatography in using hexane:ethylacetate (3:1) eluent. A clear liquid was obtained

0.58 g (52.5%); The product was the mixture of isomers in a ratio of exo/endo [1.19:1];

1 (exo,syn and exo,anti isomers) H NMR (400 MHz, CDCl3) δ 6.42 (t, J = 1.72 Hz, 2H),

4.08 (q, J = 7.03 Hz, 2H), 30.3 (s, 2H), 2.22 (t, J = 7.42 Hz, 1H), 1.40 (d, J = 7.52 Hz,

2H), 1.33-1.24 (overlapping triplet, 3H), 0.99 (d, J = 9.5 Hz, 1H), 0.90 (d, J = 10.40 Hz,

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13 1H); C NMR (100 MHz, CDCl3) δ 141.96, 60.72, 42.16, 40.27, 39.16, 31.90, 14.25;

1 (endo, syn and endo, anti isomers); H NMR (400 MHz, CDCl3) δ 6.48 (t, J = 1.64 Hz,

2H), 4.16 (q, J = 7.13 Hz, 2H), 2.91 (s, 2H), 2.53 (t, J = 2.46 Hz, 1H), 1.65 (d, J = 2.0 Hz,

2H), 1.33-1.24 (overlapping quartet, 3H), 1.12 (d, J = 9.44 Hz, 1H), 1.06 (d, J = 10.52

13 Hz, 1H); C NMR (100 MHz, CDCl3) δ 140.97, 60.24, 41.85, 39.88, 32.24, 28.41, and

14.07

3,3,3-trifluoro-1-(4-fluorophenyl)-2-hydroxy-2-methylpropan-1-one (4.68)

A solution of TMSCN (2.5 g, 25.5 mmol), 1,1,1-trifluoroacetone (3.4 g, 30.35 mmol) and dry THF (25 mL) was prepared in an oven dried flask at 0ºC. A catalytic amount of n-

BuLi (2.5 M in hexane, 0.15 mL) was added with a syringe at 0ºC. After stirring at room temperature for 4 hrs, vacuum was applied to remove the excess trifluoroacetone. In a separate flask, a solution of 4-bromofluorobenzene (10.8 g, 61.5 mmol) in dry THF (20 mL) was added dropwise to a stirred mixture of Mg chunk (1.25 g, 51.5 mmol), dry THF

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(5 mL) and one drop of 1,2-dibromoethane at room temperature. This mixing was exothermic and the temperature of the reaction mixture was moderated by using an ice bath. The addition was finished in half an hour and the stirring was continued for one more hour. The clear solution from the first flask was then transferred to the second flask containing Grignard reagent via syringe. The addition was slightly exothermic. After 6 hrs of stirring, HCl (39 mL, 6N) was carefully added into the mixture at 0º-5ºC. The stirring was continued for another two more hours until TLC showed a major spot and then saturated NaHCO3 was added to neutralize the excess acid. The solution was filtered through a celite pad to remove any solid present in it. The organic phase in the filtrate was extracted with ethylacetate and dried over MgSO4. The excess solvent was removed under reduced pressure which afforded a yellowish crude liquid. The mixture was separated in a column chromatography using hexane and ethyl acetate (9:1). The first

1 fraction gave a clear liquid (4.3 g, 73%); HNMR (400 MHz, CDCl3) δ 8.11 (dd, , J =

9.0, 5.3 Hz, 2H), 7.19-7.14 (m, 2H), 4.44 (s, 1H), 1.82 (s, 3H); 13C NMR (100 MHz,

CDCl3) δ 192.0, 163.2 (d, J = 251.2 Hz), 133.2, 133.1 (d, J = 9.3 Hz), 129.9-118.6 (q, J =

284.3 Hz), 116.0,115.7 (d, 21.6 Hz), 80.3-79.2 (q, J = 28.3 Hz), 21.05; 19FNMR (75

MHz, CDCl3) δ -77.3 (s, 3F), 102.6 (tt, J = 8.2, 5.1 Hz, 1F)

(4-(diallylamino)phenyl)-3,3,3-trifluoro-2-hydroxy-2-phenylpropan-1-one (4.71)

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A mixture of 3,3,3-trifluoro-1-(4-fluorophenyl)-2-hydroxy-2-phenylpropan-1-one (2.45 g, 10.37 mmol) and N,N-diallylamine (3.93 mL, 40.0 mmol) in DMSO (12.0 g) was stirred at 165ºC for 16 hours. The reaction mixture was monitored by TLC which indicated the presence of starting material (fluorinated hydroxyketone) even after 16 hrs of refluxing so the reaction was further refluxed six hours more. The reaction mixture was diluted with ethyl acetate followed by addition of water (200 mL). The organic phase was separated in a separatory funnel. The organic phase was washed again with water, then brine and dried over MgSO4. The excess solvent was removed under reduced pressure. The concentrated crude mixture was further separated by Kugelrohr distillation.

The first fraction was obtained at 80ºC/75µ which was the starting material (0.26 g, 10.6

1 % unreacted); H NMR (400 MHz, CDCl3,) δ 8.15-8.12 (m, 2H), 7.21-7.17 (m, 2H), 1.84

13 (s, 3H); C NMR (100 MHz, CDCl3) δ 194.9, 166.1 (d, J = 251.1 Hz), 133.2 (d, J = 9.2

Hz), 130.1-119.6 (q, J = 284.5 Hz), 115.2 (d, 21.57 Hz), 67, 21.03 and the second

1 fraction was obtained at 140ºC/75µ (2.23 g, 76%); HNMR (400 MHz, CDCl3) δ 8.01

(dd, J = 7.37, 1.85 Hz, 2H), 7.10 (dd, J = 7.24, 2.09 Hz, 2H), 5.89-5.79 (m, 2H), 5.27-

13 5.14 (m, 4H), 4.02-4.01 (m, 4H), 3.02 (s, 3H); C NMR (100 MHz, CDCl3) δ 175.7,

146.5, 136.9-133.2 (d, J = 364.5 Hz), 130.5, 123.2, 120.4, 114.2, 111.3, 98.4, 51.0, 4.8

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2-(3-cyano-4-(4-(diallylamino)phenyl)-5-methyl-5-(trifluoromethyl)furan-2(5H)- ylidene)malononitrile (4.72)

A solution of clean 1-(4-(diallylamino)phenyl)-3,3,3-trifluoro-2-hydroxy-2- methylpropan-1-one (1.0 g, 3.0 mmol) in malononitrile (0.98 g, 15.0 mmol) in pyridine

(10.0 mL) was refluxed for four hours. The reaction mixture turned into yellow. The reaction was monitored by TLC. The TLC indicated distinct two spots and other three small spots, yellow spot corresponds to the reactant at the top, pink spot corresponds to product in the middle and dark brown and blue spots correspond to some polar substances at the bottom. The reaction was run another one hour more. The pink color became dark pink but the starting material was still present there. After seven hours of running, the pink spot become larger than the starting material. One equivalent of malononitrile was added to the mixture and ran for another hour but the starting material was still there however the pink spot become litter bigger comparatively. Then the reaction was left overnight stirring. The solution turned to dark pink with black materials in it. The reaction was stopped and dissolved in ethyl acetate and washed several times with water to remove excess malononitrile and pyridine. The organic phase was further washed with brine and dried over MaSO4. The excess solvent was removed under reduced pressure and the mixture was separated by column chromatography. The unreacted starting

1 material was eluted out first (0.24 g, 24.0%); HNMR (400 MHz, CDCl3) δ 8.1 (dd, J =

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7.33 Hz, 1.85 Hz, 2H), 7.30 (dd, J = 7.24 Hz, 2.09 Hz, 2H), 5.89-5.79 (m, 2H), 5.27-5.14

(m, 4H), 4.02-4.01 (m, 4H), 3.02 (s, 3H). The desired compound was eluted in the second

1 fraction (0.11 mg, 14%); H NMR (400 MHz, CDCl3,) δ 7.97 (d, J = 9.4 Hz, 2H), 6.77 (d,

J = 9.48 Hz, 2H), 5.29 (dd, J = 10.32 Hz, 0.83 Hz, 2H), 5.19 (dd, J = 17.16 Hz, 0.78 Hz,

13 2H), 4.09-4.08 (m, 4H), 2.09 (s, 3H), 2.11 (3H); C NMR (100 MHz, CDCl3) δ 175.7,

164.1, 154.1, 132.8, 130.8, 123.6, 120.7, 117.7, 113.9, 112.6, 112.0, 111.2, 110.5, 53.0,

20.7

2-(4-(4-aminophenyl)-3-cyano-5-methyl-5-(trifluoromethyl)furan-2(5H)- ylidene)malononitrile (4.73)

In a Schlenk tube containing tetrakis(triphenylphosphine)palladium (16.33 mg, 10-2 molar equivalent per allyl group to be removed) and N,N’dimethylbarbituric acid (0.65 g,

4.23 mmol, 3 equivalent per allyl group) under nitrogen was added a solution of 2-(3- cyano-4-(4-(diallylamino)phenyl)-5-methyl-5-(trifluoromethyl)furan-2(5H)- ylidene)malononitrile (0.25 g, 0.7 mmol) in dry degassed DCM (4 mL) with a syringe.

The mixture was stirred vigorously for two hours at 35ºC. The reaction was monitored by

TLC. After 3 hrs of stirring at room temperature, three spots were observed; pink spot- starting material at the top, and the bottom two yellow spots of mono and di deallylated

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compound. The reaction was continued overnight at 35ºC. The starting material did not go away and the size of the yellow spot remains unchanged. To make the reaction go forward, additional amount of tetrakis(triphenylphosphine)palladium (16.33 mg) and

N,N’dimethylbarbituric acid (0.65 g) were added. The reaction mixture was checked by

TLC after six hours of run. The reaction mixture was left for another six hours stirring at

40ºC. The reaction was checked by TLC. There was a trace of faint pink at the top

(starting material). The reaction was stopped. After cooling, the excess DCM was removed by rotary evaporation and the residue was dissolved in diethyl ether and filtered through a Celite pad. The ethereal solution was washed with saturated NaHCO3 to remove the unreacted, N,N’dimethylbarbituric acid and dried over MgSO4. The crude product was separated by silica gel column chromatography using gradient ethyl acetate and hexane as eluent. A trace of unreacted staring material was obtained in the first fraction as confirmed by HNMR. The monodeallyation product was obtained in the second fraction (0.026 g, 11%); 1H NMR (400 MHz, DMSO) δ 7.98 (d, J = 9.4 Hz, 2H),

6.72 (d, J = 9.42 Hz, 2H), 5.32 (dd, J = 10.31 Hz, 0.81 Hz, 1H), 5.01 (dd, J = 17.16 Hz,

0.78 Hz, 2H), 4.08-4.05 (m, 2H), 2.01 (s, 3H). The desired compound was obtained in the third fraction (0.112 g, 55.4%); mp 246ºC; FTIR 3447.65, 3347.42, 3234.56, 2228.67,

746.18; 1H NMR (400 MHz, DMSO) δ 7.95 (d, J = 8.8 Hz, 2H), 6.76 (d, J = 9.2 Hz, 2H),

2.20 (s, 3H); 19FNMR (75 MHz, DMSO) δ -76.85

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2-(4-(4-azidophenyl)-3-cyano-5-methyl-5-(trifluoromethyl)furan-2(5H)- ylidene)malononitrile (4.47)

A solution of NaNO2 (0.047 g, 0.68 mmol) in water (1.0 mL) was added dropwise to a solution of 2-(4-(4-aminophenyl)-3-cyano-5-methyl-5-(trifluoromethyl)furan-2(5H)- ylidene)malononitrile (0.11 g, 0.34 mmol) in concentrated HCl (6.0 mL) and water (1.0 mL) at 0º-5ºC. After stirring the mixture at the same temperature for 1 hour, a solution of

NaN3 (0.036 g, 0.54 mmol) in water (1.0 mL) was added dropwise to the mixture and stirred for 1 hour below 5ºC and then overnight at room temperature in the dark room.

The precipitate was filtered off, washed with water and air dried to obtain a yellow mass

(0.073 g, 60%); FTIR 2235.99, 2121.08, 2096.89, 1586.22, 710.9; 1H NMR (400 MHz,

DMSO) δ 7.90 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.8 Hz, 2H), 2.22 (s, 3H); 19FNMR (75

MHz, DMSO) δ -76.83

N,N-diallyl-4-bromoaniline (4.75)

To a solution of ethanol (57 mL) and water (14 mL) in a round bottom flask, were added

4-bromoaniline (3g, 17.45 mmol), allylbromide (3.54 mL, 41 mmol), and Na2CO3 (1.88

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g, 17.8 mmol). The mixture was refluxed overnight. The reaction mixture was checked by TLC which indicated no sign of starting material. Two spots were observed-one nonpolar big spot and the other less polar small spot. The crude product was extracted with diethyl ether and concentrated under reduced pressure to obtain yellowish oil. It was separated in a column chromatography. The desired product was obtained in the first

1 fraction as colorless oil (3.7 g, 84%); HNMR (400 MHz, CDCl3) δ 7.23-7.21 (m, 2H),

6.54-6.53 (m, 2H), 5.85-5.76 (m, 2H), 5.15-5.11 (m, 4H), 3.87-3.86 (m, 4H); 13C NMR

(100 MHz, CDCl3) δ 147.5, 133.3, 131.5, 116.0, 113.8, 108.0, 52.7.

2-hydroxy-2-methyl-1-(3,4,5-trifluorophenyl)propan-1-one (4.82)

To a solution isopropyl magnesium chloride (5.0 mL, 2M in THF) in anhydrous THF (5.0 mL) at 0ºC was added dropwise 2-bromo-1,3,5-trifluorobenzene (2.11 g, 1.2 mL, 10.0 mmol) in dry THF (5.0 mL) under nitrogen. The addition was finished in five minutes and stirred for two more hours at room temperature. A solution of 2-methyl-2-

(trimethylsilyloxy)propanenitrile (1.84 mL, 1.57 g, 10 mmol) in THF (5.0 mL) was added dropwise by a syringe to the Grignard reagent. The addition was slightly exothermic.

After 12 hrs of stirring, HCl (20.0 mL, 6N) was carefully added into the mixture at 0º-

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5ºC. The solution was stirred for another 12 hours. The TLC showed two distinct spots.

One very polar stayed at the bottom and other relatively less polar at the top (eluent-

Hexane:Ethylacetate, 9:1). A solution of saturated solution of NaHCO3 was added to neutralize the excess acid. The solution was filtered through a celite pad to remove any solid present in it. The organic phase in the filtrate was extracted with ethylacetate and dried over MgSO4. The excess solvent was removed under reduced pressure which afforded light yellow crude. The TLC of this crude still showed two spots. The mixture was separated in a short silica pad with ethyl acetate. The excess solvent was removed under reduced pressure. The HNMR indicated presence of more than one component although in TLC there was only one spot. Two compounds with similar Rf values were present in the mixture. A column chromatography was set to separate the mixture using hexane to ethylacetate:hexane (1:9) as eluent which again could not separate the mixture.

A Kugelrohr distillation was done (80ºC, 75μ) which afforded a clear liquid leaving behind a yellowish stain in the flask. The TLC of the resulting liquid showed a single spot. However HNMR indicated a mixture of other components. Again another distillation was run at 35ºC, 75μ; nothing came out in 3 hrs. Temperature was increased to 50ºC, 75μ then a clear liquid came out in the first fraction (0.3 g, 14%); 1H NMR (400

MHz, DMSO) δ 6.73 (m, 2H), 1.47 (s, 6H)

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2-phenyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-2-ium tetrafluoroborate (4.92)

To a solution of pyrrolidin-2-one (0.473 mL, 0.473 mL, freshly distilled) in anhydrous

DCM (50.0 mL) was added trimethyoxynium tetrafluoroborate (1.00 g, 6.75 mmol) all at once and stirred at room temperature overnight. To this clear solution was added phenyl hydrazine (0.61 mL, 6.11 mmol) and stirred for two days. The solution turned into wine red color. After 48 hrs, the mixture was concentrated in a vacuo affording a dark red paste residue which was dissolved in anhydrous MeOH (3.4 mL). To this solution was added triethyl orthoformate (13.5 mL) and the solution was refluxed overnight at 100ºC.

A yellow precipitate was obtained at room temperature which was filtered and recrystallized in methanol to give desired triazolium salt (1.11, 60%); mp156-159°C; 1H

NMR (400 MHz, DMSO) δ 10.68 (s, 1H), 7.90-7.72 (m, 2H), 7.72-7.68 (m, 2H), 7.65-

7.61 (m, 1H), 4.42 (t, J = 7.41 Hz, 2H), 3.21 (t, J = 7.48 Hz, 2H), 2.80-2.74 (tt, J = 7.45

Hz, 2H); 13C NMR (100 MHz, DMSO) δ 163.4, 138.8, 136.0, 130.8, 130.7, 121.7, 47.4,

27.0, and 21.7

3,3,3-trifluoro-1-(4-fluorophenyl)-2-hydroxy-2-phenylpropan-1-one (4.88)

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A solution of precatalyst 2-phenyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-2-ium tetrafluoroborate (217.5 mg, 0.8 mmol, 10 mol%), DBU (241.7 mg, 1.61 mmol, 20 mol%, 4-fluorobenzaldehyde (1.0 g, 8.05 mmol) and 2,2,2-trifluoro-1-phenylethanone

(2.8 g, 16.11, 2 equiv) in anhydrous THF (25.0 mL) was stirred for 18 hrs. The TLC indicated a new spot. The reaction was stopped and was directly mounted in the column for the purification. In the first fraction unreacted excess 2,2,2-trifluoro-1- phenylethanone was obtained. A yellowish clear liquid was obtained in the second fraction however on standing a room temperature it was solidified which was crushed and washed with hexane to obtain a white solid (1.73 g, 72%); mp 77ºC (sharp); 3435.36,

1675.9, 1595.7, 1056.8; 19FNMR-73.31 (s, 3F) and -102.59 (m, 1F); 1H NMR (400 MHz,

DMSO) δ 7.80-7.76 (m, 2H), 7.55-7.52 (m, 2H), 7.45-7.43 (m, 3H), 6.99-6.95 (m, 2H);

13C NMR (100 MHz, DMSO) δ 191.9, 165.9 (d, J = 264.3 Hz), 133.9, 133.8, 129.7,

129.1, 126.6, 120.5 (q, J = 286.9 Hz), 115.7, 115.5 and 92.1 (q, J = 29.13 Hz)

1-(3,4-difluorophenyl)-3,3,3-trifluoro-2-hydroxy-2-phenylpropan-1-one (4.96)

A solution of precatalyst 2-phenyl-6,7-dihydro-5H-pyrrolo[2,1-c][1,2,4]triazol-2-ium tetrafluoroborate (217.5 mg, 0.8 mmol, 10 mol%), DBU (241.7 mg, 1.61 mmol, 20 mol%, 3,4-difluorobenzaldehyde (1.14 g, 8.05 mmol) and 2,2,2-trifluoro-1-

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phenylethanone (2.8 g, 16.11, 2 equiv) in anhydrous THF (25.0 mL) was stirred for 18 hrs. The TLC showed the complete consumption of the aldehyde and new spot was observed. The reaction was stopped and was directly mounted in the column for the purification. In the first fraction, unreacted excess 2,2,2-trifluoro-1-phenylethanone was obtained. A yellowish clear liquid was obtained in the second fraction utilizing hexane:ethylacetate (9:1). The yellow liquid was kept in the refrigerator for days which eventually solidified and scrapped off the wall with ice cold hexane. The white solid was the desired compound (1.7 g, 67%); mp 78.3ºC (sharp); FTIR 3576.34, 3438.15, 1669.01,

1 1034.77; H NMR (400 MHz, CDCl3) δ 7.66 (ddd, J = 7.65, 2.2, 2.19, 2H), 7.60-7054

13 (m, 3H), 7.48-7.46 (m, 3H); C NMR (100 MHz, CDCl3) δ 191.0, 153.87 (dd, J = 12.57

Hz, 259.21 Hz), 149.79 (dd, J = 13.35 Hz, 250.57 Hz), 133.28, 129.9, 129.2, 128.32 (dd,

J = 3.61 Hz, 7.62 Hz), 126.46, 123.29 (q, J = 286.59 Hz), 120.39 (d, J = 19.25 Hz),

119.01, 117.30 (d, J = 17.80 Hz), and 82.18 (q, J = 27.8 Hz)

Diethyl 4-fluorobenzoylphosphonate by Arbuzov reaction (4.100)

To anhydrous triethyphosphite (5.76 g, 5.95 mL, 17.18 mmol, 1.1 equiv) was added 4- fluorobenzoylchloride (5.0 g, 3.72 mL, 15.62, 1 equiv) by dropwise fashion with vigorous stirring under nitrogen. The reaction was exothermic with the evolution of ethylchloride. The mixture became yellow and the temperature was maintained below

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25ºC by placing the reaction flask in an ice/water bath. The stirring was continued at room temperature under a gentle stream of nitrogen overnight. The reaction was monitored by TLC which indicated a single spot for the product. The product was a

1 yellowish liquid and isolated in quantitative yield; H NMR (400 MHz, CDCl3) δ 8.35-

8.32 (m, 2H), 7.21-7.16 (m, 2H), 4.32-4.24 (m, overlapping quartet, 4H), 1.39 (t, J = 7.06

13 Hz, 3H), 1.38 (t, J = 7.10 Hz, 3H); C NMR (100 MHz, CDCl3) δ 197.33 (d, J =

177.04), 166.70 (d, J = 258.33 Hz), 132.79 (d, J = 9.89 Hz), 128.02, 116.16 (d, J = 22.07

Hz), 64.11, 64.04, 16.37, 16.32;

Diethyl 1,1,1-trifluoro-3-(4-fluorophenyl)-3-oxo-2-phenylpropan-2-yl phosphate (4.102)

To a solution of diethyl 4-fluorobenzoylphosphonate (8.2 g, 31.53 mmol, 1 equiv) in anhydrous DMF (65.0 mL) were added 2,2,2-trifluoroacetophenone (7.70 g, 6.04 mL,

34.68 mmol, 1.1 equiv) and KCN (10 mol%, 0.5 g, 3.15 mmol). The reaction was monitored by TLC. The reaction was left overnight at room temperature which turned into dark brown solution. The reaction mixture was diluted with diethyl ether and water

(300 mL, 1:1 by volm). The organic phase was separated and aqueous phase was extracted with diethyl ether (3 x 100 mL). The combined organic phase was washed with

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brine, separated and dried over MgSO4. The organic phase was concentrated under reduced pressure. A yellow clear solution was obtained which upon standing at room temperature for several hours white crystals were obtained. The white crystal was washed with heptane. The remaining mother liqueur still contain the product which was separated in a column chromatography since it could not precipitated out under the same condition due presence of other impurities (11.2 g, 80%); mp 112.2ºC (sharp); 1H NMR (400 MHz,

CDCl3) δ 7.76-7.7 (m, 2H), 7.57-7.55 (m, 2H), 7.46-7.43 (m, 3H), 6.96-6.92 (m, 2H),

4.16-4.05 (m, 2H), 3.92-3.82 (m, 2H), 1.29 (dt, J = 7.02 Hz, 1.14Hz, 3H), 1.19 (dt, J =

13 7.02, 1.14Hz, 3H); C NMR (100 MHz, CDCl3) δ 188.05, 166.35 (d, J = 256.11 Hz),

133.0 (d, J = 9.37 Hz), 131.99 (d, J = 10.40 Hz), 130.17, 129.0, 126.37, 121.96 (q, J =

274.46 Hz), 115.28 (d, J = 21.96 Hz), 64.89 (d, J = 6.10 Hz), 64.35 (d, J = 5.61), 15.83

(dd, J = 12.23 Hz);

3,3,3-trifluoro-1-(4-fluorophenyl)-2-hydroxy-2-phenylpropan-1-one (4.88)

A mixture of diethyl 1,1,1-trifluoro-3-(4-fluorophenyl)-3-oxo-2-phenylpropan-2-yl phosphate (1.0 g, 2.30 mmol), Et2NH (23.0 mL) and water (2.3 mL) was stirred for 18 hours. The reaction mixture was monitored by TLC which indicated a major single spot other than the starting material. The reaction was stopped and the mixture was diluted with water (50.0 mL). The organic phase was extracted with diethyl ether and dried over

338

MgSO4. The mixture was filtered through a thin silica pad and the pad was washed with

5% diethyl ether and hexane. The solvent and the excess Et2NH was removed under reduced pressure which afforded a white solid. The solid was crashed with hexane and filtered to obtain a white mass (0.62 g, 91%); mp 77.2ºC (sharp); 1H NMR (400 MHz,

CDCl3) δ 7.80-7.76 (m, 2H), 7.55-7.52 (m, 2H), 7.43-7.39 (m, 3H), 6.97-6.93 (m, 2H);

13 C NMR (100 MHz, CDCl3) δ 188.05, 166.35 (d, J = 256.11 Hz), 133.0 (d, J = 9.37

Hz), 131.99 (d, J = 10.40 Hz), 130.17, 129.5, 129.0, 126.37, 121.96 (q, J = 274.46 Hz),

115.28 (d, J = 21.96 Hz), and 92.1 (q, J = 29.13 Hz)

1-(3,4-difluorophenyl)-3,3,3-trifluoro-2-hydroxy-2-phenylpropan-1-one] (4.96)

Step 1 [3,4-difluorobenzoyl chloride]

To a solution of 3,4-difluorobenzoic acid (2.0 g, 12.65 mmol) in dry DCM (25.0 mL) was added oxalyl chloride (1.75 mL, 20.12 mmol) followed by DMF (0.25 mL, 3.2 mmol) at

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0C. As soon as the DMF was added, a vigorous effervescence was observed. The bubbling was stop after 15 min. The reaction was stirred for overnight. The complete conversion of the acid to acid chloride was confirmed by IR spectra where typical OH absorption peak was completely disappeared. The yellowish reaction mixture was filtered through thin pad of silica gel (oven-dried at 130°C overnight). The silica gel was washed with dry DCM. The desired product was obtained as a faint yellow liquid after removal of the solvent under reduced pressure (quantitative). The compound was taken to further step without further purification as acid chloride is very moisture sensitive.

Step 2 [diethyl 3,4-difluorobenzoylphosphonate]

To a solution of anhydrous triethyphosphite (2.39 g, 13.98 mmol) in anhydrous toluene

(40.0 mL) was added 3,4-difluorobenzoylchloride (2.23 g, 12.65 mmol) by dropwise fashion with vigorous stirring under nitrogen at 0ºC. The reaction was exothermic with the evolution of ethylchloride. The mixture became pale yellow. The stirring was continued at room temperature under a gentle stream of nitrogen overnight. The reaction was monitored by TLC using hexane: ethyl acetate (1:1) as eluent. The TLC indicated a major single spot for the product and other small spots. The crude was separated by column chromatography (hexane:ethylacetate/1:1). The desired product was obtained as

1 faint yellowish liquid in the first fraction (2.1 g, 59.7% %); H NMR (400 MHz, CDCl3)

δ 8.17-8.16 (m, 1H), 8.11-8.06 (m, 1H), 7.34-7.27 (m, 1H), 4.32-4.25 (overlapping of quartet, 4H), 1.39 (overlapping of triplets, J = 7.06 Hz, 6H). The compound was not very

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pure and due to the moisture sensitive nature of this compound, further purification was not done and directly taken to the next step.

Step 3 [3-(3,4-difluorophenyl)-1,1,1-trifluoro-3-oxo-2-phenylpropan-2-yl diethyl phosphate]

To a solution of diethyl 3,4-difluorobenzoylphosphonate (1.38 g, 4.95 mmol, 1 equiv) in anhydrous DMF (10.0 mL) were added 2,2,2-trifluoroacetophenone (0.95 mL, 5.45 mmol, 1.1 equiv) and KCN (10 mol%, 0.078 g, 0.49 mmol). The solution turned pale yellow as KCN was added. The reaction was monitored by TLC. The reaction was left overnight at room temperature. The TLC of the mixture gave three major spots (eluent

4:1 hexane/ethylacetate). The first top spot corresponds to the starting material (2,2,2- trifluoroacetophenone). The second spot was the desired product and the third spot was the unreacted phosphonate ester. Another batch of KCN (0.078 g) was added and the reaction mixture left stirring further six hours. A major spot was observed in the TLC.

The reaction mixture was diluted with diethyl ether and water (150 mL, 1:1 by volm). The organic phase was separated and aqueous phase was extracted with diethyl ether (2 x 100 mL). The combined organic phase was washed with brine, separated and dried over

MgSO4. The organic phase was concentrated under reduced pressure. A yellow clear solution was obtained. The crude was separated in a column chromatography using hexane:ethyl acetate (4:1). The desired product was obtained in the second fraction (0.8

1 g, 36%); mp 110.6ºC (sharp); HNMR (400 MHz, CDCl3) δ 7.54-7.46 (m, 7H), 7.09-7.02

(m, 1H), 4.18-4.12 (m, 2H), 3.95-3.8 (m, 2H), 1.33 (t, J = 1.2 Hz, 3H), 1.22 (t, J = 1.2

13 Hz, 3H); C NMR (100 MHz, CDCl3) δ 191.07, 153.92 (dd, J = 259.74 Hz, 12.88 Hz),

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149.73 (dd, J = 250.11 Hz, 12.82 Hz) 133.3, 129.9, 129.23, 128.95, 128.29, 126.46,

123.22 (q, J = 281.66), 120.29 (d, J = 19.14), 117.30 (d, J = 17.81 Hz); 82.20 (q, J =

31 27.11 Hz), 65.83, 60.42, 15.23, 14.17; PNMR (32.46 MHz, CDCl3) δ -595194.9 (s, 1P);

19 FNMR (75 MHz, CDCl3) δ -73.31 (s, 3F), -128.84 to -128.95 (s, 1F), -136.02 to -

136.13 (s, 1F)

Step 4 [1-(3,4-difluorophenyl)-3,3,3-trifluoro-2-hydroxy-2-phenylpropan-1-one]

A mixture of 3-(3,4-difluorophenyl)-1,1,1-trifluoro-3-oxo-2-phenylpropan-2-yl diethyl phosphate (0.67 g, 1.474 mmol), Et2NH (15 mL) and water (1.5 mL) was stirred for 12 hours. The reaction mixture was monitored by TLC which indicated a major single spot other than the starting material. The desired product was separated in a silica pad using

5% diethyl ether and 95% hexane as clear sticky liquid which upon standing at room

1 temperature solidified (0.466 g. 100%); mp 79°C (sharp); H NMR (400 MHz, CDCl3) δ

7.63 (ddd, J = 11.04, 7.70, 2.20, 2H), 7.57-7.51 (m, 3H), 7.46-7.40 (m, 3H); 13C NMR

(100 MHz, CDCl3) δ 191.0, 153.87 (dd, J = 12.57 Hz, 259.21 Hz), 149.79 (dd, J = 13.35

Hz, 250.57 Hz), 133.28, 129.9, 129.2, 128.32 (dd, J = 3.61 Hz, 7.62 Hz), 126.46, 123.29

(q, J = 286.59 Hz), 120.39 (d, J = 19.25 Hz), 119.01, 117.30 (d, J = 17.80 Hz), and 82.18

(q, J = 27.8 Hz)

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1-(4-azido-3-fluorophenyl)-3,3,3-trifluoro-2-hydroxy-2-phenylpropan-1-one (4.105)

To a solution of sodium azide (0.36 g, 5.48 mmol) in DMSO (9.0 mL) was added 1-(3,4- difluorophenyl)-3,3,3-trifluoro-2-hydroxy-2-phenylpropan-1-one (1.3 g, 4.11 mmol) and stirred for 5 hrs at 40°C. The reaction was monitored by TLC. The reaction mixture was poured into the water. A sticky substance was crashed out. The mixture was stirred for 2 more hours. The sticky substance was dissolved in Ethyl acetate and separated from water in a separatory funnel. The organic phase was washed with brine and dried over

MgSO4. The excess solvent was removed under reduced pressure and the yellow oily mixture was further separated by column chromatography using ethyl acetate:hexane

(1:9) as eluent. The titled compound was obtained as a clear faint yellow liquid. The liquid was left inside a refrigerator overnight. The liquid solidified and was crushed with a glass rod and washed with hexane to afford a white solid. (1.1 g, 87%); FTIR 3409.22,

2130.39, 1691.87, 1072.16; 1H NMR (400 MHz, MeOH) δ 7.6-7.53 (m, 4H), 7.47-7.45

(m, 3H), 6.97 (dd, J = 7.13 Hz, 1H), 4.64 (s, 1H); 13C NMR (100 MHz, MeOH) δ 191.0,

153.9 (d, J = 250.57 Hz) , 133.57, 1293.83, 129.20, 128.15, 128.12, 126.52, 123.25 (q, J

= 292.23Hz) 120.50, 119.25, 119.04; 19FNMR (75 MHz, MeOH) δ -73.76 (s, 3F) and -

124.63-124.69 (m, 1F)

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1-(4-(diallylamino)-3-fluorophenyl)-3,3,3-trifluoro-2-hydroxy-2-phenylpropan-1-one (4.108)

A mixture of 1-(3,4-difluorophenyl)-3,3,3-trifluoro-2-hydroxy-2-phenylpropan-1-one

(3.40 g. 10.75 mmol), a crystal of p-TsOH and N,N-diallylamine (1.4078 mL, 14.33 mmol) in DMSO (15.0 mL) was stirred at 165ºC for 24 hours under nitrogen. The mixture was poured into water and the organic phase was extracted with water. The organic phase was washed again with water, then brine and dried over MgSO4. The excess solvent was removed under reduced pressure. The concentrated crude was further separated by column chromatography using hexane (70%) and ethyl acetate (30%). The desired compound was obtained in the first fraction as a yellowish liquid (2.88 g, 68%);

1 HNMR (400 MHz, CDCl3) δ 7.65-7.63 (m, 2H), 7.50-7.48 (m, 2H), 7.40-7.37 (m, 3H),

6.48-6.46 (m, 2H), 5.81-5.72 (m, 2H), 5.50 (s, 1H), 5.18-5.08 (m, 4H) 3.93-3.61 (m, 4H);

13 C NMR (100 MHz, CDCl3) 190.14, 136.34, 134.04, 131.97, 128.97, 128.87, 126.98,

116.84, 110.67, 52.48

2-(4-fluorophenyl)-1,3-dithiane (4.110)

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A reaction mixture of 4-fluorobenzaldehyde (1.0 mL, 1.175 g, 9.47 mmol), 1,3- propanedithiol (1.045 mL, 10.41 mmol), and silica- sulfuric acid (473.43 mg) in acetonitrile (19 mL) was stirred at room temperature. The reaction was monitored by

HNMR indicating the disappearance of CHO proton for the completion of reaction. After completion of reaction in 1 hour, the excess acetonitrile was removed under reduced pressure and residue solid mass was combined with DCM and filtered. The catalyst was washed with DCM. The organic layer was washed with NaHCO3 (100 mL, 10%), followed by water (100 mL) and brine (50 mL). The organic layer was dried over MgSO4 and filtered. The excess solvent was removed under reduced pressure and the pure product (white solid) was obtained quantitatively (2.47 g); mp 105.5ºC; 1HNMR (400

MHz, CDCl3) δ 7.46-7.42 (m, 2H), 7.03-6.99 (m, 2H), 5.14 (s, 1H), 3.04-3.287 (m, 2H),

12 2.92-2.87 (m, 2H), 2.17 (m, 1H), 1.92-1.89 (m, 1H); CNMR (100 MHz, CDCl3) δ

162.53 (d, J = 247.35 Hz), 135.02, 129.54 (d, J = 8.28 Hz), 115.65 (d, J = 21.63 Hz),

50.50, 32.07, 24.99.

1,1,1-trifluoro-2-(2-(4-fluorophenyl)-1,3-dithian-2-yl)propan-2-ol (4.112)

To a degassed and stirred solution of freshly distilled anhydrous diisopropylamine (0.72 mL, 5.15 mmol) in dry THF (15 mL) at -78ºC was added n-BuLi, 1.6 M in hexane (3.22

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mL, 5.15 mmol) dropwise by a syringe pump. The solution was stirred at -78ºC for half an hour, 0ºC for 20 min and cooled back to -78ºC. To this stirred solution of LDA was added a solution of 2-(4-fluorophenyl)-1,3-dithiane (1.0 g, 4.67 mmol) in dry THF (35 mL) via syringe pump very slowly so the temperature remain constant and the reaction mixture was stirred at -78ºC for 2 hours. To this reaction mixture was added dropwise a solution of 1,1,1-trifluoroacetone (0.46 mL, 5.15 mmol) in THF (5.0 mL) and left stirring overnight at -78ºC to room temperature. The reaction was checked by TLC, there were two distinct spots for unreacted dithiane and the desired product. The reaction mixture was poured into a saturated NH4Cl (70 mL) and extracted with ethyl acetate (3x100 mL).

The combined organic phase was washed with water, brine and dried MgSO4. The crude was filtered through a thin pad of silica gel and separated in column chromatography

(hexan:ethyl acetate 9:1). The unreacted dithiane was obtained in first fraction and the desired product was obtained in the second fraction as yellow oil (0.55g, 36%); 1H NMR

(400 MHz, CDCl3) δ 8.07-8.04 (m, 2H), 7.11-7.07 (m, 2H), 2.71-2.62 (m, 4H), 1.89-1.65

12 (m, 2H), 1.65 (d. J = 1.04 Hz, 3H); C NMR (100 MHz, CDCl3) δ 162.27 (d, J = 249.13

Hz), 133.51 (d, J = 8.13 Hz), 131.87, 125.33 (q, J = 289.3Hz), 115.31 (d, J = 21.37 Hz),

79.57 (q, J = 26.85 Hz), 27.56 (d, J = 5.53 Hz), 24.21, 19.01

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3,3,3-trifluoro-1-(4-fluorophenyl)-2-hydroxy-2-methylpropan-1-one (4.68)

To a stirred solution of 1,1,1-trifluoro-2-(2-(4-fluorophenyl)-1,3-dithian-2-yl)propan-2-ol

(1.52 g, 4.67 mmol), pyridine (0.56 mL) in DCM (28.0mL) and water (5.0 mL) was added pyridine tribromide (2.22g, 6.9 mmol) followed by tetraethyl ammonium bromide

(98.0 mg, 0.47 mmol). The resulting mixture stirred for 24 hours at room temperature.

The orange slowly faded away. The reaction was monitored by TLC. The reaction mixture was poured into water (70.0 mL) and extracted with DCM (3x50 mL). The combined organic fractions were washed with water (250 mL), brine (250 mL), and dried over MgSO4, filtered and evaporated to give yellow oil which was further separated in column chromatography (hexane:ethyl acetate/4:1). The desired product was obtained in

1 the second fraction as a yellow oil (1.1, quantitative); HNMR (400 MHz, CDCl3) δ 8.13-

12 8.09 (m, 2H), 7.19-7.15 (m, 2H), 4.46 (s, 1H), 1.82 (s, 3H); C NMR (100 MHz, CDCl3)

δ 163.16 (d, J = 248.86 Hz), 138.35, 131.54, 127.88, 122.86 (q, J = 261.21 Hz), 115.45

(d, J = 21.79 Hz), 67.76 (q, J = 31.27 Hz), 41.12.

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3 (E)-2-(4-fluorobenzylidene)-1,1-dimethylhydrazine (4.116)

To a vigorously stirred solution of 4-fluorobenzaldehyde (5.35 g, 50 mmol) in dry toluene (10 mL) was added drop wise N,N-dimethylhydrazine (4.2 mL, 55 mmol). The reaction was monitored by TLC and stirring was continued for 4 hrs until there was a single spot for the product. The mixture was dried over MgSO4. The solvent was removed under reduced pressure to afford a white solid (8.42 g, 91%); mp 44.5-46ºC;

1 HNMR (400 MHz, CDCl3) δ 7.55-7.49 (m, 2H), 7.18 (s, 1H), 7.02-6.96 (m, 2H), 2.93

13 (d, J = 0.4 Hz, 6H); C NMR (100 MHz, CDCl3) δ 163.4, 161.0 (d, J = 244.8 Hz), 133.1,

131.6, 127.1, 127.0 (d, J = 8 Hz), 115.4, 115.2 (d, J = 21.7 Hz), 42.8; 19FNMR (376.5

MHz, CDCl3) δ -114.8 to -114.85 (m, 1F)

(Z)-3-(2,2-dimethylhydrazono)-1,1,1-trifluoro-3-(4-fluorophenyl) propan-2-one

(4.117)

A solution of 3 (E)-2-(4-fluorobenzylidene)-1,1-dimethylhydrazine (4.0 g, 24.06 mmol) and 2,6-distilled dry lutidine (7.74 g, 8.4 mL, 72.2 mmol) was prepared in dry CHCl3 (96 mL) at 0ºC. To this well stirred solution was added drop wise TFAA (50.43 g, 33.9 mL,

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240.6 mmol, 10 equiv). The mixture was stirred for one and half hours at 0ºC and stirred for additional one hour at room temperature. The mixture was monitored by TLC which showed the complete consumption of the starting material resulting four spots (Hexane:

EtOAc/1:1). The mixture was poured into 0.1N HCl, extracted with DCM, washed with water and then with saturated aqueous Na2CO3 solution. The solution was dried over

MgSO4 and the excess solvent was removed under reduced pressure. The mixture was separated by column chromatography using Hexane: EtOAc:Hexane (1:1); 1H NMR (400

13 MHz, CDCl3,) δ 7.23-7.19 (m, 2H), 7.11-7.06 (m, 2H), 3.08 (s, 6H); C NMR (100 MHz,

CDCl3) δ 198.2, 163.09 (d, J = 249.34Hz), 130.82 (d, J = 8.25Hz), 126.37, 115.64 (q, J =

287.71Hz), 115.41 (d, J = 22.13Hz), 84.54, 39.48

(E)-1,1-dimethyl-2-(4-nitrobenzylidene)hydrazine (4.121)

To a vigorously stirred solution of 4-nitrobenzaldehyde (2.0 g, 13.23 mmol) in dry toluene (10 mL) was added drop wise N,N-dimethylhydrazine (1.1 mL, 14.5 mmol). The reaction was monitored by TLC and stirring was continued for 4 hrs until there was a single spot for the product. The mixture turned into a yellow solution. The mixture was dried over MgSO4 and the solvent was removed under reduced pressure to afford a

1 yellow solid (2.4 g, 94%); mp 112.5ºC (reported 111.0 ºC); HNMR (400 MHz, CDCl3) δ

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8.15-8.13 (m, 2H), 7.63-7.61 (m, 2H), 7.09 (s, 1H), 3.096 (s, 3H), 3.095 (s, 3H); 13C

NMR (100 MHz, CDCl3) δ 145.8, 143.7, 126.9, 125.1, 124.0, 42.4

(Z)-3-(2,2-dimethylhydrazono)-1,1,1-trifluoro-3-(4-nitrophenyl)propan-2-one (4.122)

A solution of (E)-1,1-dimethyl-2-(4-nitrobenzylidene)hydrazine (2.0 g, 10.35 mmol) and

2,6-distilled dry lutidine (3.6 mL, 31.0 mmol) was prepared in dry CHCl3 (41 mL) at 0ºC.

To this well stirred solution was added drop wise TFAA (24.7 g, 14.6 mL, 34.103.5 mmol). The mixture was stirred for one and half hours at 0ºC and stirred for additional one more hour at room temperature. The mixture was monitored by TLC which showed the complete consumption of the starting material resulting three spots (DCM as eluent).

The mixture was poured into 0.1N HCl, extracted with DCM, washed with water and then with saturated aqueous Na2CO3 solution. The solution was dried over MgSO4 and the excess solvent was removed under reduced pressure. The mixture was separated by column chromatography using DCM as eluent to afford the desired product in 2nd fraction

1 as yellow solid (1.96 g, 65%); mp 111.5ºC (reported 116°C); HNMR (400 MHz, CDCl3)

δ 8.23 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 8.8 Hz, 2H), 3.10 (s, 6H); 13C NMR (100 MHz,

CDCl3) δ 176.5 (q, J = 124 Hz), 146.7, 139.1, 130.6, 127.7, 121.8, 116.7 (q, J = 1157.6

19 Hz) 46.8, FNMR (376 MHz, CDCl3) δ -6.9.17 (s, 3F).

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2-hydroxy-N-methoxy-N,2-dimethylpropanamide (4.134)

To a solution of 2-hydroxy-2-methylpropanoic acid (4.0 g, 40.0 mmol) in 80.0 mL anhydrous DCM was added 1,1’-carbonyldiimidazole (8.42 g, 52.0 mmol) all at once.

The solution was stirred for 20 minutes, then N,O-dimethylhydroxylamine HCl (5.12 g,

52.0 mmol) was added in one portion. The reaction was allowed to stir at room temperature overnight. Diethyl ether (100.0 mL) was added and after stirring for 5 min the clear solution decanted. The residue was again stirred with ethyl ether (40 mL), which as decanted off. The combined organic solvent was washed with saturated NaHCO3, brine and dried over MgSO4. The crude product was purified by column chromatography using hexane:ethyl acetate with increasing polarity. The desired product was obtained as

1 a clear liquid (3.25 g. 57.5%); HNMR (400 MHz, CDCl3) δ 3.72 (s, 3H), 3.28 (s, 3H),

13 1.47 (s, 6H); C NMR (100 MHz, CDCl3) 177.25, 72.14, 61.02, 33.66, 26.5

N-methoxy-N, 2-dimethyl-2-(trimethylsilyloxy)propanamide (4.135)

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To an ice chilled solution of 2-hydroxy-N-methoxy-N,2-dimethylpropanamide (3.0 g,

20.4 mmol) in pyridine (1.95 g, 1.98 mL, 24.7 mmol) was added TMSCl (3.2 mL, 25.0 mmol) drop wise. The mixture was stirred for some time at 0ºC then stirred overnight at room temperature. The mixture was poured into saturated NaHCO3 solution (100 mL) and diethyl ether (100 mL). The mixture was stirred for 1 hour. The organic layer was separated in a separating funnel, washed with brine and dried over MgSO4. The excess solvent was removed under reduced pressure. The resulting pale yellow solution was

1 distilled under vacuum to give the clear liquid (2.51 g, 56%); HNMR (400 MHz, CDCl3)

13 δ 3.73 (s, 3H), 3.29 (s, 3H), 1.47 (s, 6H); C NMR (100 MHz, CDCl3) 174.29, 76.5,

60.5, 28.3, 25.9, 2.1

N,N-diallylthiazol-2-amine (4.137)

To a solution of ethanol (65.0 mL) and water (18.7 mL) in a round bottom flask, were added 2-aminothiazole (10.0 g, 100.0 mmol), allylbromide (20.27 mL, 234.2 mmol), and

Na2CO3 (11.66 g, 109.97 mmol). The mixture was refluxed overnight. The reaction mixture was monitored by TLC. An additional amount of allylbromide (1.0 mL) was added since the reaction was not complete. There was always presence of three spots- titled compounds, monoallylated intermediate and starting materials. The crude was

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extracted with diethyl ether and concentrated under reduced pressure to obtain dark brown oil. It was separated in a column chromatography using hexane:ethylacetate (1:1) as eluent. The desired product was obtained in the second fraction as colorless oil (10.02

1 g, 57%); HNMR (400 MHz, CDCl3) δ 6.5 (d, J = 4.93 Hz, 1H), 6.01-5.88 (m, 2H), 5.86

(d, J = 4.92 Hz, 1H), 5.27 (dq, J = 16.8 Hz, 1.88 Hz, 1H), 5.19 (dq, J = 24.4 Hz, 1.2 Hz,

1H), 5.21-5.19 (m, 1H), 5.09 (dq, J = 10.0 Hz, 1.6 Hz, 1.88 Hz, 1H), 4.36 (t, J = 1.6 Hz,

1H), 4.35 (t, J = 1.6 Hz, 1H), 3.74 (t, J = 2.0 Hz, 1H), 3.73 (t, J = 1.6 Hz, 1H); 13C NMR

(100 MHz, CDCl3) δ 158.7, 136.0, 132.9, 126.8, 117.6, 114.8, 96.8, 57.2, 48.1

5-bromothiazol-2-amine (4.141)

To a solution of 2-aminothiazole (4.0 g, 40 mmol) in 306 mL of glacial acetic acid was added a solution of 2.06 mL (40.0 mmol) of bromine in glacial acetic acid (14.0 mL) drop wise at 40°C. The mixture was heated to 65°C, stirred for 10 min and then left overnight at room temperature. The excess acetic acid was removed under reduced pressure. The residue was treated with NH4OH and the organic phase was extracted with dichloromethane which was then washed with brine and dried over MgSO4. The excess solvent was removed under reduced pressure and purified in a column chromatography using ethyl acetate:hexane (1:1) to obtain 2.6 g (36%); mp 102°C; 1HNMR (400 MHz,

13 CDCl3) δ 6.98 (s, 1H), 5.02 (s, 2H); C NMR (100 MHz, CDCl3) δ 168.1, 139.7, 95.9

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Experimental of Chapter 5

N,N-diethyl-3-hydroxy-4-nitrosobenzenaminium chloride (5.2)

To a solution of 3-diethylaminophenol (1.47 g, 8.9 mmol) in HCl (9 mL, 10 M) and water

(4.5 mL) at 0ºC in an ice bath, was added a solution of NaNO2 (0.92 g, 13.3 mmol) in water (11.2 mL) by a syringe pump at the rate of 0.07 mL per minute. The mixture was stirred for 2.5 hrs. at 0°C and filtered to remove the residual impurities. A yellow solid was obtained (1.23 g, 60%); mp 173°C (reported 175°-177°C); 1H NMR (400 MHz,

CD3OD,) δ 7.73 (d, J = 10.4 Hz, 1H), 7.22 (dd, J = 8.0, 2.4 Hz. 1H), 6.42 (d, J = 2.4 Hz,

1H), 3.97 (q, J = 7.2 Hz, 2H), 3.89 (q, J = 7.2 Hz, 2H), 1.41 (t, J =6.4 Hz, 6H). 13C NMR

(100 MHz, CD3OD) δ 165.7, 162.6, 144.4, 123.4, 119.5, 97.2, 13.2, 11.7

Phenol Nile Red (5.4)

To a solution of 4-nitroso-3-hydroxy-N,N-diethylaniline hydrochloride (0.51 g, 2.22 mmol) in MeOH (20 mL) and HCl (0.38 mL, 12 M) was added 1,6-

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dihydroxynaphthalene (0.35 g, 2.22 mmol) all in one portion. The reaction mixture was refluxed for 5 hrs. The solution became dark green. In a TLC test, three spots were observed. The middle significant bright pink spot may be Nile red which was red fluorescence under UV. The excess solvent was removed under reduced pressure and the residue was dissolved in DCM, impregnated with silica gel and ran a silica gel column chromatography to separate the desired product using DCM:EtOAc (1:1) ratio of eluent to afford a dark green solid (0.44 g, 56%) mp 275º-280ºC); 1H NMR (400 MHz, DMSO)

δ 10.4 (s, 1H), 7.95 (d, J = 8.6 Hz, 1H), 7.87 (d, J = 2.4 Hz), 7.55 (d, J = 9.08 Hz, 1H),

7.08 (dd, J = 8.6 Hz, 2.48 Hz. 1H), 6.78 (dd, J = 9.12 Hz, 2.48 Hz, 1H), 6.61 (d, J = 2.52

Hz, 1H), 6.13 (s, 1H), 3.48 (q, J = 6.93 Hz, 4H), 1.15 (t, J = 7.0 Hz, 6H). 13C NMR (100

MHz, DMSO) δ 181.6, 160.6, 151.5, 150.6, 146.3, 138.6, 133.7, 130.8, 127.5, 123.8,

118.3, 109.9, 108.2, 107.9, 104.1, 96.0, 44.4, 12.4

9-(diethylamino)-2-(2-(2-(2-ethoxyethoxy)ethoxy)ethoxy)-5H-benzo[a]phenoxazin-5- one [Nile red PEG (P-189)]

To a solution of 9-(diethylamino)-2-hydroxy-5H-benzo[a]phenoxazin-5-one (Nile red phenol) 0.2 g, 0.60 mmol and KOH (0.60 g, 10 mmol) in dry DMF (20.0 mL) was added

2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (0.12 g, 0.6 mmol) in DMF

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(3.5 mL) at 0°C under nitrogen dropwise. After fifteen minutes the reaction was heated to

80°C for 6h. The reaction was checked by TLC which indicated that the reaction was incomplete. A drop of 2-(2-(2-ethoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate was added and the reaction was run overnight. The starting material was not gone completely.

The reaction was stopped and the solvent was removed under high vacuum. The solid residue was washed with hexane. It was then further dissolved in chloroform and impregnated on silica gel and a column chromatography was run using EtOAc (80%) and

Hexane (20%) as eluent. The starting material Nile red phenol was recovered as the first fraction (0.045 g, 22%). The desired product was obtained in the second fraction (0.075

1 g, 48%); mp 87°C (sharp); H NMR (400 MHz, CDCl3,) δ 8.22 (d, J = 8.8 Hz, 1H), 8.08

(d, J = 2.8 Hz, 1H), 7.61 (dd, J = 9.2 Hz, 1.2 Hz, 1H), 7.20 (dd, J = 8.6 Hz, 2.4 Hz, 1H),

6.67 (dd, J = 9.2 Hz, 2.8 Hz, 1H), .6.47 (d, J = 2.72 Hz, 1H), 6.30 (s, 1H); 13C NMR (100

MHz, CDCl3) δ 183.4, 161.4, 150.7, 150.7, 146.9, 134.1, 131.0, 127.7, 125.8, 124.9,

118.5, 109.5, 106.6, 105.3, 96.3, 70.9, 70.7, 70.6, 69.8, 69.6, 67.8, 66.6, 45.0, 15.1, 12.6

Potassium 4-(9-(diethylamino)-5-oxo-5H-benzo[a]phenoxazin-2-yloxy)butane-1- sulfonate [Nile red SO3K (P-193)]

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To a solution of hydroxy Nile red (0.1 g, 0.3 mmol) in t-BuOH (1.0 mL) at 35 ºC was added t-BuOK (1 M in t-BuOH, 0.3 mL) and butane sultone (0.03 mL, 0.04 g. 0.3 mmol).

The reaction was run for 3h and monitored by TLC. A small amount of t-BuOH (0.3 mL), t-BuOK (0.1 mL) and butane sultone (9µL, 0.1 mmol) was added as the TLC indicated that the reaction was incomplete. The reaction was run for another 3h. The excess solvent and reagents were removed under reduced pressure and remaining solid was scraped off the wall the washed with hot Et2O to afford the desired product (0.092 g,

60%); mp>250ºC;.1H NMR (400 MHz, DMSO) δ 8.03 (d, J = 8.8 Hz, 1H), 7.95 (d, J =

2.4 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.27 (dd, J = 8.6 Hz, J = 2.4 Hz, 1H), 6.82 (dd, J =

9.2 Hz, J = 2.4 Hz, 1H), 6.19 (s, 1H), 4.16 (t, J = 6.0 Hz, 2H) 3.50 (q, 6.8 Hz, 4H), 3.34-

3.32 (m, 2H), 1.87-1.84 (m, 2H), 1.79-1.75 (m, 2H), 1.16 (t, J = 6.8 Hz, 6H); 13C NMR

(100 MHz, DMSO) δ 181.9, 161.8, 152.2, 151.2, 146.9, 138.7, 134.0, 131.5, 127.6,

125.2, 124.4, 118.4, 110.5, 106.7, 104.5, 96.4, 74.8, 68.3, 65.3, 55.3, 51.5, 49.0, 44.9,

28.3, 22.3, 12.9; MS (ESI negative mode) [M-] 469, 334, 284, 153, 133.

8-hydroxy-7-nitrosojulolidine (5.5)

In a 100 mL pear shaped flask was added 8-hydroxyjulolidine (1.3 g, 6.87 mmol), ice

(2.5 g), and HCl (12M, 3 mL). The flask was immersed in an ice bath and the mixture was stirred until a homogenous solution was formed. To that stirred solution, a solution

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of NaNO2 (7.33 g, 106.2 mmol) in water (5 mL), precooled to 5ºC, was added with vigorous stirring at such a rate that temperature of the reaction mixture did not rise above

5ºC. After the addition was complete, the reaction mixture was stirred for additional 6h and filtered in a Buchner funnel. The solid was washed with ice cold water and vacuum dried at room temperature overnight to afford dark brown desired product as

1 hydrochloride salt (0.98 g, 56.0%); H NMR (400 MHz, CDCl3,) δ 11.9 (s, 1H), 6.82-6.81

(m, 1H), 3.46-3.33 (m, 4H), 2.74-2.62 (m, 4H), 2.06-1.95 (m, 4H); 13C NMR (100 MHz,

CDCl3) δ 165.3, 147.9, 146.7, 133.1, 127.6, 121.8, 59.25, 59.1, 31.4, 31.1, 29.5, 22.3

3-cyano-2-dicyanomethylen-4,5,5-trimethyl-2,5-dihydrofuran (5.13)

To a solution of 3-hydroxy-3-methylbutan-2-one 20 (4.75 g, 42.95 mmol) in pyridine (25 mL) was added malononitrile (6.15 g, 93 mol), one drop of acetic acid and stirred at room temperature for 24 hours. The reaction temperature was controlled without exceeding the room temperature by the use of an ice bath at the beginning of the reaction. The reaction mixture was then poured into 400 ml ice water with vigorous stirring. The precipitate was collected by vacuum filtration and recrystallized from ethanol to give light green solid

1 13 (6.3 g, 75%); mp 200.5° C; H NMR (400 MHz, CDCl3) δ 2.38 (s, 3H), 1.63 (s, 6 H); C

NMR (100 MHz, CDCl3) δ 14.22, 24.46, 58.63, 99.89, 105.0, 109.09, 110.56, 111.16,

175.31, 182 69 ppm.

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(E)-2-(3-cyano-4-(3-ethoxy-4-hydroxystyryl)-5,5-dimethylfuran-2(5H)- ylidene)malononitrile (DCDHF 203)

A mixture of 3-ethoxy-4-hydroxybenzaldehyde (0.59 g, 3.54 mmol) and 2- dicyanomethylen-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (0.67 g, 3.54 mmol) in 11.0 mL of ethanol was refluxed in a 100 mL RB flask. The reaction mixture was monitored by TLC. For the complete reaction, the mixture was refluxed overnight. From the cooled mixture, a red precipitate was obtained in a vacuum filtration and further washed with hexane and then by diethyl ether to afford the desired product as a brick red solid (0.91 g,

74.0 %); mp>250 ºC; FTIR 3285.95, 2988.48, 2935.11, 2231.42, 2213.24, 1618.69,

-1 1 1372.75, 1269.83 cm ; H NMR (400 MHz, DMSO,) δ 10.23 (s, 1H), 7.88 (d, J = 16.4

Hz, 1H), 7.5 (d, J = 2.0 Hz, 1H), 7.42 (dd, J = 8.4 Hz, 2 Hz, 1H), 7.03 (d, J = 16.0 Hz,

1H), 6.92 (d, J = 8.4 Hz, 1H), 1.78 (s, 6H); 13C NMR (100 MHz, DMSO) δ 177.7, 176.2,

152.8, 149.3, 147.9, 126.6, 125.9, 116.6, 114.4, 113.4, 112.6, 112.3, 111.7, 99.4, 64.6,

25.8, 15.0

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(E)-2-(3-cyano-5,5-dimethyl-4-(2,4,6-trihydroxystyryl)furan-2(5H)- ylidene)malononitrile

Yield: Brick red solid (0.95 g, 74%); mp>250ºC; FTIR 3285.95, 2988.48, 2935.11,

2231.42, 2213.24, 1618.69, 1372.75, 1269.83 cm-1; 1H NMR (400 MHz, DMSO) δ 10.84

(s, 1H), 8.17 (d, J = 16.4 Hz, 1H), 7.84 (dd, J = 7.8, Hz, 1.6 Hz, 1H), 7.42 (d, J = 16.4

Hz, 1H), 7.39-7.35 (m, 1H), 7.39-7.35 (m, 1H), 6.97-6.91 (m, 2H), 1.77 (s, 6H); 13C

NMR (100 MHz, DMSO) δ 177.8, 176.7, 158.8, 144.4, 134.5, 131.0, 121.7, 120.2, 117.0,

115.5, 113.3, 112.4, 111.7, 99.5, 97.9, 95.6, 25.6

(E)-2-(3-cyano-4-(3,4-dihydroxystyryl)-5,5-dimethylfuran-2(5H)- ylidene)malononitrile (DCDHF 202)

Yield: Brown solid (0.67 g, 60%); mp>250 ºC; FTIR 3355.38, 3165.02, 2997.44,

2785.71, 2234.2, 2221.31, 1601.69, 1378.81, 1269.24 cm-1; 1H NMR (400 MHz, DMSO)

δ 10.62 (s, 1H), 8.16 (d, J = 16.4 Hz, 1H), 7.73 (d, J = 9.2 Hz, 1H), 7.19 (d, J = 16.0 Hz,

1H), 6.40-6.38 (m, 2H), 1.74 (s, 6H); 13C NMR (400 MHz, DMSO) δ 186.2, , 177.7,

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164.8, 161.8, 145.7, 133.4, 114.5, 113.7, 112.7, 111.9, 111.3, 109.8, 102.9, 101.8, 98.9,

25.9, 23.6

(E)-2-(3-cyano-4-(4-hydroxystyryl)-5,5-dimethylfuran-2(5H)-ylidene)malononitrile

Yield: Red solid (0.65 g, 64%), mp >270ºC; FTIR 3353.9, 2223.7, 1552.4, 1377.9,

1161.2 cm-1; 1H NMR (400 MHz, DMSO) δ 10.6 (s, 1H), 7.9 (d, J = 16.4 Hz, 1H), 7.81

(d, J = 8.8 Hz, 2H), 7.02 (d, J = 16.0 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 1.78 (s, 6H); 13C

NMR (400 MHz, DMSO) δ 177.7, 176.3, 162.7, 148.8, 132.8, 126.1, 116.9, 113.4, 113.4,

112.5, 112.2, 111.7, 99.5, 97.0, 25.7

(E)-4-(2-(4-cyano-5-(dicyanomethylene)-2,2-dimethyl-2,5-dihydrofuran-3-yl)vinyl)- 2-ethoxyphenyl acetate (P-221)

In an oven dried round bottom flask, a solution of (E)-2-(3-cyano-4-(3-ethoxy-4- hydroxystyryl)-5,5-dimethylfuran-2(5H)-ylidene)malononitrile (0.57 g, 1.65 mmol) and pyridine (0.2 mL) in dry acetonitrile (2.5 mL) was prepared. To this solution, acetyl

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chloride (0.23 mL, 3.3 mmol) was added dropwise stirring under nitrogen at 0ºC in an ice bath. The dye DCDHF was not very soluble in acetonitrile but as soon as the acetyl chloride was added the brick red color immediately changed to clear yellowish solution.

The reaction was left stirring overnight afforded a yellow precipitate. The reaction mixture was checked by TLC. The yellow precipitate was collected by a vacuum filtration (0.47 g, 73%); mp 237ºC (sharp); FTIR 3060.1, 2986.1, 2229.7, 1769.4, 1573.5,

1260.4, 1104.3 cm-1; 1H NMR (400 MHz, DMSO) δ 7.89 (d, J = 16.8 Hz, 1H), 7.66 (d, J

= 0.07 Hz, 1H), 7.54 (dd, J = 8.4, 2 Hz, 1H), 7.24 (d, J = 8 Hz, 1H), 7.22 (d, J = 16.4 Hz,

1H) 4.15 (d, J = 6.8 Hz, 1H), 2.28 (s, 3H), 1.81 (s, 6H), 1.33 (t, J = 6.8 Hz, 3H); 13C

NMR (400 MHz, DMSO) δ 177.5, 175.5, 168.7, 151.0, 147.2, 143.2, 133.7, 124.0, 122.8,

116.0, 114.8, 113.1, 112.2, 111.2, 100.0, 99.9, 64.8, 25.5, 20.8, 14.9

(E)-2-(2-(4-cyano-5-(dicyanomethylene)-2,2-dimethyl-2,5-dihydrofuran-3- yl)vinyl)phenyl acetate

Product: Yellow solid (0.56 g, quantitative); mp 222.2ºC; FTIR 3050.4, 2989.5, 2226.0,

-1 1 2212.6, 1761.5, 1619.5, 1106.9 cm ; H NMR (400 MHz, CDCl3,) δ 8.09 (d, J = 16.5 Hz,

1H), 7.80 (dd, J = 7.96, 1.36 Hz, 1H), 7.56-7.52 (m, 1H), 7.38-7.34 (m, 1H), 7.23 (dd, J =

8.18, 1.12 Hz, 1H), 6.94 (d, J = 16.5 Hz, 1H); 13C NMR (400 MHz, DMSO) δ 194.1,

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173.5, 167.1, 154.5, 145.5, 133.6, 127.4, 126.7, 123.7, 115.7, 111.4, 110.7, 110.0, 100.4,

97.9, 44.5, 26.0, 21.0

(E)-4-(2-(4-cyano-5-(dicyanomethylene)-2,2-dimethyl-2,5-dihydrofuran-3- yl)vinyl)phenyl acetate (P-222)

Product: Yellow solid (0.56 g, quantitative); mp 226.2ºC; FTIR 3060.4, 2995.0, 2232.0,

1767.6, 1615.5, 1281.7, 1107.2 cm-1; 1H NMR (400 MHz, DMSO) δ 7.67 (d, J = 8.8 Hz,

2H), 7.63 (d, J = 16.4 Hz, 1H), 7.24 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 16.4 Hz, 1H), 2.34

(s, 3H), 1.8 (s, 6H); 13C NMR (100 MHz, DMSO) δ 193.2, 173.4, 168.7, 154.0, 146.0,

131.3, 130.2, 122.9, 114.9, 111.4, 110.7, 110.0, 100.4, 97.6, 44.5, 26.4, 21.1

4-(6-phenyl-1,2,4,5-tetrazin-3-yl)phenol (5.20)

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An oven dried 200 mL round bottom flask was charged with 4-hydroxybenzonitrile (0.6 g, 5.00 mmol), benzonitrile (2.6 mL, 25.0 mmol), Zn(OTf)2 (0.908 g 5 mol%) anhydrous hydrazine (4.0 mL) and stirred at 90°-110°C for six hours. A heavy yellow precipitate of mixture of dihydrotetrazine was formed. The reaction was monitored by TLC. It was filtered and washed with water and dried under suction. In the next step, the yellow precipitate of mixture of dihydrotetrazine was dissolved in glacial acetic acid (20 mL) and NaNO2 (4.0 g) in water (10 mL) was added slowly. The reaction mixture was stirred.

The color of the mixture turned to pink. The stirring was continued for half an hour until the brown fumes of NO2 was ceased. The reaction was stopped and pink precipitate was filtered under suction washing with water. The mixture tetrazine was dissolved in DCM and mounted in a column chromatography. The first fraction was side product symmetric tetrazine with no hydroxyl group which was separated out in DCM. The next fraction desired fraction was eluted down with ethyl acetate:DCM (1:1) 0.55 g (47%); mp 249°C

(sharp); FTIR 3265.77, 3055.07, 1607.31, 1391.15, 1170.35 cm-1; 1H NMR (400 MHz,

DMSO) δ 10.42, (s, 1H), 8.51-8.49 (m, 2H), 8.41 (d, J = 8.84 Hz, 2H), 7.69-7.68 (m,

3H), 7.05 (d, J = 4.80 Hz, 2H); 12CNMR (100 MHz, DMSO) δ 163.60, 163.24, 162.29,

132.74, 132.53, 130.10, 129.87, 127.70, 122.82, 116.88

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4,4’-(1,2,4,5-tetrazine-3,6-diyl)phenol (5.19)

A mixture of 4-hydrobenzonitrile (2.0 g, 16.8 mmol) and NH2-NH2 monohydrate (10.0 mL) was heated for 12 h to 90°C behind a blast shield with continuous stirring. A heavy yellow precipitate was observed after 2 hours and the heating was continued for 6 hours.

The mixture was allowed to cool to room temperature yellow precipitate was isolated by filtration, washed with cold water and dried under vacuum. In the next step, the dihydrotetrazine was dissolved in acetic acid (20.0 mL) and aqueous solution of NaNO2

(4.0 g in 10 mL water) was added slowly. The reaction mixture was continued stirring until, the brown fumes of NO2 ceased. The precipitate in the solution turned brick red which was filtered and washed several times with water (1.25 g, 94%); mp >250°C; 1H

NMR (400 MHz, DMSO) δ 10.42, (s, 1H), 8.35 (d, J = 8.80 Hz, 2H), 7.023 (d, J = 8.80

Hz, 2H); 12CNMR (100 MHz, DMSO) δ 163.04, 162.17,129.69, 122.87, 116.82

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(E)-2-(3-cyano-4-(4-fluorostyryl)-5,5-dimethylfuran-2(5H)-ylidene)malononitrile (5.28)

A mixture of 4-fluorobenzaldehyde (1 mL, 1.15 g, 9.32 mmol) and 2-dicyanomethylen-3- cyano-4,5,5-trimethyl-2,5-dihydrofuran (1.86 g, 9.32 mmol) in 35.0 mL of ethanol was refluxed in a 100 mL RB flask. The reaction mixture was monitored by TLC. For the complete reaction, the mixture was refluxed overnight. From the cooled mixture, a yellow precipitate was obtained in a vacuum filtration and further washed with hexane and then by diethyl ether to afford the desired product as a yellow solid with no further purification required (quantitative); mp>250ºC; 231.42, 2213.24, 1618.69, 1372.75,

1269.83 cm-1; 1H NMR (400 MHz, DMSO) δ 8.04-8.00 (m, 2H), 7.92 (d, J = 16.56 Hz,

1H), 7.37 (t, J = 8.84 Hz, 2H), 7.20 (d, J = 16.48 Hz, 1H), 1.80 (s, 6H); 13C NMR (100

MHz, DMSO) δ 186.23, 176.68 (d, J = 222.79), 146.40, 132.53, 117.00, 116.78, 115.78,

113.11,112.71, 112.28, 111.98, 110.46, 101.82, 25.54, 23.68

4-((2-hydroxyethyl)(methyl)amino)benzaldehyde (5.30)

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A mixture of 4-fluorobenzaldehyde (2.0 mL, 2.3 g, 18.53 mmol) and 2-

(methylamino)ethanol (5.92 mL, 74.12 mmol) with no solvent was heated at 90°-110°C stirring for 36 hours. The reaction was monitored by 1H NMR. The completion of reaction was determined by the absence of typical H-splitting next to F. The reaction mixture was then poured into the ice cold water to obtain the desired product as yellow precipitate. The compound did not require further purification (2.89 g, 87%); mp 67°C

1 (Lit mp 63°-80.7°C); H NMR (400 MHz, CDCl3) δ 9.68 (s, 1H), 7.69 (d, J = 9.0 Hz,

2H), 6.75 (d, J = 9.0 Hz, 2H), 3.86 (t, J = 5.76 Hz, 2H), 3.61 (t, J = 5.74 Hz, 2H), 3.11 (s,

13 3H); C NMR (100 MHz, CDCl3) δ 190.042, 153.91, 132.16, 125.32, 111.18, 60.06,

54.35, 39.20

(E)-2-(3-cyano-4-(4-((2-hydroxyethyl)(methyl)amino)styryl)-5,5-dimethylfuran- 2(5H)-ylidene)malononitrile (5.29)

A mixture of 4-((2-hydroxyethyl)(methyl)amino)benzaldehyde (1.0 g, 5.58 mmol), 2-(3- cyano-4,5,5-trimethylfuran-2(5H)-ylidene)malononitrile (1.11 g, 5.58 mmol) in EtOH

(21.0 mL) was refluxed overnight. The reaction mixture was monitored by TLC. A bluish precipitate was obtained. The precipitate was filtered through and washed with cold

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ethanol to afford pure product (1.58 g, 78%); mp >250°C; 1H NMR (400 MHz, DMSO) δ

7.92 (d, J = 15.8 Hz, 1H), 7.75 (d, J = 9.04 Hz, 2H), 6.86 (d, J = 15.92 Hz, 1H), 6.85 (d, J

= 8.72 Hz, 2H), 3.60-3.58 (m, 4H), 3.12 (s, 3H), 1.75 (s, 6H); 13C NMR (100 MHz,

DMSO) δ 177.83, 175.88, 153.94, 149.89, 133.44, 122.29, 114.0, 113.16, 112.81, 112.53,

108.61, 98.58, 92.20, 58.77, 54.49, 51.25, 26.14

(E)-2-(3-cyano-5,5-dimethyl-4-(4-(methyl(2-(4-(6-phenyl-1,2,4,5-tetrazin-3- yl)phenoxy)ethyl)amino)styryl)furan-2(5H)-ylidene)malononitrile (5.31)

In a 100 mL oven dried round bottom flask, (E)-2-(3-cyano-4-(4-((2-hydroxyethyl)

(methyl)amino)styryl)-5,5-dimethylfuran-2(5H)-ylidene)malononitrile (0.13 g, 0.355 mmol), 4-(6-phenyl-1,2,4,5-tetrazin-3-yl)phenol (0.1 g, 0.4 mmol), DIAD (0.12 mL,

0.595 mmol) were dissolved in anhydrous THF (2.0 mL). The resulting solution was cooled in an ice bath for 30 minutes. To this cold mixture was added a solution of Ph3P

(154.11 mg, 0.595 mmol) in anhydrous THF (2.0 mL) dropwise during 5 min with stirring. The mixture was stirred at room temperature overnight. The solvent THF was removed under reduced pressure and the crude mixture was separated in a column

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chromatography using ethyl acetate:hexane (1:4). The desired product was obtained in the second fraction (0.084 g, 24%). mp>250 ºC; FTIR 3204.13, 2914.10, 2220.83,

-1 1 1589.49, 1166.05 cm ; H NMR (400 MHz, CDCl3,) δ 8.52-8.48 (m, 4H), 7.93 (d, J =

15.88 Hz, 1H), 7.80 (d, J = 9.04 Hz, 2H), 7.79-7.67 (m, 3H), 7.22 (d, J = 5.12 Hz, 2H),

6.96 (d, J = 9.12 Hz, 2H), 6.91 (d, J = 15.88 Hz, 1H), 4.37 (t, J = 5.24 Hz, 2H), 4.01 (t, J

= 5.18 Hz, 2H), 3.21 (s, 3H), 1.7 (s, 6H).

5-Bromo-2-cyanopyridine (5.33)

To a solution of 2,5-dibromopyridine (5.0 g, 21.0 mmol) in anhydrous DMF (50 mL) was added CuCN (1.6 g, 18 mmol), NaCN (0.89 g, 18 .0 mmol). The reaction mixture was stirred for 7 hours at 150°C. After being cooled to room temperature, the reaction mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with water, brine, and dried over MgSO4. The excess solvent was removed under reduced pressure to obtained the title compound (2.0 g, 51%), mp 131°C (sharp); 1H NMR (400

MHz, CDCl3,) δ 8.81-8.81 (m, 1H), 8.02 (dd, J = 8.26, 2.30 Hz, 1H), 7.62 (dd, J = 8.24,

13 0.72 Hz, 1H); C NMR (100 MHz, CDCl3) δ 152.61, 139.79, 132.08, 129.25, 125.11,

116.58

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3-(5-Bromopyridin-2yl)-6-(pyridine-2-yl)-1,2,4,5-tetrazine (5.37)

An oven dried 200 mL round bottom flask was charged with 5-bromo-2-cyanopyridine

(0.92 g, 5.00 mmol), benzonitrile (2.6 mL, 25.0 mmol), Zn(OTf)2 (0.908 g 5 mol%) anhydrous hydrazine (4.0 mL) and stirred at 90°-110°C for six hours. A heavy dark brown solution of mixture of dihydrotetrazine was formed. The reaction was monitored by TLC. In the next step, the dark brown solution of mixture of dihydrotetrazine was diluted with glacial acetic acid (5.0 mL) and NaNO2 (2.0 g) in water (5.0 mL) was added slowly. The reaction mixture was stirred. The color of the mixture turned pink. The stirring was continued for half an hour until the brown fumes of NO2 was ceased. The reaction was stopped and pink precipitate was filtered under suction washing with water.

The mixture tetrazine was dissolved in DCM and mounted in a column chromatography.

The first fraction was by product symmetric tetrazine with no bromo group on it. It was separated out with DCM. The next fraction desired fraction was eluted down with ethyl acetate:DCM (1:1) 0.50 g (31%); mp >250°C (sharp); FTIR 3362.77, 3058.05, 1607.31,

1392.2 cm-1; 1H NMR (400 MHz, DMSO) δ 9.03 (dd, J = 2.30, 0.62 Hz, 1H), 9.00 (ddd,

J = 4.72, 1.66, 0.86 Hz, 1H), 8.77 (t, J = 1.00 Hz, 1H), 8.75 (t, J = 0.98 Hz, 1H), 8.67 (d,

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J = 0.68 Hz, 1H), 8.02 (dt, J = 7.79, 1.74 Hz, 1H), 7.60 (ddd, J = 7.63, 4.75, 1.15 Hz,

1H); 12CNMR 100 MHz, DMSO) δ 163.87, 163.54, 152.31, 151.12, 150.57, 150.01,

148.45, 140.22, 137.52, 126.70, 125.45, 124.94, 124.62

5-(4-Methoxybenzyloxy)-2-cyanopyridine (5.41)

To a suspension of NaH (7.2 g, 60%, 0.18 mmol) in anhyd DMF (100.0 mL) was added

4-methoxybenzylalcohol (1.26 g, 9.14 mmol) at 0°C. After 25 minutes at 0°C the mixture was warmed to room temperature and stirred for additional 30 minutes. To this resulting solution was added 2-cyano-5-bromopyridine (1.4 g, 7.93 mmol) in one portion. The reaction was exothermic and stirred for 10 minutes before it was cooled to room temperature. The mixture was diluted with 500 mL ethyl acetate washed with water (500 mL). The first aqueous phase was extracted with DCM (500 mL). The combined DCM phase was washed with water (500 mL). The combined organic phase was dried over

MgSO4 and filtered through a thin silica pad. The excess solvent was removed under reduced pressure to afford the desired product as a colorless thick liquid (1.31 g, 76%);

1 H NMR (400 MHz, CDCl3,) δ 8.4 (dd, J = 2.88, 0.48 Hz, 1H), 7.62 (dd, J = 8.60, 3.06

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Hz, 1H), 7.33 (d, J = 8.76 Hz, 2H), 7.28 (dd, J = 8.64, 2.88 Hz, 1H) 6.93 (d, J = 8.72,

13 2H), 5.09 (s, 2H), 3.82 (s, 3H); C NMR (100 MHz, CDCl3) δ 160.03, 157.07, 140.64,

129.53, 129.45, 126.74, 125.39, 120.84, 117.48, 114.31, 70.65, 55.36

5-Hydroxy-2-cyanopyridine (5.42)

To a solution of 5-(4-Methoxybenzyloxy)-2-cyanopyridine (1.20 g, 4.99 mmol) in DCM

(20.0 mL) was added Et3SiH (0.8 mL, 4.99 mmol). The reaction mixture was stirred at room temperature for 10 minutes followed by addition of CF3CO2H (2.08 mL). The reaction mixture was stirred for 3 hours at room temperature under nitrogen. The solvent was removed under reduced pressure. The residue was taken up in DCM and chromatographed on silica gel eluting with EtOAc:Hexane (1:1) to afford the titled

1 compound ( 0.47 g, 78%); mp 192 (sharp); H NMR (400 MHz, CDCl3,) δ 11.20 (s, 1H),

8.25 (dd, J = 2.78, 0.42 Hz, 1H), 8.25 (dd, J = 2.78 Hz, 1H), 7.28 (dd, J = 8.64, 2.88 Hz,

13 1H ) 6.93 (d, J = 8.72, 2H), 5.09 (s, 2H), 3.82 (s, 3H); C NMR (100 MHz, CDCl3) δ

157.48, 140.83, 130.888, 123.03, 122.92, 118.52

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tert-butyl 2-(2-(6-cyanopyridin-3-yloxy)ethoxy)ethylcarbamate (5.45)

To a suspension of NaH (0.317 g, 60%, 7.937 mmol) in anhyd DMF (6.5 mL) was added tert-butyl 2-(2-hydroxyethoxy)ethylcarbamate (1.87 g, 9.13 mmol) at 0°C. After 25 minutes at 0°C the mixture was warmed to room temperature and stirred for additional 30 minutes. To this resulting solution was added 2-cyano-5-bromopyridine (1.4 g, 7.93 mmol) in one portion. The reaction was exothermic and stirred for 10 minutes before it was cooled to room temperature. The mixture was diluted with 500 mL ethyl acetate washed with water (500 mL x 3). The first aqueous phase was extracted with DCM (500 mL x 2). The combined DCM phase was washed with water (500 mL x 3). The combined organic phase was dried over MgSO4 and filtered through a thin silica pad. The excess solvent was removed under reduced pressure to afford the desired product as a colorless

1 thick liquid (quantitative); H NMR (400 MHz, CDCl3,) δ 8.41-8.40 (m, 1H), 7.66-7.63

(dd, J = 8.64, 0.48 Hz, 1H), 7.27 (dd, J = 2.92, 3.09 Hz, 1H), 4.85 (s, 1H), 4.24-4.22 (m,

2H), 3.87-3.84 (m, 2H), 3.6 (t, J = 5.28 Hz, 2H), 3.36-3.32 (m, 2H), 1.44 (s, 9H); 13C

NMR (100 MHz, CDCl3) δ 157.15, 156.0, 140.36, 129.49, 125.60, 120.57, 117.42, 79.56,

710.61, 68.95, 68.20, 40.23, 28.40

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tert-butyl 2-(2-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3- yloxy)ethoxy)ethylcarbamate (5.40)

An oven dried 200 mL round bottom flask was charged with tert-butyl 2-(2-(6- cyanopyridin-3-yloxy)ethoxy)ethylcarbamate (2.145 g, 6.98 mmol), 2-cyanopyridine

(3.36 mL, 34.90 mmol), Zn(OTf)2(1.288 g 5 mol%) anhydrous hydrazine (5.7 mL, 176 mmol) and stirred at 90°-110°C for six hours. A dark brown solution mixture of dihydrotetrazine was obtained. The reaction was monitored by TLC. It was filtered and washed with water and dried under suction. In the next step, the dark brown precipitate of mixture of dihydrotetrazine was dissolved in glacial acetic acid (20 mL) and NaNO2 (4.0 g) in water (10 mL) was added slowly. The reaction mixture was stirred. The color of the mixture turned pink. The stirring was continued for half an hour until the brown fumes of

NO2 was ceased. The reaction was stopped and pink precipitate was filtered under suction washing with water. The mixture tetrazine was dissolved in DCM and mounted in a column chromatography. The first fraction was side product symmetric pyridyl tetrazine with no functionalized tail which was separated out in DCM. The next fraction desired fraction was eluted down with ethyl acetate:DCM (1:1) 1.2 g (40%); mp 175°C, FTIR

3348.82, 3059.11, 2979.65, 1744, 1682.33, 1128.11 cm-1; 1H NMR (400 MHz, DMSO) δ

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8.99 (m, 1H), 8.75-8.72 (m, 2H), 8.65-8.64 (m, 1H), 8.01 (dt, J = 7.74, 1.74 Hz, 1H),

7.59-7.55 (m, 1H), 7.45 (dd, J = 8.80, 2.92 Hz, 1H), 4.26-4.24 (m, 2H), 4.01 (t, J = 5.88

Hz, 2H), 3.85-3.83 (m, 2H), 3.56 (t, J = 5.88 Hz, 1H), 1.63 (s, 9H); 12CNMR (100 MHz,

DMSO) δ 163.55, 163.38, 157.41, 157.98, 150.27, 139.93, 137.51, 137.45, 126.62,

126.41, 125.69, 124.29, 121.25, 85.49, 68.89, 68.12, 67.24, 39.47, 28.03

2-(2-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yloxy)ethoxy)ethanamine (5.41)

To a solution of tert-butyl 2-(2-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3- yloxy)ethoxy) ethylcarbamate (0.5 g, ) in DCM (25 mL) was added trifluoroacetic acid

(0.5 mL) and stirred for 1 hour. The excess solvent was removed under reduced pressure and excess trifluoroacetic acid was removed under a Kugelrohr distillation at 35°C

(105µ). The sticky residue was stored in a refrigerator under diethyl ether. After 3 hours, the solid was crushed and filtered to obtain solid pink desired product. 1.2 g (40%); mp

175°C, FTIR 3348.82, 3059.11, 2979.65, 1744, 1682.33, 1128.11 cm-1; 1H NMR (400

MHz, DMSO) δ 8.94 (d, J = 3.84 Hz, 1H), 8.66-8.58 (m, 3H), 8.16 (dt, J = 7.72, 1.28 Hz,

1H), 7.90 (broad, 3H), 7.76-7.72 (m, 2H), 4.40 (m, 2H), 3.89 (m, 2H), 3.71 (t, J = 5.06

375

Hz, 2H), 3.04 (t, J = 5.04 Hz, 2H); 12CNMR (100 MHz, DMSO) δ 163.50, 158.35 (q, 30

Hz), 157.48, 151.06, 150.64, 149.04, 142.57, 139.90, 138.28, 135.71, 127.05, 126.06,

124.60, 121.96, 69.13, 68.39, 67.31

4- Methylaminobutanoic acid hydrochloride (5.47)

A mixture of N-methylpyrrolidin-2-one (NMP) (5.0 mL, 5.135 g, 51.79 mmol) and concentrated hydrochloric acid (5.0 mL) was heated at 110°C overnight. A thick white slurry was obtained which was washed with diethyl ether to get the pure title compound (quantitative); mp 121°C; 1H

NMR (400 MHz, H20) δ 2.99 (t, J = 15.48 Hz, 2H), 2.63 (s, 3H), 2.43 (t, J = 7.22 Hz,

2H), 1.88 (quint, J = 7.49 Hz, 2H), 12CNMR (100 MHz, DMSO) δ 176.76, 48.16, 32.69,

30.43, 20.

4((4-Formylphenyl)(methyl)amino)butanoic acid (5.48)

A suspension of 4- methylaminobutanoic acid hydrochloride (8.11 g, 52.79 mmol), 4- fluorobenzaldehyde (5.66 mL, 52.79 mmol), KOH (5.92 g, 105.58 mmol) and water (5.5 mL) was refluxed at 110°C for 3 days. The reaction was monitored by HNMR and TLC.

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The reaction was treated with HCl (10.0 mL, 6N) and stirred for 4 hours at room temperature. The reaction mixture was extracted with DCM. The aqueous layer was washed with DCM and the combined DCM was dried under reduced pressure. The excess solvent was removed under reduced pressure. The solid was cooled in a refrigerator and the solid chunk was broken into fine powder while stirring in hexane and filtered. The desired product further washed with ether (5 g, 40%); mp 112°C; FTIR 2908.99,

2841.83, 2524.83, 1720.29, 1631.22, 1581.92, 1171.89 cm-1; 1H NMR (400 MHz,

CDCl3) δ 9.71 (s, 1H), 7.73 (d, J = 8.96 Hz, 2H), 6.73 (d, J = 8.96 Hz, 2H), 3.5 (dd, J =

7.50 Hz, 2H), 3.07 (s, 3H), 2.44 (t, J = 7.04 Hz, 2H), 1.96 (quint, J = 7.27 Hz, 2H),

12CNMR (100 MHz, DMSO) δ 190.49, 178.33, 153.44, 132.29, 125.18, 110.99, 51.42,

38.59, 30.91, 21.87.

(E)-4-((4-(2-(4-cyano-5-(dicyanomethylene)-2,2-dimethyl-2,5-dihydrofuran-3- yl)vinyl)phenyl)(methyl)amino)butanoic acid (5.49)

A mixture of 4((4-Formylphenyl)(methyl)amino)butanoic acid (1.0 g, 4.52 mmol), 2-(3- cyano-4,5,5-trimethylfuran-2(5H)-ylidene)malononitrile (0.9, 4.52 mmol) in EtOH (20.0 mL) was refluxed overnight. The reaction mixture was monitored by TLC. A greenish

377

precipitate was obtained. The precipitate was filtered through and washed with cold ethanol to afford pure product (1.62 g, 89.06%); mp >250°C; FTIF 1H NMR (400 MHz,

DMSO) δ 12.16 (s, 1H), 7.92 (d, J = 15.80 Hz, 1H), 7.77 (d, J = 9.04 Hz, 1H), 6.88 (d, J

= 15.84 Hz, 1H), 6.86 (d, J = 9.08 Hz, 1H), 3.50 (dd, J = 7.59 Hz, 2H), 3.08 (s, 3H), 2.29

(t, J = 7.16 Hz, 2H), 1.75 (s, 6H); FTIR 2911.44, 2217.03, 1705.42, 1490.9 1155.86; 13C

NMR (100 MHz, DMSO) δ 177.85, 175.95, 174.57, 153.50, 149.86, 133.51, 122.37,

113.15, 112.67, 112.49, 108.79, 98.65, 92.43, 51.34, 49.05, 38.70, 31.03, 26.11, 22.33

(E)-2,5-dioxopyrrolidin-1-yl 4-((4-(2-(4-cyano-5-(dicyanomethylene)-2,2-dimethyl- 2,5-dihydrofuran-3-yl)vinyl)phenyl)(methyl)amino)butanoate (5.50)

To a solution of (E)-4-((4-(2-(4-cyano-5-(dicyanomethylene)-2,2-dimethyl-2,5- dihydrofuran-3-yl)vinyl)phenyl)(methyl)amino)butanoic acid (0.52 g, 1.3 mmol), and N- hydroxy succinimide (0.16 g, 1.41 mmol) in anhydrous DCM (10 mL) was added DCC

(0.3 g, 1.41 mmol) and DMAP (0.06 g, 0.072 mmol) and stirred for 36 hours at room temperature. The reaction mixture was monitored by TLC which indicated the complete consumption of the starting materials. The solid crude was washed with several times

378

with THF which was then column chromatographed with DCM to afford a bluish solid

(0.45 g, 68.54 %), FTIR 3269.23, 2929.56, 2859.63, 2221.3, 1648.25, 1552.3, 1156.25;

1HNMR δ7.92 (d, J = 15.80 Hz, 1H), 7.78 (d, J = 9.04 Hz, 2H), 6.88 (d, J = 15.96 Hz,

2H), 6.90 (d, J = 8.12 Hz, 1H), 3.58 (t, J = 14.96 Hz, 2H), 3.11 (s, 3H), 2.83 (s, 4H),

2.781 (t, J = 7.24 Hz, 2H), 1.92 (quint, J = 7.2 Hz, 2H), 1.76 (6H), and 12CNMR 177.85

176.00, 170.74, 169.28, 153.32, 149.82, 133.45, 122.53, 113.96, 113.12, 112.69, 112.44,

108.99, 98.72, 50.77, 47.96, 33.81, 28.02, 26.10, 25.92, 24.93, 22.18

(E)-4-((4-(2-(4-cyano-5-(dicyanomethylene)-2,2-dimethyl-2,5-dihydrofuran-3- yl)vinyl)phenyl)(methyl)amino)-N-(2-(2-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3- yl)pyridin-3-yloxy)ethoxy)ethyl)butanamide (5.51)

To a solution of ((E)-2,5-dioxopyrrolidin-1-yl 4-((4-(2-(4-cyano-5-(dicyanomethylene)-

2,2-dimethyl-2,5-dihydrofuran-3-yl)vinyl)phenyl)(methyl)amino)butanoate (100.0 mg,

0.2 mmol) and 2-(2-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3- yloxy)ethoxy)ethanamine trifluoroacetic acid (90.67 mg, 0.2 mmol) in DMF (15.0 mL) was added Hunig’s base (0.52 mL, 0.3 mmol) and stirred at rt for 5 hours. The reaction

379

was monitored by TLC which indicated the consumption of both starting materials. The

DMF was removed under reduced pressure in a Kugelrohr distillation at 40°C and 200µ.

The blue solid was dissolved in DCM and washed with water to remove the remaining salt. The excess solvent was removed under reduced pressure in rotatory evaporator. The blue solid was scrapped off from the wall with checked with HNMR. There was presence of some impurities which was separated in a column chromatography using eluent MeOH

(5%), EtOAc (50%) and DCM (45%) to afford a dark blue solid (35 mg, 24.22%), FTIR

3272.04, 3059.39, 2928.98, 2889.89, 2223.95, 1638.17, 1558.06, 1518.17, 1156.23

1HNMR δ 8.93 (d, J = 4.12 Hz, 1H), 8.61 (d, J = 2.8 Hz, 1H), 8.58 (d, J = 17.96 Hz, 2H ),

8.15 (dt, J = 7.8 Hz, 1.64 Hz 1H), 7.98 (t, J = 5.44 Hz, 1H), 7.87 (d, J = 15.80 Hz, 1H),

7.74-7.71 (m, 3H), 7.68 (dd, J = 8.90 Hz, 2.90 Hz, 1H), 6.82 (d, J = 15.68 Hz, 1H), 6.81

(d, J = 9.12 Hz, 1H), 4.34 (t, J = 4.02 Hz, 2H), 3.82 (t, J = 4.10 Hz, 1H), 3.53 (t, J =

11.20 Hz, 2H), 3.44 (t, J = 7.22 Hz, 2H), 3.28 (q, J = 7.22 Hz, 4H) 3.06 (s, 3H), 2.15 (t, J

= 6.84 Hz, 2H), 1.74 (s, 6H), and 13CNMR 177.80, 175.84, 172.10, 163.45, 163.22,

157.51, 153.47, 151.04, 150.62, 149.82, 142.43, 139.84, 138.24, 133.51, 127.03, 125.96,

124.57, 122.28, 121.84, 114.01, 113.14, 112.63, 112.48, 108.62, 55.38, 51.56, 38.92,

38.32, 32.43, 26.11, 22.87

380

Experimental of Chapter 6

The naphthalene, anthracene, and phenanthrene are commercial grade obtained from

Sigma Aldrich whereas tetracene was prepared by Alex. Perfluorophenazine (PFP) was synthesized according the literature procedure 304. It was recrystallized from 1-propanol

(MP 215ºC, reported melting points range from 230°C 305 to 260°C 306). The purity of all these compounds was checked by using NMR (400 MHz, Bruker) and GC-MS (Finnigan

Trace GC Ultra equipped with mass detector (Finnigan Polaris Q) and used without further purification. The melting was obtained from Nikon eclipse E600 POL with temperature controller (Mettler FP90).

X-ray crystallography of all the co-crystals was performed by attaching each crystal onto a thin glass fiber from a pool of FluorolubeTM and immediately placing it under a liquid N2 stream, on a Bruker AXS defractometer. The radiation used was optimized from a least-squares calculation on carefully centered reflections. Lattice determination, data collection, structure refinement, scaling, and data reduction were carried out using APEX2 version 1.0-27 software package. Each structure was solved using different methods. This procedure yielded a number of the C and F atoms.

Subsequent Fourier synthesis yielded the remaining atom positions. The hydrogen atoms were fixed in positions of ideal geometry and refined within the XSHELL software.

These idealized hydrogen atoms had their isotropic temperature factors fixed at 1.2 or 1.5 times the equivalent isotropic U of the C atoms to which they were bonded. The final refinement of each compound included anisotropic thermal parameters on all non- hydrogen atoms.

381

Co-crystal parameters

-1 Crystal data for PFP (1.1): C12 F8 N2, MW = 324.14 g mol , crystal dimensions

0.25 x 0.10 x 0.09 mm, monoclinic, space group P21/n, a = 5.688 (2) Å, b = 8.834 (3) Å,

3 -3 c = 10.408 (4) Å, β = 99.606 (5)º, V = 515.7 (3) Å , Z = 2, ρcalc = 2.088 mg cm , Bruker

SMART APEX II diffractometer, 3.04º < θ < 25.04º, Mo(Kα) radiation (λ = 0.71073 Å),

ω scans, T = 100(2)K; of 3875 measured reflections 913 were independent with I > 2σ(I),

-6 < h < 6, -10 < k <10, -12 < l < 12; R1 = 0.0323, wR2 = 0.0748, GOF = 2.710 for 100

-3 parameters, Δρmax = -0.252 e. Å . The structure was solved by direct methods (SHELXS-

97) and refined by full matrix least-squares procedures (SHELXL-97), Lorentzian and polarization corrections and absorption correction SADABS were applied, µ = 0.229 mm-

-1 Crystal data for PFP naphthalene: C22 H8 F8 N2, MW = 452.30 g mol , crystal dimensions 0.58 x 0.15 x 0.12 mm, monoclinic, space group P21/n, a = 7.580(3) Å, b =

3 6.140 (2) Å, c = 19.065 (7) Å, β = 100.559(7)º, V = 872.3(6) Å , Z = 2, ρcalc = 1.722 mg cm-3, Bruker SMART APEX II diffractometer, 2.17º < θ < 25.05º, Mo(Kα) radiation (λ =

0.71073 Å), ω scans, T = 100(2)K; of 7759 measured reflections 1763 were independent with I > 2σ(I), -14 < h < 14, -17 < k < 17, -14 < l < 14; R1 = 0.0396, wR2 = 0.1183, GOF

-3 = 0.946 for 163 parameters, Δρmax = -0.213 e. Å . The structure was solved by direct methods (SHELXS-97) and refined by full matrix least-squares procedures (SHELXL-

97), Lorentzian and polarization corrections and absorption correction SADABS were applied, µ = 0.152 mm-1.

382

-1 Crystal data for PFP anthracene: C26 H10 F8 N2, MW = 502.36 g mol , crystal dimensions 0.34 x 0.15 x 0.12 mm, triclinic, space group P1, a = 7.0691(11) Å, b =

7.2780 (11) Å, c = 10.1984 (4) Å, α = 85.402 (3)º, β = 99.606(5)º, γ = 77.949 (3)º, V =

3 -3 494.87(13) Å , Z = 1, ρcalc = 1.686 mg cm , Bruker SMART APEX II diffractometer,

2.07º < θ < 25.05º, Mo(Kα) radiation (λ = 0.71073 Å), ω scans, T = 100(2)K; of 3981 measured reflections 1749 were independent with I > 2σ(I), -8 < h < 8, -8 < k <8, -12 < l

-3 < 12; R1 = 0.0457, wR2 = 0.1088, GOF = 0.898 for 163 parameters, Δρmax = -0.167 e. Å .

The structure was solved by direct methods (SHELXS-97) and refined by full matrix least-squares procedures (SHELXL-97), Lorentzian and polarization corrections and absorption correction SADABS were applied, µ = 0.153 mm-1.

-1 Crystal data for PFP phenanthrene: C26 H10 F8 N2. MW = 502.36 g mol , crystal dimensions 0.56 x 0.20 x 0.18 mm, monoclinic, space group C2/c, a = 12.0383(17) Å, b

3 = 14.314(2) Å, c = 12.2772(17) Å, β = 110.203(2)º, V = 1985.5(5) Å , Z = 4, ρcalc = 1.681 mg cm-3, Bruker SMART APEX II diffractometer, 2.30º < θ < 25.05º, Mo(Kα) radiation

(λ = 0.71073 Å), ω scans, T = 100(2)K; of 6597 measured reflections 1540 were independent with I > 2σ(I), -9 < h < 9, -7 < k < 7, -22 < l < 22; R1 = 0.0347, wR2 =

-3 0.1183, GOF = 1.004 for 145 parameters, Δρmax = -0.213 e. Å . The structure was solved by direct methods (SHELXS-97) and refined by full matrix least-squares procedures

(SHELXL-97), Lorentzian and polarization corrections and absorption correction

SADABS were applied, µ = 0.163 mm-1.

-1 Crystal data for PFP tetracene: C26 H10 F8 N2, MW = 552.42 g mol , crystal dimensions 0.55 x 0.21 x 0.10 mm, triclinic, space group P1, a = 6.854(3) Å, b = 7.080

383

(3) Å, c = 12.010 (5) Å, α = 94.898 (3)º, β = 105.801(7)º, γ = 95.977(7)º, V = 553.8(4)

3 -3 Å , Z = 1, ρcalc = 1.656 mg cm , Bruker SMART APEX II diffractometer, 1.77º < θ <

25.05º, Mo(Kα) radiation (λ = 0.71073 Å), ω scans, T = 100(2)K; of 4317 measured reflections 1957 were independent with I > 2σ(I), -8 < h < 8, -8 < k <8, -14 < l < 14; R1 =

-3 0.0635, wR2 = 0.1322, GOF = 0.669 for 181 parameters, Δρmax = -0.192 e. Å . The structure was solved by direct methods (SHELXS-97) and refined by full matrix least- squares procedures (SHELXL-97), Lorentzian and polarization corrections and absorption correction SADABS were applied, µ = 0.145 mm-1

-1 Crystal data for PFP DTT C28 H8 F8 N2 S6, MW = 716.72 g mol , crystal dimensions 0.15 x 0.08 x 0.07 mm, monoclinic, space group P21/n, a = 7.199 (3) Å, b =

3 21.573 (9) Å, c = 8.779 (4) Å, β = 101.151 (7)º, V = 1337.6 (10) Å , Z = 2, ρcalc = 1.780 mg m-3, Bruker SMART APEX II diffractometer, 1.89º < θ < 25.05º, Mo(Kα) radiation (λ

= 0.71073 Å), ω scans, T = 100(2)K; of 10274 measured reflections 2379 were independent with I > 2σ(I), -8 < = h < 8, -25 < = k < = 25, -10 < = l < = 10; R1 = 0.0591,

-3 wR2 = 0.0980, GOF = 1.038 for 199 parameters, Δρmax = -0.352 e. Å . The structure was solved by direct methods (SHELXS-97) and refined by full matrix least-squares procedures (SHELXL-97), Lorentzian and polarization corrections and absorption correction SADABS were applied, µ = 0.594 mm-1.

384

PFP synthesis (6.2)

Lead tetraacetate 25.0g (55.0 mmol) was added to 5.0 g (27.0 mmol) of pentafluoroaniline in 75 mL of toluene at room temperature. The solution turned to orange red as soon as the lead tetraacetate was added. The mixture was heated under reflux for 1 hour at 110°C. The reaction was incomplete when checked by TLC [EtOAc

20% and Hexane-80%]. It was heated for another 4 hours with the addition of three equivalent of lead tetraacetate to complete the reaction. Two distinct spot were observed with no traces of starting materials when checked by TLC. The reaction mixture was filtered through a silica pad to remove undissolved particles. Toluene (175 ml) was used to elude the product, then the organic mixture was washed with aqueous 50% acetic acid, saturated NaHCO3 and several times with water in a separatory funnel and finally dried with anhydrous magnesium sulfate. The resulting mixture was impregnated on silica gel

(25g) which was placed on the top of a silica gel which was eluted with pure hexane. The

19 first orange fraction was 1.2 (1.84g, 37.44%); mp 110.5C°; F NMR (75MHz, CDCl3) δ

(ppm) – 148.75 (m, 6F), -160.69 (m, 4F) and second yellow fraction was the desired

19 product 1.1 (0.86 g, 20.0%); mp 215°C; FNMR (75MHz, CDCl3) δ -146.47 to -146.51

(m, 4F) and -49.41 to -149.45 (m, 4F)

385

Co-crystallization of PFP and naphthalene 342

Perfluorophenazine 0.0815g (0.25 mmol) and naphthalene 0.0322g (0.2513mmol) were added to dichloromethane (4ml) and a homogenous solution (faint yellow) was prepared by gentle heating below boiling point of the solvent. The mixture was kept in Dewar flask overnight for evaporation. During the cooling process small cubes formed at the bottom of the container. The dichloromethane was evaporated completely leaving small cube like crystals. The presence of both components in the crystalline product was confirmed by

19FNMR, 1HNMR and by GC-MS. The melting point of the co-crystal is 173°C, whereas the melting point of 1.1 is 215°C and naphthalene is 80.25°C.

Co-crystallization of PFP and anthracene

Perfluorophenazine 0.0626g (0.1931 mmol) and anthracene (0.03442g (0.1931 mmol) was added to dichloromethane (4.0 mL) and a homogenous orange solution was prepared by gentle heating below boiling point of the solvent. The mixture was kept in Dewar flask overnight for evaporation. During the cooling process fine need shaped orange crystals were observed floating on the surface of the solution. The dichloromethane was

386

evaporated completely and skinny elongated orange crystals were harvested. The presence of both components in the crystalline product was confirmed by 19FNMR,

1HNMR and by GC-MS (mp 226ºC). The melting point of anthracene is 218°C.

Co-crystallization of PFP and phenanthrene

Perfluorophenazine 0.0626g (0.1931 mmol) and phenanthrene (0.03442g (0.1931 mmol) was added in dichloromethane (4.0 mL) and a homogenous orange solution was prepared by slow heating below boiling point of the solvent. The mixture was kept in Dewar flask overnight for evaporation. During the cooling process fine needle shaped orange crystal were observed floating on the surface of the solution. The dichloromethane was evaporated completely and skinny elongated orange crystals were harvested. The presence of both components in the crystalline product was confirmed by 19FNMR,

1HNMR and by GCMS (mp 117ºC). The melting point of phenanthrene is 101°C.

387

Co-crystallization of PFP and tetracene

Tetracene 0.011g (0.046 mmol) was added to 1-octanol (4.0 ml) and heated in an oil bath at 80ºC to dissolve it. In a separate vessel, a solution of perfluorophenazine 0.015g (0.046 mmol) in 1-octanol (2ml) was prepared by heating the mixture at 80ºC. The perfluorophenazine-octanol solution was added to the tetracene-octanol solution and heated in oil bath for some time. The solution was left overnight at room temperature.

Very skinny dark brown crystals were obtained. The presence of both components in the crystalline product was confirmed by 19FNMR, 1HNMR and by GCMS (mp 289ºC). The melting point of tetracene is 357°C.

Co-crystallization of PFP and DTT

Perfluorophenazine 0.125 g (0.386 mmol) and DTT 0.0193 g (0.1931 mmol) were dissolved in dichloromethane (4ml) and homogenous solution (faint yellow) was prepared at room temperature. The mixture was kept in Dewar flask overnight for evaporation. Dichloromethane was evaporated completely next day leaving behind yellowish crystals adhered on the beaker which was very carefully separated from excess

388

DTT under a microscope. The presence of both components in the crystalline product was confirmed by 19FNMR, 1HNMR and by GCMS (mp 289ºC). The melting point of point of co-crystal is 169°C, whereas melting point of 1 is 215°C and DTT is 65°C-67°C.

Experimental of Chapter 7

(E)-2,6-dimethyl-N-(perfluorobenzylidene)aniline (7.5)

A neat mixture of perfluorobenzaldehyde and 2,6-dimethylaniline was stirred at room temperature for half an hour. A yellow solid was precipitated out which was washed with hexane (quantitative); mp 114.5°C (sharp), FTIR cm-1 2964.37, 1649.15, 1495.52; 1H

NMR (400 MHz, CDCl3) δ 9.35 (s, 1H). 7.08 (d, J = 7.6 Hz, 2H), 7.00-6.97 (m, 1H), 2.16

13 19 (s, 6H); C-NMR (75 MHz, CDCl3) δ, ppm 156.2, 142.5, 129.6, 1263, 124.6, 17.48; F-

NMR (282.4 MHz, CDCl3) δ, -145.3 (m, 2F), -147.17 (m, 2F), 160.56 (m, 1F)

1,2,3,4-tetrafluorobenzo[b]acridine (7.3)

389

To a solution of (E)-2,6-dimethyl-N-(perfluorobenzylidene)aniline (1.0 g, 3.34 mmol) in xylene (25 mL) was added 2-naphthylamine (0.48 g, 3.34 mmol). The mixture was refluxed until all the starting materials were consumed (36 hours). A yellow precipitate was produced when the reaction mixture was cooled which was filtered under vacuum to afford of the title product (0.45 g, 44.5 %); mp 283.5°C; FTIR cm-1 3049.36, 1678.76,

1492.66; 1H NMR (400 MHz, DMSO) δ 9.85 (s, 1H), 9.02 (d, J = 8.4 Hz, 1H), 8.21 (d, J

= 9.6 Hz, 1H), 8.07-8.05 (m, 1H), 7.97 (d, J = 9.2 Hz, 1H), 7.03 (dq, J = 6.8 Hz, 1.2 Hz,

1H); 19F-NMR (75 MHz, DMSO) δ, -149.43 (t, J = 3.75 Hz, 1F), -152.64 (t, J = 3.75 Hz,

1F), 154.91 (t, J = 3.75 Hz, 1F), 159.26 (t, J = 3.75 Hz, 1F).

Dibenzo[a,c]phenazine (7.8)

Solid 9,10-phenanthrenequinone 0.625g, (3.0 mmol) was slowly added to 20 ml of glacial acetic acid, and it was warmed until everything was dissolved. Next, 0.324g, (3.0 mmol) of o-phenylenediamine was slowly added to this solution of phenanthrenequinone resulting in the immediate formation of yellowish solid. The reaction mixture was checked by TLC (mixture of EtOAc and hexane as eluent). It showed incomplete reaction. So, it was heated for 2 hours maintaining temperature of 120°C. The solid was filtered in the vacuo, then recrystallized from DMF (0.61 g, 70.2%); mp 224.5°C; 1H

390

NMR (300 MHz, CDCl3) δ, ppm 9.39 (d, J = 5.4 Hz, 2H). 8.5 (dd, J = 6 Hz, 0.6 Hz, 2H),

13 8.31 (m, 2H), 7.83 (m, 2H), 7.75 (m, 4H); C-NMR (75 MHz, CDCl3) δ, ppm 142.42,

142.16, 132.04, 130.30, 129.75, 129.46, 127.93, 126.28, 122.91.

11-Fluorodibenzo[a,c]phenazine (7.9)

Phenanthrenequinone (0.625 g, 3.0 mmol) was dissolved in acetic acid (20.0 mL) with warming and 4-fluoro-1,2-diaminobenzene (0.378 g, 3.0 mmol) was slowly added. There was instant formation of yellow precipitate. The reaction was checked by TLC, stirred and left it overnight at 80°C with the addition of 4-fluoro-1,2-diaminobenzene (0.022 g,

0.174 mmol). A brown solid product was filtered off and washed with glacial acetic acid several times. The product was recrystallized from DMF (0.73 g, 84%); mp 216°C; FTIR

-1 1 cm 3012.37, 1675.25, 1485.12; H NMR (400 MHz, CDCl3) δ 9.38 (dt, J = 6 Hz, 1.2Hz,

2H), 8.58 (d, J = 6.3Hz, 2H), 8.36 (dd, J = 6.9Hz, 4.5 Hz, 1H), 7.96 (dd, J = 6.3Hz,

13 2.1Hz, 1H), 7.82 (m, 4H), 7.68(m, 1H); C-NMR (100 MHz, CDCl3) δ 162.90 (d, J =

252.37), 142.98, 142.84, 142.71, 141.94, 139.51, 132.29, 131.92, 131.80, 131.70, 130.92,

130.62, 130.16, 128.31, 126.53, 126.20, 123.18, 121.06, 120.80, 112.48 (d, J = 21.15Hz);

19 F-NMR (75Hz, CDCl3): δ (ppm): -108.512 (s, 1F).

391

2,3,4,5-tetrafluoro-6-nitroaniline (7.12)

To a solution of pentafluoronitrobenzene (1.82 mL, 3.01 g, 14 mmol) in diethyl ether

(200 mL), with magnetic stirring, ammonium hydroxide (5 ml, 4.5 g, 35.67 mmol, 2.55 equiv) was added dropwise by a syringe. The color of the reaction mixture changed to faint yellow and the reaction was monitored by TLC. After 24 hours, the reaction was complete with two products present. The solid was separated by vacuum filtration and dissolved in ethyl acetate and washed with water and brine. The solution was then dried with MgSO4. The yellow clear solution was impregnated on 25g of silica gel and purified on a silica gel column using 15% ethyl acetate and 85% hexane mixture solvent as eluent.

The desired product was separated in the first fraction (1.607 g, 54%); mp 43.6°C;

19 FNMR (282 MHz, CDCl3): δ ppm -145.3 (m, 1F), -147.17 (m, 1F), 160.56 (d, 1F), -

172.26 (t, 1F)

3,4,5,6-tetrafluorobenzene-1,2-diamine (7.7)

392

A solution of 6-nitro-2,3,4,5 tetrafluoro-1-aniline, 3.013 g (7.3 mmol) was prepared by dissolving it in ethyl alcohol (30ml) with stirring. Stannous chloride (26.51 g) was added slowly to 29 ml of hydrochloric acid (12N) in a separate beaker. The stannous chloride solution was then added to the perfluoronitroaniline solution in ethyl alcohol. An orange precipitate was formed, but after a short time it disappeared and changed to pale yellow solution. After two hours, the solution became colorless. The mixture was refluxed overnight at 85°C. After twelve hours, the reaction mixture was checked by TLC; there was no starting material present. The product was more polar than the starting material.

The color of the solution turned to faint yellow. The mixture was poured in 800 ml of water without any precipitation. The solution was neutralized by addition of sodium bicarbonate. The organic compound (perfluoro-1,2-benzenediamine) was extracted several times with ethyl acetate which was washed with water and dried over MgSO4 and filtered. The excess solvent was removed under reduced pressure to afford off white solid of 3,4,5,6-tetrafluorobenzene-1,2-diamine (2.346 g, 78.2%); mp 115°C; 19FNMR (282

MHz, CDCl3): δ ppm -160.51 (m, 2F), -1172.21 (m, 2F)

Dibenzo[a,c]-2,3,4,5-tetrafluorophenazine (7.10)

Solid 9,10-phenanthrenequinone 0.62 g (3.0 mmol) was slowly added to 20mL of glacial acetic acid and it was heated (100oC) until everything was dissolved with magnetic

393

stirring. Next, 0.54 g (3.0 mmol) of 1,2-diamino-3,4,5,6-tetrafluorobenzene was slowly added (a few crystals of diamine at a time) to the solution of phenanthrenequinone resulting in the immediate formation of yellowish solid in each addition of diamine. The reaction mixture was checked by TLC (mixture of EtOAc and hexane as eluent), which indicated the presence of both starting materials. The reaction mixture was further heated for 30 minutes at 110°C. The reaction was checked again by TLC, which showed the completion of reaction with no indication of starting materials. The precipitate was separated by vacuum filtration (1.03 g, 97%); mp >250°C; FTIR cm-1 3022.47, 1668.25,

1460.121H NMR (400 MHz, DMSO) δ 9.32 (d, J = 7.8 Hz, 2H), 8.77 (d, J = 7.96 Hz,

13 2H), 7.95 (d, J = 7.18 Hz, 2H), 7.85 (d, J = 7.30 Hz, 2H); C-NMR (75 MHz, CDCl3) δ,

19F-NMR (75 MHz, DMSO) δ, -153.27 (d, J = 17.35Hz, 2F), -154.49 (d, J = 15.43Hz,

2F).

Pyrene-4,5-dione (7.15):

Sodium periodate (10.0g, 46.8 mmol), water (50.0 mL), and ruthenium trichloride

(hydrated), 0.2g (0.96 mmol) were added to a solution of pyrene (2.02 g, 10 mmol) in

CH2Cl2 (40.0 mL) and acetonitrile (40.0 mL). The solution immediately turned dark brown. After a short time, a yellowish precipitate was observed which turned dark brown after a while. The dark brown suspension was stirred at room temperature overnight. The

394

reaction mixture was monitored by TLC, which indicated the presence of four different products in DCM. The mixture was poured into 500 mL of water and the organic phase was separated in separating funnel. The aqueous phase was further extracted with dichloromethane (3 x 50 mL). The CH2Cl2 extracts were combined with the organic phase and washed with H2O (3 x 200 mL) to afford a dark orange solution. The excess solvent was removed under reduced pressure to afford a dark orange solid. The mixture was separated using DCM as eluent in column chromatography. The desired pure product was isolated in the first fraction as bright orange crystals (0.76 g, 32%); mp >250°C; 1H

NMR, (400 MHz, CDCl3) δ 8.51, (dd, J = 7.2 Hz, 1.2 Hz, 2H), 8.18 (dd, J = 9.2 Hz, 1.2

12 Hz, 2H), 7.86 (s, 2H), 7.76 (t, J = 7.6 Hz, 2H); CNMR, (100 MHz, CDCl3) δ 179.65,

135.35, 132.00, 130.98, 129.05, 128.35, 128.17, 127.56.

Pyrene-4,5,9,10-tetraone (7.17)

Sodium periodate (17.5g, 81.8 mmol), water (50.0 mL), and ruthenium trichloride

(hydrate), 0.25g (1.2 mmol) were added to a solution of pyrene (2.02 g, 10.0 mmol) in

CH2Cl2 (40.0 mL) and acetonitrile (40.0 mL). The solution immediately turned to dark brown. After a short time around 10 minutes yellowish precipitate was observed which turned into dark brown after a while. The dark brown suspension was stirred at room

395

temperature and kept overnight. The mixture was poured into 200 mL of water, and the solid (dark green) was removed by vacuum filtration. After the dark green product was washed with 500 mL of water, the organic phase was separated in a separatory funnel.

The aqueous phase was extracted with dichloromethane (3 x 50 mL). The CH2Cl2 extracts were combined with the organic phase and washed with H2O (3 x 200 mL) to give an orange solution. The solvent was evaporated under reduced pressure to afford a dark orange solid. Thin layer chromatography, using an ethylacetate /hexanes (2/5) mixture indicated the presence of two products- one in small quantity and the other in larger quantity. The crude mixture was separated using ethyl acetate as eluent to afford bright orange crystals of pyrene-4,5,9,10-tetraone (0.26 g, 14.6%); mp > 250°C; 1H NMR, (400

12 MHz, DMSO-d6): δ (ppm) 8.33 (d, J = 7.6 Hz, 4H), 7.74 (t, , J = 7.6 Hz, 2H); CNMR,

(100 MHz, DMSO-d6) δ 178.11, 135.12, 134.57, 132.03, 130.90

Tribenzo[a,b,c]-2,3,4,5-tetrafluorophenazine (7.16)

Pyrene-4,5-dione (0.625g, 3.0 mmol) was slowly added to 30 mL of glacial acetic acid and it was heated to 120°C with magnetic stirring until everything dissolved. Next, 0.54 g

(3.0 mmol) of 1,2-diamino-3,4,5,6-tetrafluorobenzene was slowly added (a few crystals of diamine at a time) to this solution of pyrene-4,5-dione resulting in the slow formation

396

of a yellowish solid with each addition of diamine. The reaction mixture was checked by

TLC (mixture of EtOAc and hexane as eluent), which indicated the presence of both starting materials. The reaction mixture was further heated for 30 minutes at 120°C. The reaction mixture was cooled and the resulting precipitate was separated by vacuum filtration (1.08 g, 98%); mp >250°C; FTIR cm-1 30892.27, 1669.51, 1435.12; 1H NMR,

19 (400 MHz, DMSO-d6): δ (ppm) 8.33 (d, J = 7.6 Hz, 4H), 7.74 (t, , J = 7.6 Hz, 2H); F-

NMR (75 MHz, DMSO) δ, -153.7 (d, J = 18.29 Hz, 2F), -154.11 (d, J = 22.85 Hz, 2F).

10,11,12,13,-Tetrafluorophenanthro[4,5-abc]phenazine-4,5-dione (7.18)

Pyrene-4,5,9,10-tetraone (0.13 g (0.496 mmol) was slowly added to 30 mL of glacial acetic acid and it was heated (120°C) with magnetic stirring until everything was dissolved. Next, 0.089 g (0.496 mmol) of 1,2-diamino-3,4,5,6-tetrafluorobenzene was slowly added (a few crystals of diamine at a time) to this solution of pyrene-4,5,9,10- tetraone resulting in the slow formation of yellowish solid in each addition of diamine.

The reaction mixture was checked by TLC (mixture of EtOAc and hexane as eluent), which indicated the presence of both starting materials. The reaction mixture was further heated for 30 minutes at 120°C. The precipitate was separated by vacuum filtration (0.15

1 g, 51.3%); H NMR, (400 MHz, DMSO-d6) δ 9.34 (d, J = 7.68 Hz, 2H), 8.48 (dd, J =

397

7.46 Hz, 1.26 Hz, 2H), 8.00 (t, J = 7.74 Hz, 2H); 19F-NMR (75 MHz, DMSO) δ, -152.61

(m, 4F).

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