Ghulam Shabir

Multichromic Rylene, Azo, Xanthene Scaffolds, Synthesis and Studies

Islamabad

A dissertation submitted to the Department of Chemistry, Quaid-i-Azam University Islamabad, in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Organic Chemistry

Ghulam Shabir

Department of Chemistry Quaid-i-Azam University Islamabad 2015

Dedicated To My Father and My Mother

For their Prayers and Encouragement And To all my respected teachers especially Prof. Dr. Aamer Saeed and Dr. Ghulam Hussain For imparting me knowledge

Table of Contents Title Page Acknowledgments i List of Figures iii-v List of Schemes v-vii List of Tables viii-x List of Abbreviations xi Abstract xii-xiii

PART I Chapter -1 INTRODUCTION 02 1.1 Colorants 02 1.2 Rylene Dyes 03 1.3 Perylene Based Dyes and Pigments 03 1.4 Synthesis and Optical Tuning of Perylene Based Dyes 05 1.4.1 Diamidine PBIs 05 1.4.2 Halogenated PBIs 06 1.4.3 Core substituted PBIs 07 1.4.4 Perylenemonoimides 09 1.4.5 Perylene Tetraesters and Diesters 10 1.5 Water-soluble Rylene Dyes 11 1.6 Higher Rylene Dyes 13 1.7 Summary of Applications of Perylene Chromophore 15 Objective and Plan of Research 16 Chapter -2 RESULTS AND DISCUSSION 2.1 Synthesis and Characterization of Perylene Dianhydride Azo Hybrid Dyes 18 (5a-j) 2.1.1 Fluorescence Studies of Perylene Dianhydride Azo Hybrid Dyes (5a-j) 23 2.2 Synthesis and Characterization of Perylene Diimide Azo Hybrid Dyes 25 (6a-g) 2.2.1 Fluorescence Studies of Perylene Diimide Azo Hybrid Dyes (6a-g) 30 2.2.2 Luminescence Studies 32 2.2.3 Electrochemical Studies 33 2.3 Synthesis and Characterization Perylene Dianhydride Alkoxy Derivatives 35 (7a-h) 2.3.1 Optical Properties 39 2.3.1.1 Maximum Extinction Coefficients (ϵmax) 39 2.3.1.2 Fluorescence Investigations 39 2.3.1.3 Singlet Energies (Es) 41 2.3.1.4 Oscillator Strengths (f) 41 2.3.1.5 Theoretical Radiative Lifetimes (Ƭo) 43 2.3.1.6 Fluorescence Rate Constants (kf) 43 2.3.2 Electrochemical Properties 43 2.3.2.1 Redox Potentials (E1/2) 43 2.3.2.2 Lowest Unoccupied Molecular Orbitals (LUMO) 44 2.3.2.3 Band Gap Energy Value (Eg) 45 2.3.2.4 Highest Occupied Molecular Orbitals (HOMO) 45 2.3.3 Thermal Properties 46

2.4 Synthesis and Characterization of Perylene Schiff Bas Diimide Dyes 47 (13a-e) 2.4.1 Electrochemical Properties 52

Chapter- 3 EXPERIMENTAL

3.1 Materials 55 3.2 Purification of Solvents 55 3.3 Instrumentation 56 3.4 Chromatographic Techniques 56 3.4.1 Thin Layer Chromatography (TLC) 56 3.5 Experimental Procedures: 57 3.5.1 General Procedure for Synthesis of Phenolic Azo Dyes (3a-j) 57 3.5.2 General Procedure for Synthesis of Perylene Dianhydride Azo Hybrid 57 Dyes (5a-j) 60 6a-g 3.5.3 General Procedure for Synthesis of Perylene Diimide Azo Hybrid Dyes 3.7 General Procedure for Synthesis of Perylene Alkoxy Derivatives (7a-h) 62 3.8 General Procedure for Synthesis of Perylene Schiff base diimide Dyes 64 (13a-e) PART II Chapter -4 INTRODUCTION 68 4.1 Azo Dyes 68 4.2 Classification of Azo Dyes 68 4.2.1 Direct Dyes 69 4.2.2 Acid Dyes 70 4.2.3 Reactive Dyes: 70 4.2.4 Disperse Dyes 71 4.2.5 Metal Complex Dyes 71 4.2.6 Mordant Dyes 72 4.2.7 Formazan Dyes 72 4.3 Derivatives of Azo Dyes 73 4.3.1 Reactive Dyes 73 4.3.2 Acid Dyes 74 4.3.2.1 Copper Complexes 75 4.3.2.2 Chromium Complexes 76 4.3.2.3 Cobalt Complexes 76 4.4 Applications of Azo Dyes 77 4.4.1 Dosimetric Indicators 77 4.4.2 Dyeing of Protein, Polyester and Cellulosic fibers 77 4.4.3 Food Colorants 78 4.4.4 Cosmetic Colorants 79 4.4.5 Staining of Biological Tissues 79 4.4.6 Solar Cell Sensitizers 80 4.4.7 Medicinal Potential 80 4.5 Summary of Applications and Derivatization of Azo Dyes 81 Objective and Plan of Research 83 Chapter-5 RESULTS AND DISCUSSION 84 5.1 Synthetic Pathway to the Reactive Azo Dyes (9a-j) 84

5.2 Spectral Properties of Reactive Dyes (9a-j) 87 5.3 Dyeing Properties of Reactive Dyes (9a-j) 90 5.3.1 Exhaustion and Fixation Study of Reactive Dyes (9a-j) 91 5.3.2 Fastness Properties of Reactive Dyes (9a-j) 92 5.4 Synthetic Pathway to the Reactive Azo Dyes (10a-h) 94 5.5 Spectral Properties of Reactive Azo Dyes (10a-h) 96 5.6 Dyeing Properties of Reactive Azo Dyes (10a-h) 98 5.6.1 Exhaustion and Fixation Study of Reactive Azo Dyes (10a-h) 98 5.6.2 Fastness Properties of Reactive Azo Dyes (10a-h) 99 5.7 Synthetic Pathway to the Reactive Azo Dyes (15a-h) 100 5.8 Spectral Properties of Reactive Azo Dyes (15a-h) 103 5.8 Dyeing Properties of Reactive Azo Dyes (15a-h) 106 5.9.1 Exhaustion and Fixation Study of Reactive Azo Dyes (15a-h) 106 5.9.2 Fastness Properties of Reactive Azo Dyes (15a-h) 106 5.10 Synthetic Pathway to the Heterocyclic Dyes 19a-i 107 5.11 Spectral Properties of Dyes 19a-i 108 5.11.1 Dyeing Properties of Dyes 19a-i 111 5.11.2 Exhaustion and Fixation Study of Dyes 19a-i 111 5.11.3 Fastness Properties of Dyes 19a-i 113 5.12 Synthesis of Calix [4] Resorsoniarene Azo Dyes (23a-f) 114 5.13 Characterization of Dyes (23a-f) 115 5.14 Applications of calix [4] Resorsoniarene Azo Dyes (23a-f) 117

Chapter -6 EXPERIMENTAL 119 6.1 Materials 119 6.2 Purification of Solvents 119 6.3 Instrumentation 119 6.4 Chromatographic Techniques 120 6.4.1 Thin Layer Chromatography (TLC) 133 6.5 Experimental Procedures: 120 6.5.1 General Procedure for the Synthesis of bisanilines (1k-t) 120 6.5.2 General Procedure for the Synthesis of Reactive Azo Dyes (9a-j) 122 6.5.3 Dyeing method 126 6.5.4 General Procedure for the Synthesis of Reactive Azo Dyes (10a-h) 127 6.5.5 General Procedure for the Synthesis of γ-acid Based Reactive Azo Dyes 130 (15a-h) 6.5.6 General Procedure for the Synthesis of Heterocyclic Acid Dyes 133 6.5.7 Dyeing Method 136 6.5.8 General Procedure for Synthesis of Calix [4] resorsoniarene Azo Dyes 137 (23a-f)

PART III Chapter-7 INTRODUCTION 7.1 Xanthene Dyes 141 7.2 Photophysical Properties 142 7.3 Derivitzation of Xanthene Dyes 142 7.3.1 Fluorescein Derivatives 143

7.3.1.1 Fluorinated Benzo [c]Xanthene Dyes 144 7.3.1.2 Benzoxanthene Derivatives 145 7.3.1.3 Imidazole Derivative–Fluorescein 146 7.3.1.4 Fluorescein-Based N-glycosylamines 147 7.3.2 Rhodamines Derivatives 148 7.4 Applications of Xanthene Dyes 150 7.4.1 pH Sensors 150 7.4.2 Metal Ion Detectors 151 7.4.3 Fluorescence Imaging 151 7.4.4 Xanthene Dyes as Dye Lasers 152 7.5 Summary of Applications and Derivatives of Xanthene Dyes 153 Objective and Plan of Research 154 Chapter-8 RESULTS AND DISCUSSION 8.1 Synthesis of Biphenyl-3,3’,4,4’-tetracarboxylic dianhydride Based 155 Xanthene Dyes (26a-e) 8.1.1 Spectral Characterization of Biphenyl-3,3’,4,4’-tetracarboxylic 156 dianhydride Based Xanthene Dyes (26a-e) 8.1.2 Electrochemical Studies of Biphenyl-3,3’,4,4’-tetracarboxylic dianhydride 158 Based Xanthene Dyes (26a-e) 8.1.2.1 Redox Potentials (E1/2) 158 8.1.2.2 Lowest Unoccupied Molecular Orbital (LUMO) 159 8.1.2.3 Band Gap Energy (Eg) 160 8.1.2.4 Highest Occupied Molecular Orbital (HOMO) 160 8.1.3 Fluorescence Studies of Biphenyl-3,3’,4,4’-tetracarboxylic dianhydride 161 Based Xanthene Dyes (26a-e) 8.2 Synthesis of Benzophenone-3,3’,4,4’-tetracarboxylic dianhydride Based 162 Xanthene Dyes (28a-e) 8.2.1 Spectral Characterization of Benzophenone-3,3’,4,4’- tetracarboxylic 163 dianhydride Based Xanthene Dyes (28a-e) 8.2.2 Electrochemical Studies of Benzophenone-3,3’,4,4’- tetracarboxylic 166 dianhydride Based Xanthene Dyes (28a-e) 8.2.2.1 Redox Potentials (E1/2) 167 8.2.2.2 Lowest Unoccupied Molecular Orbital (LUMO) 167 8.2.2.3 Band Gap Energy (Eg) 168 8.2.2.4 Highest Occupied Molecular Orbital (HOMO) 168 8.2.3 Fluorescence Studies of Benzophenone-3,3’,4,4’-tetracarboxylic 169 dianhydride Based Xanthene Dyes (28a-e) 8.3 Synthesis of 4, 4'-Oxydiphthalic anhydride Based Xanthene Dyes (30a-e) 170 8.3.1 Spectral Characterization of 4, 4'-Oxydiphthalic anhydride Based 171 Xanthene Dyes (30a-e) 8.3.2 Electrochemical Properties of 4, 4'-Oxydiphthalic anhydride Based 173 Xanthene Dyes (30a-e) 8.3.3 Fluorescence Studies of 4, 4'-Oxydiphthalic anhydride Based Xanthene 175 Dyes (30a-e)

8.4 Synthesis of 1,4,5,8-Naphthalene tetracar-boxylic dianhydride Based 176 Xanthene Dyes (32a-e) 8.4.1 Spectral Characterization of 1,4,5,8-Naphthalene tetracarboxylic 178 dianhydride Based Xanthene Dyes (32a-e) 8.4.2 Electrochemical Properties of 1, 4, 5, 8-Naphthalenetetracarboxylic 181 dianhydride Based Xanthene Dyes (32a-e) 8.4.3 Fluorescence Studies of 1,4,5,8-Naphthalenetetracarboxylic dianhydride 182 Based Xanthene Dyes (32a-e) 8.5 Synthesis of Xanthene Schiff Bases (35a-j) 183 8.5.1 Characterization of Xanthene Schiff Bases (35a-j) 184 8.6 Applications of Xanthene Dyes 185 Chapter-9 EXPERIMENTAL 187 9.1 Materials 187 9.2 Purification of Solvents 187 9.3 Instruments Used 187 9.4 Chromatographic Techniques 187 9.4.1 Thin Layer Chromatography (TLC) 187 9.5 Experimental Procedures 188 9.5.1 General Procedure for Synthesis of Biphenyl-3,3’,4,4’-tetracarboxylic 188 dianhydride Based Xanthene Dyes (26a-e) 9.5.2 General Procedure for Synthesis of Benzophenone- 3,3’,4,4’- 189 tetracarboxylic dianhydride Based Xanthene Dyes (28a-e) 9.5.3 General Procedure for Synthesis of 4, 4'-Oxydiphthalic anhydride Based 191 Xanthene Dyes (30a-e) 9.5.4 General Procedure for Synthesis of 1,4,5,8-Naphthalenetetracarboxylic 193 dianhydride Based Xanthene Dyes (32a-e) 9.5.5 General Procedure for Synthesis of Xanthene Schiff Bases (35a-j) 195 References 200 Conclusions 202

Acknowledgments

All praise and glory to ALLAH SUBHANA’O TAALA the only creator who helped and guided me in all fields of life. All the respects for HOLY PROPHET HAZRAT MUHAMMAD (P.B.U.H) whose life is an ideal pattern of success for us.

I feel great pleasure and privileges to express my profound sense of gratitude and earnest appreciations to my worthy supervisor Prof. Dr. Aamer Saeed, Department of Chemistry, Quaid- i-Azam University, Islamabad, for enthusiastic encouragement, astonishing suggestions and financial support. I am thankful to Prof. Dr. Muhammad Siddiq, Chairman, Department of Chemistry, Quaid- iAzam University Islamabad for providing me all necessary research facilities. I cordially thanks to Prof. Dr. Shahid Hameed, Head of Organic Chemistry Section, Department of Chemistry, Quaid-i-Azam University, Islamabad, and all teachers of Organic Chemistry Section especially Prof. Dr. Muhammad Farman for his moral and ethical support. Many thanks to staff members of this department, especially Mr. Mahmood, Mr. Sharif Chohan, Mr. Shams, Mr. Aamir, Mr. Shabbir, Mr. Iliyas, Mr. Matloob, Mr. Saqib, Mr. Mustafa, Mr. Irfan Sabir, Mr. Tayyab, Mr. Rashid, Mr. Liaqat, Rana Tahir, Shahid Naeem and Mr. Arif for their all time devotion. I also owe my recognition to my lab fellows Dr. Madiha Irfan, Dr. Madiha Kazmi, Dr. Hummera Rafique, Dr. Aliya Ibrar, Mr. Ali Bahadur, Mr. Pervaiz Ali, Ms. Mobeen Arif, Ms. Asma Khurshid, Ms. Aliya Shahzadi, Mr. Jamal ul din, Mr. Shamsul Mahmood, Ms. Iram Batool, Mr. M. Qasim, Mr. M. Arif, Muhammad Imran and Muhammad Attique for their help at crucial time of my research work and all my seniors and juniors for their encouragement. Special thanks to my college teacher Prof. Ghulam Hussain and a great scholar for his prayers and support in providing dye intermediates and also in every difficult time. I am also thankful to Dr. Muhammad Arshed (PS; Pinstech) and Dr. Khalid Masood (PS; NLP) for their support in my research. Finally, I would like to thanks my parents for their support, love and everything they given to me and last but not least to all my family members and those who prayed for my prosperity.

Ghulam Shabir

List of Figures

PART I Figure 1.1 The main component in ancient purple, 6,6´-dibromoindigo. 2 Figure 1.2 Rylene chromophores and their mono and diimide derivatives 3 Figure 1.3 Structures of different colored PBI pigments. 4 Figure 1.4 Representation of H-Type aggregates and their effect on fluorescence 12 Figure 1.5 Different core unsubstituted PBIs bearing ionic and non ion imide 12 substituents Figure 1.6 Different core unsubstituted PBIs bearing ionic and non ion imide 13 substituents Figure 1.7 Water soluble derivatives of terrylene (1, 2, 3) and quaterrylene 14 Figure 1.8 Water soluble derivatives of terrylene (1, 2, 3) and quaterrylene 15 Figure 2.1 U.V Visible spectra of (5a-j) 22 Figure 2.2 1H-NMR spectrum of 5a 22 Figure 2.3 13C-NMR spectrum of 5a 23 Figure 2.4 Fluorescence Spectra of Dyes 5a-j 24 Figure 2.5 UV-Visible spectra of 6a-g in water and ethanol 28 Figure 2.6 FTIR spectrum of Perylene diimide azo hybrid dye 6a 29 Figure 2.7 1H-NMR spectrum of Perylene diimide azo hybrid dye 6a 30 Figure 2.8 13C-NMR spectrum of Perylene diimide azo hybrid dye 6a 30 Figure 2.9 Fluorescence Spectra of perylene diimide azo hybrid dyes, 6a-g 31 Figure 2.10 UV-Visible spectra of dyes 7a-h in water 37 Figure 2.11 Fluorescence spectra of dyes 7a-h in water 40 Figure 2.12 Cyclic Voltammogram of 7a-h in water 44 Figure 2.13 UV-Visible Spectra of perylene schiff base diimide dyes 13a-e in DMSO 49 Figure 2.14 Fluorescence spectrum of compounds (13a-e) 51 Figure 2.15 1H-NMR spectrum of 13d 52 Figure 2.16 13C-NMR spectrum of 13d 52 Figure 2.17 Cyclic Voltammogram of perylene schiff base diimide azo dyes 13a-e in 53 DMSO PART II Figure 4.1 Structure of a typical azo dye. 68 Figure 4.2 Examples of anionic direct azo dyes (1, 2 and 3) 69 Figure 4.3 Examples of cationic azo dyes (4 and 5) 69 Figure 4.4 Examples of anionic acid dyes (6, 7 and 8) 70 Figure 4.5 Examples of cationic Acid dyes (9 and 10) 70 Figure 4.6 Reactive dyes base on diazine and vinylsulphone para ester 71 Figure 4.7 Typical examples of disperse azo dyes 71 Figure 4.8 Metal complex azo dyes 72 Figure 4.9 Typical examples of mordant azo dyes 72 Figure 4.10 Basic structure of formazan azo dyes 72

Figure 4.11 1, 5- substituted examples of formazan azo dyes 73 Figure 4.12 Different reactive functionalities in reactive dyes 74 Figure 4.13 1:1 and 2:1 metal complex acid dyes 75 Figure 4.14 o,o-dihydroxyazo copper complex acid dyes 76 Figure 4.15 1: 2 and 1:1 chromium complex dyes 76 Figure 4.16 1:2 cobalt complex dyes 77 Figure 4.17 Examples of indicator Azo dyes 77 Figure 4.18 Examples of Azo dyes used for dyeing of protein, polyester and cellulosic 78 fibers Figure 4.19 Examples of azo dyes food colorants dyes 78 Figure 4.20 Examples of Azo dyes used in cosmetics 79 Figure 4.21 Examples of Azo dyes used for staining 79 Figure 4.22 Typical Example of Azo dyes used in DSSC 80 Figure 4.23 Azo derivative of 5-aminosalicylic acid immobilized on polyethylene 81 glycol Figure 4.24 Bisazo compound with anti-HIV activity 81 Figure 5.1 Combined UV-Visible spectrum of MCT Dye 9a-j 88 Figure 5.2 FTIR spectrum of synthesized reactive azo dye 9d. 89 Figure 5.3 Samples of dyes applied on cotton cloth pieces before washing 90 treatments for wash fastness. Figure 5.4 Samples of dyes applied on cotton cloth pieces after washing treatments for 90 wash fastness. Figure 5.5 Structures of methylene bisanilines (1k-r) used as linker 94 Figure 5.6 Combined UV-Visible spectrum of MCT Dye 10a-h 97 Figure 5.7 Combined UV-Visible spectrum of MCT dyes (15a-h) 103 Figure 5.8 1H-NMR spectrum of reactive dye 15c 105 Figure 5.9 1H-NMR spectrum of reactive dye 15c 105 Figure 5.10 Combined UV-Visible spectrum of heterocyclic azo dyes (19a-i) 109 Figure 5.11 FTIR spectrum of heterocyclic dye 19f 110 1 Figure 5.12 H-NMR spectrum of heterocyclic dye 19f in CDCl3 111 13 Figure 5.13 C-NMR spectrum of heterocyclic dye 19f in CDCl3 111 Figure 5.14 Application of Heterocyclic dyes on leather (19a-i) 113 Figure 5.15 Combined UV-Visible spectrum of calix [4] resorsoniarene azo dyes (23a- 116 f) Figure 5.16 Most probable metal ion interaction mechanism of calix [4] 117 resorsoniarene azo dyes. Figure 5.16 Study of different metal ion interactions with Calix [4] resorsoniarene azo 118 dyes (23a-f)

PART III Figure 7.1 Structural relationship among xanthone, xanthene and coumarin 141 chromophores Figure 7.2 Naturally occurring xanthene in plants 142

Figure 7.3 General structure of xanthene derivatives with different photophysical 142 properties.

Figure 7.4 Photophysical behavior expressed by Rhodamine 101 and Rhodamine B. 143 Figure 7.5 Two forms of fluorescein in the range of 6.31 to 6.80 phenolic pKa values 144 Figure 7.6 Structure of modified fluorescein 144 Figure 7.7 Structures of selected rhodamines 149 Figure 7.8 Mechanism of the xanthene probe response to pH changes 151 Figure 7.9 Xanthene dyes detectors for Fe+3 and Hg+2 151 Figure 7.10 Rhodamine 6G chloride solution in methanol emitting yellow light under the 152 influence of a green laser Figure 8.1 Combined UV spectrum of Biphenyl-3,3',4,4'-tetracarboxylic dianhydride base 157 xanthene dyes (26a-e) Figure 8.2 1H-NMR spectrum of xanthene dye 26e 158 Figure 8.3 LCMS spectrum of xanthene dye 26e 158 Figure 8.4 Cyclic voltammogram of xanthene dyes (26a-e) 159 Figure 8.5 Fluorescence spectrum of xanthene dyes (26a-e) 161 Figure 8.6 Combined UV spectrum of benzophenone-3,3',4,4'-tetracarboxylic dianhydride 165 base xanthene dyes (28a-e) Figure 8.7 1H-NMR of benzophenone based xanthene dye 28e 165 Figure 8.8 13C-NMR of benzophenone based xanthene dye 28e 166 Figure 8.9 LCMS of benzophenone based xanthene dye 28e 166 Figure 8.10 Combined cyclic voltammogram of1H-NMR of benzophenone based xanthene 167 dyes (28a-e) Figure 8.11 Fluorescence spectra of xanthene dyes (28a-e) in water 169 Figure 8.12 UV-Visible spectrum of xanthene dyes (30a-e) 172 Figure 8.13 LCMS spectrum of xanthene dye 30d 173 Figure 8.14 Combined cyclic voltammogram of xanthene dyes (30a-e) 174 Figure 8.15 Fluorescence spectrum of Xanthene dyes (30a-e) 176 Figure 8.16 UV-Visible spectrum of xanthene dyes (32a-e) 178 Figure 8.17 FTIR spectrum of dye (32e) 179 Figure 8.18 1H-NMR spectrum of dye (32e) 180 Figure 8.19 LCMS spectrum of xanthene dye (32e) 180 Figure 8.20 Fluorescence spectra of xanthene dyes (32a-e) in water 183

Figure 8.21 Dye 26e applied on onion cells, concentrated in cell membrane, b) Dye 26e 186 emitting yellow fluorescence under UV-light.

List of Schemes PART I Scheme 1.1 Synthetic route towards 3,4,9,10-Perylenetetracarboxibisimides 4 (PBIs). Scheme 1.2 Route towards perylene diamidines from perylene-3,4,9,10tetracarboxylic 6 dianhydride Scheme 1.3 Chlorination and bromination of Perylene-3,4,9,10-tetracarboxylic 7 dianhydride Scheme 1.4 Red shifted diphenoxylated and tetraphenoxylated PBIs 7 Scheme 1.5 Synthetic routes towards various bay substituted PBIs 8 Scheme 1.6 Synthesis of core enlarged PBIs via Diels-Alder reaction 9 Scheme 1.7 Lateral core enlarged PBIs 9 Scheme 1.8 Synthetic pathways to halogenated PMIs 10 Scheme1.9 Synthetic pathways to halogenated PMIs 11 Scheme 2.1 Synthetic route to phenolic azo dyes 18 Scheme 2.2 Synthetic scheme for tetrachloro perylene based azo dyes (5a-j) 19 Scheme 2.3 Synthetic route to tetrachloroperylene based azo dyes (6a-g) 26 Scheme 2.4 Synthetic route to tetrachloroperylene based fluorescent dyes (7a-h) 36 Scheme 2.5 Synthesis of perylene Schiff base diimide dyes (13a-e) 48

PART II Scheme 4.1 Synthesis of diazonium Salt 68 Scheme 4.2 Summary of reactions exhibited by diazonium salts 82 Scheme 5.1 Synthesis of 5,5'-methylenebis(3-aminoanilines) linkers 84 Scheme 5.2 Synthetic route to bisazo MCT reactive dyes (9a-j) 86 Scheme 5.3 Synthetic route to monosazo MCT reactive dyes (10a-h) 95 Scheme 5.4 Synthetic route to monoazo MCT reactive dyes (15a-h) 102 Scheme 5.5 Synthetic pathway to heterocyclic direct dyes (19a-i). 108 Scheme 5.6 Synthesis of Calix [4] azo dyes 23a-f 115

PART III Scheme 7.1 Synthesis of carboxy SNARF-4F dye 145 Scheme 7.2 Benzoxanthene derivatives active heterocycles synthesis 145 Scheme 7.3 Dibenzoxanthenes derivatives synthesis 145 Scheme 7.4 Dioxo-xanthenes derivatives synthesis 146 Scheme 7.5 Synthesis of 12-aryl-8,9,10,12-tetrahydrobenzo[a]xanthene-11-one 146 derivatives in ionic liquid [NMP]H2PO4. Scheme 7.6 Synthetic scheme of Nutlin-Glycine-FAM conjugate 147 Scheme 7.7 Synthesis of fluorescein-based N-glycosylamines 148 Scheme 7.8 General synthesis of rhodols and rhodamines. 149 Scheme 7.9 Synthesis of rhodamines via direct nucleophilic substitution from 150 halogenated fluoresceins.

Scheme 7.10 Synthesis of rhodol fluorophores via Buchwald-Hartwig amination 150 reaction

Scheme 7.11 Synthesis of rhodamines via Buchwald-Hartwig amination reaction 150 Scheme 7.12 Synthesis of chemosensor 152 Scheme 8.1 Synthesis of Biphenyl-3,3',4,4'-tetracarboxylic dianhydrides based 155 xanthene dyes (26a-e) Scheme 8.2 Synthesis of Benzophenon-3,3',4,4'-tetracarboxylic dianhydrides based 163 Xanthene dyes (28a-e) Scheme 8.3 Synthesis of 4,4'-Oxydiphthalic anhydride based xanthene Dyes (30a- 171 e) Scheme 8.4 Synthesis of 1,4,5,8-Naphthalenetetracarboxylic dianhydride based 177 xanthene Dyes (32a-e)

Scheme 8.5 Synthesis of fluorescein Schiff bases (35a-j) 184

List of Tables S.No. Title Page No. PART I

Table 2.1 Wavelength of maximum absorption λmax of perylene azo hybrid dyes in 20 Different solvents. Table 2.2 Fluorescence values of 5a-j azo hybrid dyes in water 25

Table 2.3 Wavelength of maximum absorption λmax of 6a-g hybrid Azo Dye in 27 Different solvents. Table 2.4 Fluorescence values of Perylene diimide Azo Hybrid Dyes 6a-g in water 32 Table 2.5 Luminescence and Fluorescence Quantum Yield (FLQ) values of 33 perylene diimide Azo Hybrid Dyes 6a-g in water Table 2.6 Oxidation Potential (Eox) of compounds perylene diimide Azo Hybrid 34 Dyes (6a-g) Table 2.7 Energy of Highest Occupied Molecular Orbital’s of perylene diimide azo 35 hybrid dyes (6a-g) Table 2.8 Wavelength of maximum absorption λmax of perylene dyes in water 37 Table 2.9 Molar extinction coefficients of perylene dyes 39 Table 2.10 Fluorescence values of Alkylated perylene dyes (7a-h) in water 40 Table 2.11 Singlet energies of dyes 41 Table 2.12 Oscillator strengths of alkylated perylene dyes (7a-h) 42 Table 2.13 Theoretical radiative lifetime of perylene dyes 42 Table 2.14 Fluorescence rate constant of perylene dyes 43

Table 2.15 Redox potential (E1/2) of perylene dyes 44

Table 2.16 Half wave potential (E1/2) and LUMO energy levels of alkylated perylene 45 dyes Table 2.17 Band gap energy and HOMO energy levels alkylated of perylene dyes 46 Table 2.18 TG and DTA data of Alkylated perylene dyes 7a-h 46 Table 2.19 Absorption Maxima of perylene Schiff base diimide dyes 50

Table 2.20 Half wave potential (E1/2) and LUMO energy levels of perylene Schiff 54 base diimide 13a-e PART II Table 5.1 Characterization data of dyes 9a-j 88 Table 5.2 Dye-bath containing materials 91 Table 5.3 Exhaustion and fixation data of the dyes 9a-j 91

Table 5.4 Fastness properties data of the dyes 9a-j 93 Table 5.5 Characterization data of dyes 10a-h 96 Table 5.6 Exhaustion and fixation data of the dyes 10a-h 99 Table 5.7 Fastness properties data of the dyes 10a-h 100 Table 5.8 Characterization data of dyes (15a-h) 102 Table 5.9 Exhaustion and fixation data of the dyes 15a-h 106 Table 5.10 Fastness properties data of the dyes 15a-h 107 Table 5.11 Exhaustion and fixation data of heterocyclic azo dyes 19a-i 112 Table 5.12 Fastness properties data of heterocyclic azo dyes (19a-i) 113 PART III Table 8.1 Physical Characteristics of Xanthene Dyes 26a-e 155 Table 8.2 Wavelength of maximum absorption λmax of xanthene dyes (26a-e) in 156 water Table 8.3 LUMO energy levels of xanthene dyes (26a-e) 159 Table 8.4 HOMO energy levels and band gap energy of xanthene dyes (26a-e) 160 Table 8.5 λmax and Emission of Xanthene Dyes 26a-e in water 162 Table 8.6 Physical Characteristics of Xanthene Dyes (28a-e) 162 Table 8.7 Wavelength of maximum absorption λmax of xanthene dyes (28a-e) in water 163

Table 8.8 LUMO energy levels of xanthene dyes (28a-e) 167 Table 8.9 HOMO energy levels and band gap energy of xanthene dyes (28a-e) 168 Table 8.10 Fluorescence data of Xanthene Dyes (28a-e) in water 169 Table 8.11 Physical Characteristics of Xanthene Dyes 30a-e 170 Table 8.12 Wavelength of maximum absorption (λmax/nm)of xanthene dyes (30a-e) in 171 water Table 8.13 LUMO/ eV energy levels of xanthene dyes (30a-e) 174 Table 8.14 HOMO/eV energy levels and band gap energy of xanthene dyes (7a-e) 175 Table 8.15 Fluorescence data of Xanthene Dyes (30a-e)in water 176 Table 8.16 Physical Characteristics of 1,4,5,8-Naphthalenetetracarboxylic 177 dianhydride based Xanthene Dyes (32a-e) Table 8.17 Wavelength of maximum absorption (λmax/nm)of xanthene dyes (32a-e) in 178 water Table 8.18 LUMO/ eV energy levels of 1,4,5,8-Naphthalenetetracarboxylic dianhydride 181 based xanthene dyes (32a-e) Table 8.19 HOMO/eV energy levels and band gap energy of xanthene dyes (32a-e) 182

Table 8.20 Fluorescence data of 1,4,5,8-Naphthalenetetracarboxylic dianhydride based 183 Xanthene Dyes (32a-e) in water

List of Abbreviations CV Cyclic voltammetry DCM Dichloromethane DMF Dimethylforamide DSC Differential scanning calorimetry DMSO Dimethyl sulfoxide Eg Energy gap

Eox Oxidation potential

E1/2 Half wave potential FLQ Fluorescence quantum yield HOMO Highest occupied molecular orbital LCMS Liquid chromatography mass spectrometry LUMO Lowest unoccupied molecular orbital MCT Monochlorotriazine PBA Perylenetetracarboxi-3,4,9,10-bisanhydride PBI Perylenetetracarboxi-3,4,9,10-bisdiimide PDI Perylene diimide PMI Perylenemonoimides

PPNs poly-perinaphthalenes PTE Perylene tetraesters QBI Quaterrylene bis-diimide QY Quantum yield TBAB Tetrabutylammonium bromide

TBAPF6 Tetrabutylammonium hexafluorophosphate TGA Thermal gravimetric analysis THF Tetrahydrofuran TICT Intramolecular charge-transfer TLC Thin-layer chromatography TEM Positron emission tomography

Abstract

This dissertation describes the investigation of the synthesis, characterization, photophysical and electrochemical studies of new rylene, azo and xanthene scaffolds. In the first part of this thesis, perylene dianhydride and perylene dimide azo hybrid chromophoric materials were designed, synthesized and characterized. It was found that the bay substitution of azo dyes on PDI affected

π-π stacking with the neighboring PDIs, and enhanced their solubility in aqueous as well as in other polar solvents. Conjugation extension of perylene dianhydride along the molecular axis did not affect the π-π stacking and solubility issue which is a major obstacle in application of these compounds remained unsolved.

The second part of this research work is concerned with the synthesis, characterization and applications of azo reactive, azo heterocyclic and azo claix [4] resorcinrene chromophores. In case of azo reactive dyes, various new bis anilines have been used as bridging groups which have enhanced the exhaustion and fixation of dyes with textile fibers without affecting the color intensity and shades. In addition to exhaustion and fixation, fastness parameters (light fastness, wash fastness and rubbing fastness) also improved which are the key parameters in applications of reactive dyes. In azo heterocyclic dyes synthetic methodology has been changed. Here at first chromophoric intermediates were synthesized and after color development heterocycles of pyrazolone type were introduced which resulted in high purity of products. Azo claix [4] resorcinrene have been synthesized by taking the advantage of nucleophilicity of carbons sandwiched between hydroxyl groups of claix [4] resorcinrene and coupling with those diazo components which have hydroxyl groups ortho to azo functionality. Combination of such type of azo components and calixarene coupler resulted in dyes which were found to be useful in cations removal from aqueous medium and complexometric titrations.

Third part of thesis is concerned with xanthene synthesis and characterization along with electrochemical, fluorescence and cells staining study of dyes. Novelty of this work lies in the synthesis of xanthene derivatives using first time the commonly available catalyst NH4Cl to synthesize the xanthene dyes from double dianhydrides.

PART I

Chapter 1 INTRODUCTION 1.1 Colorants: God is gorgeous, He likes the beauties. How nice He colors the cosmos by spreading the Sun's rays on His creatures to exhibit his own Beauty. Mere color, unspoiled by meaning and unallied with definite form, can speak to the soul in a thousand ways. Colors are a form of nonverbal communication that can speak volumes in a fraction of seconds. They can instantly set a mood, convey an emotion, invoke a physiological reaction or inspire people to take action. Colorants are characterized by their ability to absorb or emit light in the visible range (400-700 nm). Colorants may either be inorganic or organic compounds according to chemical structure, and both can be subdivided into natural and synthetic. Another classification for colorants is their division into dyes (applied to many substrates like textiles, leather, paper and hair, from a liquid in which they are completely or partly soluble), and pigments (small insoluble particles in the medium in which they are applied, and they need additional compounds like polymers to be attached to the substrates). Development and applications of dyes and pigments have always been the hub of research if we take the picture of the past history of mankind. Even in prehistoric times, man has dyed textiles, furs, fibers and other items for artistic reasons to get similarity with objects of nature. Evidence in favour of dyes and pigment applications comes from the cave paintings in the Cauvet-caves in France which have been estimated to originate 33000 years B.C. [1]. Initially dyes were obtained from natural sources. For example, ancient purple was obtained from purple snail and a very small quantity of dye was achieved from a large number of snails [2] (Figure 1.1). Less availability of this dye made it highly valuable.

Figure 1.1 The Main component in ancient purple, 6,6´-Dibromoindigo. This state of affairs has augmented interest in the expansion of economical synthetic routes to dye materials. From the eighteen century on, various synthetic goals in dye chemistry were achieved, with the synthesis of Berliner blau by Diesbach in 1706, [3] Mauveine by Perkin in 1856 [4] and

indigo by Baeyer in 1878 [5]. Consumption of natural dyes in textile field was reduced to 10% until the early 1900 [6]. The main focus on dye research was the development of high intensity colors with conceivable application methods. The current level of interest in dye development has changed from their simplistic utilization to more nominal, superior and particular topics in the fields of electronics, laser technology and medicine [7]. The present status of dyes applications deals with their color properties as well as away from their colors. 1.2 Rylene Dyes The term rylene dyes describes a family of dyes which consist of naphthalene units connected via peri- positions to each other (Figure 1.2). Therefore, if one looks at them in a polymeric way they belong to the class of the poly-peri-naphthalenes (PPNs) [8]. A common substitution pattern of these dyes is at the outer peri positions with imide functions leading to the respective monoimides or bisimides. Due to their electron withdrawing nature this increases the photostability of the chromophore drastically [9]. Other important substitution positions are the so-called bay position, providing a cis-butadiene like structure and the ortho position next to the imide function [10].

Figure 1.2 Rylene chromophores and their mono and diimide derivatives 1.3 Perylene Based Dyes and Pigments Rylene dyes and pigments are based upon perylene chromophore and are among the widely used pigments in various scientific applications ranging from organic electronics [11] over solar cell [12] to supramolecular chemistry research [13]. Rylene dyes are functional dyes which are not used because of their visual appearance, but due to their substantial or chemical properties. As the color of these materials is of secondary importance, the wavelength span of functional dyes lies in the range 200- 1500 nm. Perylenetetracarboxi-3,4,9,10-bisanhydride (PBA) 5 has been the starting point of many synthesized rylene chromophores which was developed by Kardos in 1912-13 [14] (Scheme 1.1) using acenaphtene 1 as starting material. Synthetic methodology involved oxidation with vanadium pentoxide as catalyst to provide 1,8-Naphthalene-dicarboxanhydride 2 followed by imidization with ammonia to give 1,8-Naphtalene-dicarboximide 3. Dimerization of 1, 8naphtalene-dicarboximide to Perylene-3,4,9,10-tetracarboxibisimide (PBI) 4 was made by oxidative coupling in an alkaline melt which on hydrolysis with sulfuric acid at high temperatures (220 °C) to provide perylene-3,4,9,10- tetracarboxidibisanhydride (PBA) 5.

Scheme 1.1 Synthetic route towards 3,4,9,10-Perylenetetracarboxibisimides (PBIs). Derivatization of 5 into different PBIs (6) can be achieved easily via simple coupling with aromatic or aliphatic amines and phenols at high temperatures [15]. Due to poor solubility of these derivatives in aqueous and non aqueous solvents they were mainly used as organic car pigments, lacquers and in optical switches [16]. Based on the same chromophore different colored pigments from bright red to black [17] were accessed due to the strong cystallochromic effect (Figure 1.3).

Figure 1.3 Structures of different colored PBI pigments Differences in the packing in the solid state has led to a different degree of orbital overlap and therefore to a different color and bathochromic shift in absorption are observed due to better overlap between adjacent PBI molecules. Improvement in solubility of perylenebisdianhydride PBIs was achieved by introducing sterically demanding bulky groups at the imide positions which hindered the strong aggregation among PBIs molecules. Excellent aggregation blockers are mostly, ortho substituted anilines [18] or secondary branched amines [19] which are so-called swallow tail substituents which produced PBIs with solubility in organic solvents > 100 g/l. In solution all core unsubstituted PBIs possess the same colour, absorption and emission properties resulting from S0-S1 absorption with small Stoke shift following the mirror image rule [20a,b]. A little effect of substituents at the imide position on absorption as well as the emission properties has been seen because there are nodes at the imide nitrogen atoms in the HOMO and LUMO [21]. Enhancement in solubility of PBIs extended their applications beyond their use as pigments into many other areas like dye lasers [22] or fluorescence collectors [23] to increase the efficiency of solar cells. 1.4 Synthesis and Optical Tuning of Perylene Based Dyes Optical and photophysical properties of rylene dyes have made them to be used in many different fields which can be prescribed by changing the substitution patterns on PBI chromophores. The most important examples, including their synthesis are shown in the following sections.

1.4.1 Diamidine PBIs Diamidine PBIs were prepared by treatment of perylene-3,4,9,10-Tetracarboxylic dianhydride with o-disubstituted aromatic diamines (8 and 10) which led to the synthesis of a mixture of syn- and anti-isomeric dyes 8a, 8b, 10a and 10b (Scheme 1.2) [24]. By the extension of the aromatic π-system with o-Phenylenediamine (7), bathochromic shift of ~70nm was observed in these dyes in comparison to PBIs. The substitution of PBIs with more extended aromatic diamines like 1,8Diaminophenantrene (9) resulted in red shift in absorbance of bisnaphtalene and the bisphenanthrene substituted diamidines 10a and 10b having absorption about 660 nm. However, this bathochromic shift in absorbance was not exclusively connected to extended π-system but also to substitution of conjugated systems at the bay region of perylene core which increased its solubility which was the main obstacle in the synthesis of perylene derivatives. These spectral properties have made these dyes attractive class in xerographic applications [25].

Scheme 1.2 Synthetic route towards perylene diamidines from Perylene-3,4,9,10-tetracarboxylic dianhydride 1.4.2 Halogenated PBIs Tetrachlorinated and brominated PBAs are key intermediates in the synthesis of bay substituted PBIs which are produced via direct chlorination of perylene-3,4,9,10-tetracarboxylic dianhydride in sulfuric acid with elemental chlorine [26] or in the case of bromination by adding oleum and elemental bromine to the reaction mixture (Scheme 1.3) [27]. In all halogenated PBAs purification always remained a big problem due to poor solubility of intermediates and to some extent they were purified by column chromatography. In case of brominations it was assumed the formation of dibrominated 1,7 regioisomer. This issue did not catch any further attention as the resulting

brominated bisimides were only distinguishable upon using high field (>400 MHz NMR) 1H-NMR and were in principle not separable by common purification techniques like column chromatography. Wurthner et al. in 2004 first time obtained the regioisomerically pure 1,7 dibrominated PBIs via repetitive crystallization, which still is the only way to produce large amounts of regioisomerically pure 1,7-substituted PBIs [28]. Now routes are available for the selective mono, di and tetra brominated PBIs by controlling the conditions of reaction mixture.

Scheme 1.3 Chlorination and bromination of perylene-3,4,9,10-tetracarboxylic dianhydride 1.4.3 Core Substituted PBIs One of the most common ways to increase the solubility of perylene dyes is the substitution at bay region of halogenated PBIs by phenol or derivatives of phenol. Substitution at bay position reduces the tendency of π-π self aggregation and also alters the optical properties of PBIs. [29]. For example, the di- and tetraphenoxylated PBIs [30, 31] substituted with simple phenol groups possess red-shifted absorptions in comparison to core unsubstituted PBI with absorption maximas at 540 nm and 573 nm, respectively (Scheme 1.4).

Scheme 1.4 Red shifted diphenoxylated and tetraphenoxylated PBIs Similar red shift in fluorescence of these dyes has been observed, for di and tetraphenoxylated PBIs to 571 nm and 608 nm while retaining the quantum yield of its core unsubstituted precursor with φfl=100 % and φfl= 96 %, respectively. From literature it has been inferred that the red shift is mostly ascribed to the electron donating ability of the ether oxygens instead of extended π-system [32]. Orientation of the phenoxy groups relative to the perylene core leads to a different degree of conjugation from the oxygen molecular orbitals to the perylene core which changes the optical properties [33].

Various electron donating or withdrawing substituents can also be introduced to the PBI-core at the bay positions besides phenoxylation (Scheme 1.5). Electron donating amino groups like pyrrolidine caused a red shift in absorbance as well as fluorescence to extremely high wavelength of 709 nm and 748 nm respectively [34]. In contrast to the core unsubstituted PBIs the optical transition of these bay amine substituted dyes gets a strong quadrupolar charge transfer, character resulting in a moderate solvatochromic effect [34,35]. Electron withdrawing groups like fluoro and cyano can also be substituted, but their effect on absorption and emission is very small. Carbon- carbon coupling can also be done on halogenated PBIs yielding alkynelated and arylated PBIs [36,37] which provides a way for the core extended PBIs [38]. Although the aromatic core of these new dyes is enlarged, but in most cases hypsochromic effect on absorption and emission is seen which resulted due to larger HOMO-LUMO band gap energies.

Scheme 1.5 Synthetic routes towards various bay substituted PBIs. Diels-Alder reaction provides another way to enlarge PBIs core from a core unsubstituted PBI with maleic anhydride under drastic conditions (Scheme 1.6) yielding PBI diels alder adduct [39] having an anhydride motif at the bay region. This was further transformed with primary amines or peri diaminonaphtalenes yielding PBI trisimides displaying blue shift in absorption to 465 and 464 nms respectively.

Scheme 1.6 Synthesis of core enlarged PBIs via diels-alder reaction. Lateral core extension of PBIs by two bridged donor substituents led to a strong bathochromic shift of the optical transitions due to the alpha-donor effect of this arrangement [39,40] (Scheme 1.7). Therefore the one side bay-substituted PBI has its absorbance shifted by approx. 130 nm to 650 nm and for the doubly substituted PBIs resulting in shift of about approx. 260 nm to 778 nm. Both dyes possess fluorescent properties with the maxima of fluorescence at 775 nm and 837 nm, but with no quantum yields reported. It is interesting that although these properties make these dyes interesting fluorophores in various application fields, but no other reports about alpha donor substituted PBIs can be found.

Scheme 1.7 Lateral core enlarged PBIs 1.4.4 Perylenemonoimides Halogenated 3,4-perylenemonoimides (PMI) also act as key intermediate in rylene dyes like core halogenated PBIs because of their readily accessibility (Scheme 1.8). The absorbance of core unsubstituted PMIs is slightly shifted towards shorter wavelength; to 510 nm due to the loss of one electron withdrawing imide functionality. Similar to PBIs they have high fluorescence exhibiting φfl = 90% [41]. Diversity of PMIs can be obtained from phenoxylation [42] substitution with various amines, [43] and metal catalyzed carbon-carbon couplings [44] similar to that of PBIs. Trisbrominated PMIs offer the easy possibility to functionalize the same core with different substituents, as the bromine at the peri position possesses different reactivity than the bay bromines. Diversity in this class of has enabled them in a variety of applications like PBIs.

Scheme 1.8 Synthetic pathways to halogenated PMIs. 1.4.5 Perylene Tetraesters and Diesters Besides the imide functionalization another common substitution pattern of 3,4,9,10-perylene bisanhydrides are the corresponding perylene tetraesters (PTEs). Although the first preparation of these dye materials was reported in the 1980s [45] (Scheme 1.9), but during the last decades they did not attract as much attention in research as their related PBIs. This might be due to the fact that they do not offer the easy possibility of introducing different functional groups onto the dye scaffold.

Scheme 1.9 Synthetic pathway to perylene tetra esters and diesters 1.5 Water Soluble Rylene Dyes Fluorescent dyes are gaining importance in the modern science because of their diverse applications in the area of medicine, pharmaceutical and biochemical research. Staining of certain biomolecules, regions, and aerials within a biological system to allow a deeper insight into the working principles is the main function of fluorescent dyes [46]. For example ways and functions within biological processes on a cellular level can be traced by labeling the proteins with fluorescent markers [47]. There are certain properties which are favorable for most applications of the fluorescent dyes which include high brightness, high thermal, photochemical and chemical stability, low toxicity and water solubility. Major problem associated with rylene dyes is aggregate formation which changes their optical properties [48]. There are two possible aggregates which are known by the names of their discoverers as H-aggregates and J-aggregates. These aggregates are also called as Scheibeaggregates due to G. Scheibe, who independently discovered the same phenomenon in PBIs [49]. H-aggregates are characterized by the hypsochromic shift in absorption while J- aggregates are characterized by their bathochromic shifts in comparison with their monomeric units. Haggregates in most cases are non- fluorescent while J-aggregates are fluorescent having competitive FQY with monomeric ones [50]. During aggregate formation special arrangement of molecules occurs which involve specific interactions of dipole moments leading to two different energy states (Davydov-splitting): One is higher energy state and other is lower energy state [51]. Most of the rylene dyes aggregates in an H-type manner, decrease the FQY which is exemplified from the given PBI 11 in which there is a decrease of FQY from 96 to 6% upon increasing its concentration ethanol from 10-5 to 10-3 M [52]. The tendency of aggregate formation increases in polar solvents and this was the obstacle in the way to progress toward the application of dyes in aqueous environment [53].

Figure 1.4 Representation of H-type aggregates and their effect on fluorescence Different research groups in early 1990s have made efforts to prepare water soluble PBIs by imidization of perylene with differently substituted ionic groups (12-14) [54-56] as well as bay substitution with nonionic groups (15-17, Figure 1.5).

Figure 1.5 Different core unsubstituted PBIs bearing ionic and non ion imide substituents The well known work done in synthesis of water soluble rylene dyes is of Prof. Mullen, who did the tetraphenoxylation of a PBI core with polypeptides which provided the first water soluble core substituted PBIs 18 and 19 with a reported fluorescence in water in 2001 [57]. Although no aggregation behavior was observed for these compounds, the reported FQY of the resulting material was rather low with φfl = 3% for PBI 19 [58-59]. There is no explanation available to describe the reduction in fluorescence, but looking at the structure it seems probable that it mainly arises from amplification in flexibility and non radiative deactivation pathways (Figure 1.6) [60-64].

Figure 1.6 Different core unsubstituted PBIs bearing ionic and non ion imide substituents 1.6 Higher Rylene Dyes In higher rylene dyes the rylene chromophore is extended along the molecular axis by naphthalene units on both sides of molecule. Higher rylenes have longer λmax than to their original precursor and are more suitable for biological applications to visualize the intercellular spaces, especially in vivo studies [65, 66]. First report on the synthesis of water soluble higher rylenes was based on the core substituted polypeptides higher rylenes, terrylene (1, 2, 3) and quaterrylene (Figure 1.7) [67]. In contrast to the corresponding water-soluble PBIs, however, no fluorescent properties were reported for higher rylenes. Due to lack of studies on these compounds it remained unclear that what was the main driving for fluorescence quenching. Tetraphenoxylation of terrylene and subsequent transformation into ionic groups resulted into terrylene derivatives 2 and 3 [68, 69]. It was observed that terrylene bearing ionic groups expressed no aggregation, which is seen for terrylene having non ionic chains. Although fluorescence signal was noted for 3 but very low FLQ was achieved as compared with organic solvent soluble terrylene derivatives. Water solubility of terrylene scaffolds was obtained via substitution at bay positions, but same disadvantages as associated with water soluble PBIs were observed over here also.

Figure 1.7 Water soluble derivatives of terrylene (1-3) and quaterrylene The first report about a water-soluble terrylene bearing swallow tailed PEG chains at the imide positions was only quite recently published (Figure 1.8) [70]. However, it shows a strong Haggregation behavior and is therefore non fluorescent in the aqueous phase.

Figure 1.8 Water soluble terrylene bearing PEGs at imide position

1.7 Summary of Applications of Perylene Chromophore Perylene based dyes are well suited as functional dyes due to their wavelength span which ranges from 200-1500 nm. More recently, perylene molecules and their derivatives have attracted more and more attention in the past decade due to not only their outstanding thermal and photochemical stabilities but also their large application potential in organic optoelectronic or electronic devices [3-6], such as field effect transistors, solar cells, light-harvesting arrays and light-emitting diodes. Perylene dyes are well known as the key chromophores among the metalfree dyes which have the advantage of low cost production because they donot involve the precious rare earth metals. Their solubility, absorption, and emission behavior can be efficiently controlled using a variety of synthetic procedures, which include functionalization of peri or bay-positions of perylene core. Systematic tuning of HOMO and LUMO levels of perylene dyes improve both light harvesting properties and electron injection capabilities to TiO2 conduction band for obtaining the high PCE.

Objective and Plan of Research Keeping in view the above mentioned facts, rylene dyes stand for an excellent versatile class of dyes because of their good fluorescent properties; the high thermal, chemical and photochemical stability and uncomplicated processability in order to modify the optical properties have led to the development of a multitude of functional rylene dyes. Efficient solubilization and effective site isolation of these dyes in the high polarity medium water, is still a challenging task. Therefore the following points were the central objectives in this work: 1) Synthesis and optical characterization of tetra choloroperylene bis dianhydride azo hybrid dyes, and electrochemical study of these structural motifs. 2) Extension and evaluation of the tetrachloroperylene dianhydride to bis diimides and their hybridization with azo dyes. 3) Extension of perylene dianhydride to perylene azo and Schiff base diimides along the molecular axis, and their optical and electrochemical studies.

Chapter 2 RESULTS AND DISCUSSION 2.1 Synthesis and Characterization of Perylene Dianhydride Azo Hybrid Dyes (5a-j) For the synthesis of perylene-azo hybrid dyes, the phenolic azo coupling partners were synthesized according to scheme 2.1. Thus potassium phenolate was treated with diazonium salts 2a-j of the suitably substituted anilines in aqueous medium to produce phenolic azo dyes (3a-j) in excellent yields. Diazotization of nitro, methyl, methoxy and chloro substituted anilines was carried out at low temperature 0-5oC to avoid the decomposition of thermally labile diazonium salt. The completion of diazotization was checked by absence of yellow coloration which is developed in reaction with N, N-dimethylamino benzaldehyde with unreacted aniline. The diazonium salts were treated with phenol at low temperature, maintaining the pH above 8 using K2CO3. High pH increased the nucleophilicity of phenol, made the aromatic ring more nucleophilic and more of the p-substituted product was obtained due to stability and high electron density at the para position (scheme 2.1). Synthesis of perylene dianhydride azo hybrid fluorescent dyes had been accomplished in dry distilled DMF solvent at a temperature 110oC [72, 73]. Temperature was not allowed to go beyond this limit to avoid the decomposition of DMF, which may impart its role for substitution of nitrogen in the ring in place of oxygen. Mole ratio was kept 1:4 between tetrachloro perylene dianhydride and 4-hydroxy azobenzene derivatives (scheme 2.2), for the replacement of all the four chloro groups from the perylene ring. This replacement occurred through addition- elimination mechanism. Potassium carbonate was added in excess to increase the nucleophilic character of 4- hydroxy azo benzene derivatives to complete the reaction within short period of time and to neutralize the acid HCl generated during the reaction which might hydrolyze the dianhydride functionalities in perylene ring system. Reaction mixture was filtered to isolate the product and then dissolved in minimum amount of water and acidified the medium to reprecipitate the product to remove the potassium carbonate from product.

Scheme 2.1 Synthetic route to phenolic azo dyes

Scheme 2.2 Synthetic scheme for tetrachloro perylene based azo dyes (5a-j) The structures of newly synthesized dyes were elucidated by UV, FTIR and NMR studies. The strong solvatochromic behavior was observed for dye molecules with large dipole moment changes occurring during transitions between two electronic states. The solvatochromic behavior of a dye is the shift of absorption wavelength in solvents of different polarity due to interaction between the solute and solvent molecules. The UV- Visible absorption spectra of dyes (5a-j) were obtained at room temperature in various organic solvents having different polarities at a very dilute solution (1 x 10-7M, Figure 2.1). The selected spectral data is also summarized in Tables 2.1.

Table 2.1 Wavelength of maximum absorption λmax of perylene azo hybrid dyes in different solvents.

Dyes λmax (nm) in Water λmax (nm) in Ethanol λmax (nm) in Methanol

5a 442.6 346 349

5b 444 351 356 5c 446 344 350 5d 448 387 390 5e 443 350 353 5f 439 358,426 432 5g 423 340, 423 328, 432 5h 430 349, 416 351, 428 5i 428 347, 422 348, 425 5j 280, 442 266, 426 270, 428

UV-visible spectra of dyes were taken in water, ethanol and methanol. The electronic transitions in molecules provided two absorption maxima (λmax) in aqueous solution at 260-280 and 420-440 nms and three bands were observed when ethanol or methanol was used as solvent for dyes. The λmax for all the compounds is a result of π-π* transition of the compounds indicating the presence of the C=C characteristic of benzene and other aromatic nuclei in the dyes. This is in agreement with earlier, report by Mielgo et al., as per benzenoid uv-visible absorption [74]. The λmax in range 420-440 nm is due to π-π* transitions of azo linkages N=N. Difference in λmax of the synthesized dyes is not too much high for different substituents. All hybrid azo dyes (5a-j) have λmax value maximum in water and minimum in ethanol which is in accordance with the polarity of solvents. Greater is the solvent polarity greater will be shift in λmax. Water is more polar than methanol and ethanol so it shifts π-π* transitions to higher wavelength. The compound 5a showed the lowest λmax in all solvents because of the fact that it had NO2 group at the o-position of azobenzene which decreased the possibility of π-π* transitions and produces hypsochromic shift. The dyes 5c and 5d showed highest λmax because they had OCH3 groups at o and p-positions of azobenzene ring and these were electron donating groups, which caused bathochromic shift. Dye 5j had a chloro group at p- position of phenolic azodyes which is an electron donating group by resonance caused red shift as it is obvious from figure 2.1 and its λmax was 442 nm. Dyes 5a-c exhibited the absorption maximum in the range 335-350 nm in ethanol and methanol, is due to n-π* of N=N transitions, which confirmed the presence of azo linkage in the structure of dyes. These results showed that the solvent effect on UV/visible absorption spectra of investigated perylene azo dyes hybrid was multifaceted and strongly dependent on the nature of the substituent on the aromatic rings. This phenomenon was due to the difference in the conjugational or migrating ability of the electron lone pairs on nitrogen atoms and the azo-hydrazo tautomerism of azo dyes in accordance with their structure (Table 2.1). This also indicates that the electronic behavior of the nitrogen atoms of azo group was to some extent different in different derivatives and in solvents of different polarities. The infrared spectra of the synthesized azo hybrid dyes (5a-j) showed absorption bands due to Ar-H, C=O of dianhydride, C=C and N=N, stretching and bending vibrations at 3160- 3448 cm-1, 1820 cm-1, 1760 cm-1, 1589 - 1637 cm-1,1230-1250 cm-1 and 723 - 750 cm-1 respectively. Specifically speaking, using FTIR spectrum of these rylene dyes, peak observed at 1150-1100 cm-1 was as a result of C- 39

O-C functionality. The absorption bands at 1618 cm-1 and 750 cm-1 depicted the present of C=C stretching and bending vibrations respectively. Azo linkage was confirmed by the peak at 1540- 1510 cm-1. The 1H-NMR spectrum of 5a showed four doublet signals down field in the aromatic region of the TMS scale at δ 6.61-7.53 and 7.59-7.79 ppm due to four sets of mutually coupled chemically and magnetically non equivalent protons in the substituted azobenzene ring and a singlet signal at δ 7.73 ppm due to four protons of perylene ring. These four symmetrical protons were common in all azo hybrid dyes. The signal positions in perylene ring of these dyes were not affected by different azo dyes replacing the chlorine groups of tetrachloro perylene dianhydride. The compound 5b showed 1H broad singlet at 11.0 ppm due to COOH group in the molecule and two 2H doublets at 8.33 and 8.14 ppm due to azobenzene ring attached to carboxyl group and two 2H doublets at 7.76 and 6.93 ppm because of benzene ring having oxygen atom attached. Dye 5c, showed four different signals in the range 8.14-8.33 ppm having COOH group at o-position of aromatic ring and doublet peaks at 7.76 and 6.93 ppm are due to benzene ring having oxygen atom attached. Difference from p-COOH was that, here four signals are observed; in that case two symmetrical signals were observed. Dye 5d exhibited peaks at 8.39 and 8.19 ppm due to two different types of proton of the benzene ring attached with NO2 group and two, 2H, doublets at 7.76 and 6.93 ppm were due to benzene ring having oxygen atom attached. The dye 5e showed four different signals due to non symmetrical protons attached to benzene ring having nitro group at m-position. The 13C-NMR spectrum of 5a dye showed carbonyl of dianhydride motif at 171.73 and ten aromatic carbon signals ranging from 95.7 ppm to 153.1 ppm. The other compounds had also been confirmed from their respective 1H-NMR and 13CNMR spectra (Figure 2.2 and 2.3).

Figure 2.3 13C-NMR spectrum of 5a 2.1.1 Fluorescence Studies of Perylene Dianhydride Azo Hybrid Dyes (5a-j) When a fluorophore absorbs a photon of light, an energetically excited state is formed. The fate of this species is varied, depending upon the exact nature of the fluorophore and its surroundings, but the end result is deactivation (loss of energy) and return to the ground state. The main deactivation processes which occur are fluorescence (loss of energy by emission of a photon), internal conversion and vibrational relaxation (non-radiative loss of energy as heat to the surroundings), and intersystem crossing to the triplet manifold and subsequent non-radiative deactivation [75, 76]. Fluorescence data of all the synthesized azo hybrid dyes (5a-j) are shown in Table 2. Fluorescence spectra of the dyes were recorded by selecting different excitation wavelengths of the source because excitation spectrum is the dependence of emission intensity at single wavelength, upon different excitation wavelengths (Figure 2.4). In other words it provided the intensity contribution to the observed emission at a given wavelength by different excitation wavelengths to which sample was exposed. The fluorescence spectra showed only one emission peak clearly at 500-513 nm when excited by different wavelengths in concentration of 1x10-7 M. Among these dyes 5c 41

having OCH3 at o-position of phenol azo dye showed highest fluorescence. It might be attributed to non aggregation of dyes molecules due to presence of OCH3 at oposition which hindered the parallel alignment of dye molecules making it highly fluorescent. Emission peak of high intensity at 500-513 nm were observed for different hybrid dyes corresponding to absorption peaks at 442.8, 446, 445, 448 and 443.6 nms for 5a-e respectively. This observed phenomenon of absorption and emission verified the mirror image rule [77]. Fluorescence of azo hybrid dyes (5a-j) was high in water and low in other solvents. In water high fluorescence was due to non aggregation of dye molecules because of high polarity and small size of water molecules. While other solvents have a larger size, they could not penetrate and solvate completely the individual molecules of dyes having bulky structure. Self association of dye molecules occured in solvent like ethanol, methanol and so fluorescence decreased [78a]. Stoke shift of dyes 5a and 5c was high as it depended on the rapid decay of excited electrons to the lowest vibrational energy level of the excited state and on the molecular structure which was depicted in the sharpness of the emission peak in fluorescent spectra. In both cases o-positions of azo dyes had substituents, which reduced the chances of self association and increased the Stoke shift value. 6000 5 a 5 b 5000 5 c 4000 5 d 5 e 3000 5 f 2000 5 g 5 h 1000 5 i 0 5 j 0 200 400 600 800 1000 -1000

Figure 2.4 Fluorescence spectra of dyes, (5a-j)

Stoke shift value depends upon the conjugated size of molecules, larger the delocalized structure of fluorescent molecule, higher is the stoke shift. The dye 5a had the large stoke shift value which was attributed to tetrahedral structure of sulfonic acid substituents at azo coupling partner of rylene azo hybrid dyes. Due to expanded structure and polarity of dye 5a substituents there were more chances of association of dye, which decreased fluorescence but high stoke shift value was observed for it.

Table 2.2 Fluorescence values of azo hybrid dyes (5a-j) in water Dye Absorption wavelength (nm) Emission wavelength (nm) Stoke Shift 5a 425 511 76 5b 438 505 52 5c 445 503 45 5d 447 507 43 5e 443 509 47 5f 435 502 57 5g 441 510 59 5h 430 503 63 5i 444 508 53 5j 454 513 49

2.2 Synthesis and Characterization of Perylene Diimide Azo Hybrid Dyes (6a-g) 1,6,7,12-Tetrachloro perylene 3,4,9,10 (p-nitrophenyl) diimide (3) was synthesized by treating 1,6,7,12-tetrachloro perylene 3,4,9,10-tetracarboxo dianhydride (1) with 4-nitroaniline (2) keeping mole ratio 1: 2 between tetrachloroperylene dianhydide and 4-nitroaniline in order to replace both dianhydride oxygen atoms with nitrogen. Synthesis of tetrachloroperylene diimides (3) was accomplished in 18h of continuous stirring and reflux in the presence of propanoic acid. Propanoic acid acted as acidic medium and solvent to protonate both the oxygen atoms of dianhydrides and opened the both anhydride rings to tetra carboxylic acid motif. Acidic medium protonated the oxygen atoms of carboxylic acid functional groups and facilitated the attack of the amino group of p-nitroaniline on carbonyl group of carboxyl group and elimination of four water molecules. On completion of reaction, the reaction mixture was added to ice cold water and precipitated the product. The product (3) was filtered, dried, recrystallized from absolute ethanol and determined it melting point which was found to be more than 300oC. Synthesis of azo hybrid fluorescent dyes (6a-g) has been accomplished in dry distilled DMF solvent at temperature 110oC. Temperature was not allowed to go beyond this temperature to avoid the decomposition of DMF, which might impart its role for substitution of nitrogen in the ring in place of oxygen. Mole ratio was kept 1:4 between tetrachloroperylenediimides (3) and 4hydroxy azobenzene derivative 4a-g for the replacement of all the four chloro groups from the perylene ring. This replacement occurred through addition, elimination mechanism. Potassium carbonate (K2CO3) was added in excess to increase the nucleophilic character of 4-hydroxy azo benzene derivatives to complete the reaction within short period of time and to counteract the acid HCl produced during the reaction which could hydrolyze the dianhydride functionalities in 1,6,7,12- Tetrachloro perylene 3,4,9,10-tetracarboxo dianhydride molecule. Reaction mixture was filtered

to separate the product and then dissolved in minimum amount of water and acidified the medium for re-precipitation and purification of the product.

Scheme 2.3 Synthetic route to tetrachloroperylene based azo dyes (6a-g) UV-Visible, FTIR and 1H-NMR studies were done to elucidate the structures of newly synthesized azo hybrid dyes 6a-g. Solvatochromic behavior was well-built for dye molecules which undergo large changes in dipole moment during electronic transitions between two states. Due to the interaction between the solute and solvent molecules of different polarity, shift in the absorption wavelength of hybrid azo dyes was observed. The U.V. visible absorption spectra of the 6a-g hybrid azo dyes (1×10-7 M) were obtained at room temperature in various organic solvents with different polarities (Figure 2.5) and the selected spectral data is summarized in Table 2.3.

Table 2.3 Wavelength of maximum absorption λmax of 6a-g hybrid Azo Dye in Different solvents. S. No. λmax in Water λmax in Ethanol λmax in Methanol 6a 280, 458 278,355, 434 277, 355, 409 44

6b 291, 456 280, 344, 426 278,355, 424 6c 293, 451 271, 315, 435 276, 327, 430 6d 293, 452 284, 351, 438 261, 350, 401 6e 294, 459 268,352, 429 258, 355, 419 6f 294, 4 250, 370, 438 274,366, 438 6g 293,454 258,355, 435 278,363,426

UV-visible spectra of dyes (6a-g) were taken in water, ethanol and methanol. Two absorption maxima (λmax) bands in UV-visible spectra of perylene diimide azo hybrid dyes were observed during electronic transition of dye molecules in water, first at 260-280 nm and other at 450-459 nm, and three bands were seen when ethanol or methanol were used as solvents. The λmax for all the dyes was the result of π-π* transitions of the molecules indicative of the existence of C=C characteristic of benzene and other aromatic nuclei in the dyes. The absorption λmax in range 440- 460 nm was due to π-π* and n- π* transitions of azo linkages N=N conjugated with C=C bonds of perylene ring. For dyes (6a-g) effect of substituents was not high as depicted from their λmax and was not too much different from one dye to another. All perylene diimide azo hybrid dyes exhibited highest λmax in water and lowest in ethanol, which was according to polarity of solvents. Solvents with higher dipole moments caused a larger shift in λmax of dyes. Water was more polar than methanol and ethanol so it shifted π-π* transitions to higher wavelength. All the dyes (6a-g) in ethanol and methanol showed the absorption bands in the range 335-350 nm, due to n-π* of N=N transitions, which confirmed the presence of azo linkage in the structure of dyes. While these bands in aqueous were shifted to higher wavelength and more broad bands were produced, rather than sharp peaks in ethanol and methanol. Azo linkage bands were absorbed in perylene ring band in water. These results showed that the solvent effect on UV/visible absorption spectra of synthesized rylene dyes was versatile and strongly depend on the nature of the substituent on the aromatic rings.

Figure 2.5 UV-Visible spectra of 6a-g in water and ethanol The FTIR spectra of hybrid azo dyes showed absorption bands due to different functionalities present in dyes. IR peaks were produced due to Ar-H, C=O of diimides, C=C and N=N, stretching vibrations at 3000-3160, 1680-1705, 1605-1634, 1581-1590, 1505-1520 cm-1 respectively. Peaks observed at 1150-1100 cm-1 were the result of stretching vibrations of C-O functionality in dyes. The absorption bands at 743-750 cm-1 depicted the presence of C=C bending vibrations of aromatic substituted nuclei. Azo linkage was confirmed by strong absorptions at 1505-1520 cm-1 due to asymmetric stretchings. Specifically speaking dye 6a showed peaks at 3085, 1701, 1634, 1595, 1539, 1449, 1369, 1277, 1202, 1147, 1120, 1078, 959, 834, 798, 753 cm-1 with respect to different functional groups (Figure 2.6). In this way other dyes 6b-g were also identified from their respective FTIR spectra.

Figure 2.6 FTIR spectrum of perylene diimide azo hybrid dye 6a The 1H-NMR spectrum of dye 6a showed two pairs of doublets in the aromatic region at 6.94 and 7.54 ppm, and at 6.07 and 7.41 ppm due to diazo component of phenolic azo dyes. Perylene diimide ring system exhibited singlet at 7.95 ppm (s) due to four symmetrical protons. Methoxy proton in 6a showed singlet peak at 3.78 ppm. The position of perylene ring system of these dyes was not affected much in different azo dyes. Dye 6b showed 3H singlet at 2.38 ppm because of CH3 group and doublet signal at 6.97 ppm was due to 16H ortho to azo linkage of phenolic azo dyes and 8H doublet at 7.76 ppm was caused by o-protons of methoxy group and 6.93 ppm doublet due to o- protons of oxygen attached to perylene ring. Dye 6c provided multiplet peaks in aromatic region 8.14-8.33, 7.65-7.80 ppm due to diazo component of substituted azo dyes. A broad singlet signal was seen at 11.0 ppm due to the carboxyl group in diazo component. The compound 6d provided symmetrical four doublets at 7.47, 7.86, 7.76 and 6.93 ppm (Figure 2.7). Similarly, four symmetric peaks were present in spectrum of 6f but were downfield than 6d, because here nitro group was present at p-position of the diazo component of azo dye. Three multiplets and one singlet peak was exhibited by 6e at 8.39, 7.72, 8.32 and 8.86 ppm respectively by the azo dyes substituted in the bay region of perylene diimides ring. Singlet peaks at 3.80 ppm due to OCH3 attached at o-positions of diazo component and other multiplet signals in the range 6.90-7.85 and 7.65-7.80 ppm were present in the 1H-NMR spectrum dye 6g. The 13C-NMR spectrum of dye 6a showed distinguishing signals for methoxy at 55.72 ppm and carbonyl carbon atoms in the range 162.78 ppm (Figure 2.8). In this way all dyes synthesized dyes were confirmed for their structures from NMR studies.

Figure 2.7 1H-NMR spectrum of perylene diimide azo hybrid dye 6a

Figure 2.8 13C-NMR spectrum of perylene diimide azo hybrid dye 6a 2.2.1 Fluorescence studies of Study of perylene diimide azo hybrid dyes (6a-g) When a fluorophore absorbs a photon of light, an energetically excited state is formed. The fate of this species is diverse, depending upon the exact nature of the fluorophore and its surroundings, but the final result is deactivation (loss of energy) and returning back to the ground state. The main deactivation processes which occur are fluorescence (loss of energy by emission of a photon), internal conversion and vibrational relaxation (non-radiative loss of energy as heat to the surroundings), and intersystem crossing to the triplet manifold and subsequent nonradiative deactivation [78b].

Fluorescence data of all the synthesized hybrid azo dyes (6a-g) is shown in Table 2.4. All hybrid dyes (6a-g) showed high fluorescence in water and low in other solvents. Due to high polarity and small size of water molecule aggregation of dye molecules could not occur so the fluorescence of dyes was not affected. In solvents having larger sized molecules, van der waal’s interactions of solvents with dye molecules were more common so self-association of dye molecules occured in solvent like ethanol, methanol and fluorescence was reduced. Fluorescence quantum yield of dyes (6a-g) were determined by preparing the equimolar solution (1x 10-7 M) of synthesized dyes and fluorescein, and comparing the emission intensity of dyes and reference compound. Dyes 6c and 6f exhibited lowest FLQ value 0.73 and 0.71 respectively (Figure 2.9). In these dyes electron withdrawing carboxyl and nitro groups are attached at p- position of diazo component of dyes. The dyes 6a and 6g have highest FLQ value as these dyes had methoxy groups at the m and p- position of diazo components and other dyes are found to have values in between these extremes. From the FLQ study, it might be generalized that electron withdrawing groups decrease and electron donating groups increase the FLQ values.

460 480 500 520 540 560 580 600 620 640 660 Wavelength (nm)

Figure 2.9 Fluorescence Spectra of perylene diimide azo hybrid dyes, 6a-g Table 2.4 Fluorescence values of Perylene diimide Azo Hybrid Dyes 6a-g in water Dye Excitation Emission Wavelength Emission Height Stoke Shift Wavelength (nm) (nm) 6a 468 492.5 632.02 24.5 6b 466 493 407.60 27 6c 460 491 533.77 31 6d 461 492 383.55 31 6e 469 495 404.6 26

6f 468 493 835.4 28 6g 464 496 827.94 32

2.2.2 Luminescence Studies Luminescence study was conducted by preparing the films of (6a-g) in ethanol. Luminescence was found to be of value 0.208 to 0.239 cd/m2 and was maximum for dye 6a and 6f. For rest of dyes luminescence was no much varied from each other. It was observed that π-conjugation effects appear to play a relevant role in the luminescent behavior which is strong only when substituents of the azo components are capable of pi-conjugation. Moreover, an increase of the πelectron withdrawing character of substituents significantly increased the luminescence quantum efficiency and led to a shift of the emission to lower energy. The emission was also dependent on pH, being quenched in acidic media probably due to protonation of attached substituents on the azo components of azo hybrid dyes. Hence, this study presented a good account of perylene azo hybrid dyes whose luminescence properties can be easily tuned by changing the electronic properties of the substituent of azo components and the pH of the solution and thus, provides an opportunity to investigate and establish structure based luminescence relationships. The luminescence values of hybrid dyes are given in table 2.5.

Table 2.5 Luminescence and fluorescence quantum yield (FLQ) values of perylene diimide azo hybrid dyes (6a-g) in water Dye Luminescence Value (cd/m2) FLQ 6a 0.239 0.83 6b 0.208 0.77 6c 0.208 0.73 6d 0.228 0.75 6e 0.221 0.73 6f 0.231 0.71 6g 0.210 0.82

2.2.3 Electrochemical studies The electrochemical characterization of all compounds was made in detail using cyclic voltammetry in aqueous solution containing 0.1 M TBAPF6 as a supporting electrolyte. All dyes

exhibited irreversible oxidation peaks in their voltammograms and oxidation onset potential were determined from their CV curves. Oxidation potential (Eox) of hybrid dyes were different from each other and were in the range 0.5712 to 0.7221V as shown below in the Table 2.6. Eox potential was highest for dye 6a and lowest for 6f. Dye 6a has a methoxy group at p-position of the coupling component of azo dyes, so this dye had more potential for electron donation to acceptor system. Dye 6f had nitro group in the coupling component which provided deficiency of electrons it makes the whole chromophoric system less oxidative. Dyes 6a-e and 6g have values in between the oxidation potential of 6a and 6f. From oxidation potential values of dyes it was inferred that the dyes containing electron donating groups are less prone to reduction as compared to those containing electron withdrawing groups. The Eox values were used to determine the energy levels of highest occupied molecular orbital (HOMO) by following the empirical Bredas equations.

Table 2.6 Oxidation potential (eox) of compounds perylene diimide azo hybrid dyes (6a-g)

Compounds Eox 6a 0.7221 6b 0.7093 6c 0.6717 6d 0.6299 6e 0.6131 6f 0.5712 6g 0.6088

HOMO energy levels have energy -4.9712 to -5.1221 eV, highest energy for 6f dye bearing nitro (NO2) group in the azo dyes used for replacement of chloro groups from perylene bis diimides and lowest for 6a having methoxy group in the azo component of theses hybrid dyes. It is observed from the HOMO energy level data that dyes, having electron withdrawing have HOMO level at higher energy while for those containing electron donating groups HOMO are at a lower energy. Similarly LUMO values were calculated which were in accordance with HOMO values. Dyes with highest HOMO values have highest LUMO energy levels and vice versa. LUMO energies of the investigated dyes 6a-g are presented in Table 2.7. From HOMO and LUMO levels date it is observed that energy levels can be modified by changing the substituents in the perylene chromophores keeping in mind the electron donating and electro withdrawing effect of substituents. HOMO represents the energy required to remove an electron from a molecule, which is an oxidation process, and LUMO is the energy necessary to add an electron to a molecule, thus implying a reduction process. Electron donating group will decrease the energies of HOMO and LUMO levels and electron withdrawing groups will increase the energy of these levels. The term band gap refers to the energy difference between the top of the valence band and the bottom of the 50

conduction band. Electrons are able to jump from one band to another. However, in order for an electron to jump from a valence band to a conduction band, it requires a specific minimum amount of energy for the transition. The required energy differs with different materials. Electrons can gain enough energy to jump to the conduction band by absorbing either a phonon or a photon [79]. Every solid has its own characteristic energy-band structure. This variation in band structure is responsible for the wide range of electrical characteristics observed in various materials. In semiconductors and insulators, electrons are confined to a number of energy bands, and forbidden from other regions. The optical band gap values were calculated using the following equation.

Eg = 1242 eV/λ nm

Band gap energies of perylene diimide azo hybrid dyes are given in Table 2.7. Band gap energy ranges from 2.8291 to 2.9154 eV and was highest for 6b and lowest for 6d. Dye 6d has chloro groups in the azo components which have comparable values of electron donation and electron attraction, so the energy gap was lowest. For dye 6b highest energy gap was due to azo dye component containing alkyl group which has electron donating effect due to hyper conjugation effect and this effect was not strong like resonance, so energy gap was highest. Table 2.7 Energy of Highest Occupied Molecular Orbital’s of perylene diimide azo hybrid dyes 6a-g E (HOMO) = (Eox + 4.4) eV (Bredas equation)

E (LUMO) = E(HOMO) +Eg Compounds LUMO /eV Eg /eV HOMO/eV λ (nm) 6a -2.2604 2.8617 -5.1221 434 6b -2.1939 2.9154 -5.1093 426 6c -2.2166 2.8551 -5.0717 435 6d -2.2008 2.8291 -5.0299 439 6e -2.1113 2.9018 -5.0131 428 6f -2.1161 2.8551 -4.9712 435 6g -2.1732 2.8356 -5.0088 438

2. 3 Synthesis and Characterization Perylene Dianhydride Alkoxy Derivatives (7a-h) Synthesis of alkoxy perylene derivatives (7a-h) was accomplished by condensation of tetrachloroperylene dianhydride with different alcohols (3a-h) in the presence of potassium carbonate and dry distilled DMF at 110oC. Synthesis completed in three hours of continuous stirring and heating. Temperature was not allowed to exceed 110oC to avoid the decomposition of DMF, which might result in substitution of ring oxygen by nitrogen. A molar ratio of 1:4 for tetrachloroperylene dianhydride and different aliphatic and alicyclic alcohols was applied for the substitution of all four chloro groups by an addition-elimination mechanism. The addition of excess potassium carbonate was meant to enhance the nucleophilic character of different alcohols to complete the reaction quickly and to reduce the effect of the acid generated within the reaction 51

mixture to put off the hydrolysis of dianhydride. The solids were filtered, dissolved in a minimum amount of water and acidified to afford the products free of base (Scheme 2.4). The structures of newly-synthesized perylene derivatives were elucidated by UV-Visible, FTIR and NMR spectroscopy. The solvatochromic behavior was observed for these dye molecules due to change in their dipole moment, which occured during transitions between two electronic states (π-π*) with differential solvent polarities. The UV-visible absorption spectra of the perylene dyes (1×10−7 M) were taken at room temperature in aqueous medium (Figure 2.10) and the selected spectral data are shown in Table 2.8.

Scheme 2.4 Synthetic route to tetrachloroperylene based fluorescent dyes (7a-h)

Figure 2.10 UV Visible spectra of dyes 7a-h in water

Table 2.8 Wavelength of maximum absorption λmax of perylene dyes in water Dye λmax (nm)

7a 282, 446.6 7b 282, 445.4 7c 282, 447.5 7d 282, 448 7e 282, 448 7f 282, 448.5 7g 282, 446 7h 282, 445.5

The electronic transition of UV-visible spectra of dyes in water provided two absorption maxima (λmax), first at 282 nm and the other at 445-448 nm, respectively. The λmax for all the compounds at 445-448 nm was the result of π-π* transition of the compounds due to the delocalization of conjugated electrons of perylene motif. Difference in λmax of the synthesized dyes followed a narrow range for different alkyl substituents. All dyes (7a-h) had highest λmax in water when compared with that in non-polar solvents. From UV results, it was revealed that all perylene derivatives showed approximately identical λmax. This evidence indicated that chain length of alkyl groups at bay positions of perylene ring did not affect much the λmax of dyes. Moreover, the solvent effect on UV-visible absorption spectra of dyes (7a-h) strongly dependent on the nature of the substituent on the perylene nucleus, whether the substituents’ interact with solvent molecules or not. Solvent effect was more pronounced for substituents undergoing intermolecular interaction with solvent molecules. This solvent effect had also been observed for many perylene derivatives. The FTIR spectra of (7a-h) dyes provided absorption bands due to C-H, C=O (for dianhydride), C=C and C-O, stretching and bending vibrations in the range of 2900-2980, 1805-1828, 17571777, 1150-1100 and 723-750 cm-1, respectively. In particular, the peak observed in the range of 1150- 1100 cm-1 was as a result of ether functionality. The absorption bands at 1618 cm-1 and 750 cm-1 depict the presence of C=C stretching and bending vibrations, respectively. All these stretching and bending bands identify perylene derivatives (7a-h). 1 The H NMR spectrum of 7a exhibited a high field triplet signal due to CH3 group at 0.88 ppm, a multiplet due to methylene envelope in the zone of 1.26-1.76 ppm and a triplet at 4.06 ppm due to CH2 group adjacent to oxygen atom in the decyl chain by the de-shielding caused by oxygen. Due to perylene ring there is singlet peak in the range of 7.27-7.81 ppm, common in all compounds. For compound 7b there were one triplet and one doublet signals at 0.88 and 0.96 ppm, respectively, due to two CH3 groups present in 2-octyl chain, attached in bay region of perylene. These were highly shielded protons due to high electron density around them. Hexet at 3.70 ppm was ascribed to the proton adjacent to oxygen atom and a multiplet produced due to methylene envelope in the range of 1.26-1.63 ppm. In molecule 7c, 3H triplet, 2H hexet, 2H pentet and 2H triplet were found 53

at 0.90, 1.27, 1.62 and 4.09 ppm, respectively, were attributed to butyl chain symmetrically attached to perylene ring. One triplet at 1.72 ppm and a quartet at 4.72 ppm appear due to the presence of ethyl chain in case of 7d. Dye 7e exhibited a triplet due to CH3 (adjacent to CH2 group) at 0.86 ppm and a broad multiplet at 1.27-1.33 ppm owing to methylene envelope in the n-pentyl chain. A triplet at 4.95 ppm points at CH2 adjacent to oxygen atom. Compound 7f showed triplet, hexet and triplet at 1.3, 1.90 and 4.09 ppm, respectively. These include CH3, CH2, and CH2 protons in the propyl segment of dye 7f. Dyes 7g and 7h (cyclopentyl and cyclohexyl attached to perylene ring) manifested 1H pentet (each) at 3.71 and 3.74 ppm, respectively, while other protons in these compounds exhibited multiplets in aliphatic region in the range of 1.46 to 2.02 ppm. All chemical shifts proved the substituents’ identity and existence of the perylene ring chromophore in these derivatives (7a-h). 2.3.1 Optical properties 2.3.1.1 Maximum Extinction Coefficients (ϵmax) The maximum extinction coefficient is a measurement of how strongly a chemical species absorbs light at a given wavelength depending upon the presence or absence of certain functionalities in the molecule. It is an intrinsic property which depends upon the actual absorbance, A, path length (l) and the concentration (c) of the species (Beer-Lambert law) i.e., A = ϵmax cl. Molar extinction coefficient of perylene dyes is presented in Table 2.9. Table 2.9 Molar extinction coefficients of perylene dyes

Dyes λmax(nm) A ϵmax (L mol-1 cm-1) 7a 446.6 1.5060 30120.38 7b 445.4 1.5432 30865.1 7c 447.5 0.7859 15719.28 7d 448 1.016 20331.98 7e 448 0.9863 19727.46 7f 448.5 1.178 23564 7g 446 1.09674 21934.8 7h 445.5 0.9887 19774.94

Absorption in UV- visible region by alkylated perylene dyes is higher for those dyes which had long alkyl chains substituted on perylene dianhydride chromophore and higher value of molar extinction coefficients are linked to/associated with high absorption intensity. It can be generalized that branched chain and long-chained alkyl groups have high molar extinction coefficients as these appeared larger for 7a and 7b. 2.3.1.2 Fluorescence Investigations Fluorescence data of these dyes are reproduced in Table 2.10. Fluorescence spectra were recorded by selecting different excitation wavelengths of the source as excitation spectrum is dependent on emission intensity at a single wavelength upon various excitation wavelengths (Figure 2.11) [80]. The fluorescence spectra of these dyes displayed only one fluorescence peak at 500-513 nm when 54

excited by different wavelengths in the concentration range of 1x10-7M. All dyes exhibited nearly identical emissions with the least effect of chain length on emission. The emission peaks of high intensity at 500-513 nm for different dyes corresponding to the absorption peak in the range of 445-448 nm were produced in the emission spectra of compound. This observed phenomenon of absorption and emission verifies the mirror image rule.

460 480 500 520 540 560 580 600 620 640 660 Wavelength (nm)

Figure 2.11 Fluorescence spectra of dyes 7a-h in water Fluorescence of these dyes was higher in water and lower in other solvents. Self association of dye molecules occured in solvents like ethanol, methanol and consequently, fluorescence decreased. Table 2.10 Fluorescence values of alkylated perylene dyes (7a-h) in water Dye Excitation Wavelength Emission Wavelength Emission Height Stoke Shift (nm) (nm) (nm) 7a 455 502 720 78 7b 455 510 698 59 7c 460 503 4859 81 7d 490 515 12.58 25 7e 460 513 1894 49 7f 455 470 1070 30 7g 455 480 1070 40 7h 455 490 1070 50

2.3.1.3 Singlet Energies (Es) Singlet energy is the minimum amount of energy needed for a chromophore to get excited from 5 the ground state to excited state and it can be calculated using the equation Es= 2.86 x 10 / λmax. Singlet energies were calculated by using the equation. The data are presented in Table 2.11 Table 2.11 Singlet energies of dyes (7a-h)

Dye λmax (Å) Es (kcal/mol) 7a 4466 64.3 7b 4454 64.2 7c 4475 63.91 7d 4480 62.5 7e 4480 62.5 7f 4485 63.7 7g 4460 64.1 7h 4455 64.34

From this study, it was concluded that there is small change in the singlet energies of perylene dyes. Singlet energy depends upon the wavelength of maximum absorption, which does not vary large. In order to notice bigger changes in wavelength of maximum absorption there must be delocalization of electrons through alternating single and double bonds. Since energy difference decreases between HOMO and LUMO energy levels, singlet energies reduce consequently. 2.3.1.4 Oscillator Strengths (f) Oscillator strengths were calculated by the reported procedure using the equation, f= 4.32 x 10-9 ΔV1/2 ϵmax and results were presented in Table 2.12. It was observed that oscillator strength varied from 0.311 to 0.512 and was highest for 7b, which had tetra-substituted 2-octyl chains on the perylene dianhydride. In case of compound 7b, the molar extinction coefficient was higher than other derivatives, so its oscillator strength is high. This might also be attributed to larger absorptions by branched chain alkyl group on perylene ring. Same pattern was also observed for 7a, which had second highest fluorescence rate constants in the present series.

Table 2.12 Oscillator strengths of alkylated perylene dyes (7a-h)

Dye ΔV1/2 (cm-1) ϵmax (L mol-1 cm-1) Oscillator strengths f 7a 3449 30120.38 0.448 7b 3847 30865.10 0.512 7c 4603 15719.28 0.312

7d 3755 20331.98 0.329 7e 3660 19727.46 0.311 7f 3424 23564.00 0.348 7g 3481 21934.80 0.329 7h 4294 19774.94 0.366

2.3.1.5 Theoretical Radiative Lifetimes (Ƭo)

Theoretical radiative lifetimes (Ƭo) depend upon molar extinction coefficient (ϵmax), mean 2 frequency (V max) and half-width of the selected absorption (ΔV1/2). Molar extinction coefficient (ϵmax) of these dyes was high for longer alkyl chains substituted on perylene dianhydride chromophore and varied from 4.43 to 7.30 ns (Table 2.13). The compounds 7a and 7b had larger ϵmax value and smaller values of radiative lifetime (Ƭo). From data, it can be gathered that those perylene derivatives which have larger absorptions in UV-visible region, possess lower values of radiative lifetime (Ƭo) [81]. Table 2.13 Theoretical radiative lifetime of perylene dyes (7a-h) Dyes ΔV1/2(cm-1) ϵmax (L mol-1 cm-1) Vmax (cm-1) Ƭo (ns) 7a 3449 30120.38 25918 4.98 7b 3847 30865.10 25773 4.43 7c 4603 15719.28 26012 5.49 7d 3755 20331.98 26093 6.73 7e 3660 19727.46 25768 7.30 7f 3424 23564.00 25725 6.55 7g 3481 21934.80 25737 7.04 7h 4294 19774.94 25242 6.46

2.3.1.6 Fluorescence Rate Constants (kf) The results of fluorescence rate constants of dyes are reproduced in Table 2.14. The values for these dyes vary from 1.36 to 2.25x108/s, with dye 7b displaying the highest value, which has tetra- substituted 2-octyl chains on the perylene dianhydride. In compound 7b, the radiative life time was very low, so it had high fluorescence rate constant. This might be attributed to space interaction of branched chain alkyl group with perylene electrons that decreases the radiative life time and increases the fluorescence rate constant. Same pattern was also seen for 7a which had second highest fluorescence rate constant in this series [82]. Table 2.14 Fluorescence rate constant of perylene dyes 8 Dye Ƭo (ns) kf (10 /s )

7a 4.98 2.00 7b 4.43 2.25 7c 5.49 1.82 7d 6.73 1.48 7e 7.30 1.36 7f 6.55 1.52 7g 7.04 1.42 7h 6.46 1.54

2.3.2 Electrochemical Properties The electrochemical characterization of these dyes was studied by cyclic voltammetry using water having 0.1 M TBAPF6 as a supporting electrolyte. All redox potentials, HOMO (highest occupied molecular orbital), LUMO (lowest unoccupied molecular orbital) and band gap energies (Eg) were calculated from this technique. 2.3.2.1 Redox Potentials (E1/2) For reversible processes, reduction potentials can be calculated from cyclic voltammograms according to reported procedure (Figure 2.12) [83]. Redox potentials of dyes are shown in Table 2.15.

Table 2.15 Redox potential (E1/2) of perylene dyes (7a-h) Dyes Epa (V) Epc (V) ΔEp (mV) E1/2 (V) 7a -0.0390 -1.4390 1.400 -0.739 7b -0.3445 -1.0335 0.689 -0.689 7c -0.3495 -1.0485 0.699 -0.699 7d -0.3745 -1.1235 0.749 -0.749 7e -0.2490 -1.4390 1.190 -0.696 7f 0.0790 -1.4390 1.360 -0.759 7g -0.1290 -1.4390 1.310 -0.784 7h -0.3595 -1.0785 0.719 -0.719

Figure 2.12 Cyclic Voltammogram of dyes (7a-h) in water 2.3.2.2 Lowest Unoccupied Molecular Orbitals (LUMO) In order to calculate the absolute energies of LUMO level with respect to the vacuum level, the redox data are standardized to the ferrocene/ferricenium couple which has a calculated absolute energy of – 4.8 eV. The data related to LUMO level energies of dyes are presented in Table 2.16.

Table 2.16 Half wave potential (E1/2) and LUMO energy levels of alkylated perylene dyes Dyes E1/2 (V) LUMO (eV) 7a -0.739 -4.010 7b -0.689 -4.110 7c -0.699 -4.100 7d -0.749 -4.050 7e -0.696 -4.104 7f -0.759 -4.041 7g -0.784 -4.016 7h -0.719 -4.081 It was inferred from energy range of -4.010 to -4.110 eV that there is no appreciable difference between different aliphatic and alicylic chains used for substitution on perylene ring. It was concluded that the effect of chain length of alkyl groups was same for all dyes. The energy of LUMO levels can be varied only by increasing the delocalization of electrons through alternating 59

single and double bonds and it is noticed that energy difference decreases with increasing conjugation and vice versa. 2.3.2.3 Band Gap Energy Values (Eg) The optical band gap values are calculated using the standard procedure. The band gap energy is the span of energies that lies between the valence and conduction bands for insulators and semiconductors. Every solid has its own characteristic energy-band structure. This variation in band structure is responsible for the wide range of electrical characteristics observed in various materials [84]. Band gap energy of dyes is given in Table 2.17. No significance difference was evident for different aliphatic and alicylic chains substituted on perylene ring and it was clear that chain length of alkyl group didnot affect the band gap energy. Band gap energy can be varied only by increasing the delocalization of electrons through alternating single and double bonds and energy difference decreases with increasing conjugation and vice versa. 2.3.2.4 Highest occupied molecular orbitals (HOMO) Table 2.17 depicts the highest occupied molecular orbital energy levels which are in range from 6.582 to -6.672 eV for these dyes and no large difference for various aliphatic and alicylic chains substituted on perylene ring was observed. It was concluded that there was minor effect of chain length of alkyl groups on HOMO energy levels. The HOMO energy levels can be changed only by increasing the delocalization of electron through alternating single and double bonds. Energy difference decreases with increasing conjugation and vice versa. Table 2.17 Band gap energy and HOMO energy levels alkylated of perylene dyes Dye Cut-off λ (nm) Eg (eV) HOMO (eV) 7a 482 2.572 -6.582 7b 484 2.561 -6.672 7c 490 2.530 -6.630 7d 479 2.588 -6.638 7e 488 2.540 -6.644 7f 480 2.583 -6.624 7g 475 2.610 -6.623 7h 485 2.555 -6.630

2.3.3 Thermal properties Thermogravimetric measurement of dyes was carried out to check the thermal stability [85] using inert atmosphere. The results are reproduced in Table 2.18. On the basis of TG thermograms, perylene dyes demonstrated high thermally stability. The stability was attributed to the presence of ringed structure when To (temperature at which first mass-loss is detected) is taken into account from TG results. These will not be affected by higher temperatures if the applications demand thermal stability. Table 2.18 TGA of alkylated perylene dyes (7a-h)

o Dyes TG, C (To)

7a 218 7b 195 7c 193 7d 214 7e 198 7f 189 7g 190 7h 194 2.4 Synthesis and Characterization of Perylene Schiff Base Azo Diimide Dyes (13a-e) Synthesis of perylene Schiff base diimide involved the two step procedure which consisted of synthesis of Schiff bases and their condensation with perylene dianhydride. Schiff bases synthesis was conducted by reacting p-amino acetanilide and substituted aldehydes and ketones. p-amino acetanilide (0.01 mol) was reacted with substituted aldehydes (0.01mol) in ethanol as solvent and catalyzed by acetic acid. Reaction mixture was stirred for 10-12 h at reflux temperature until its completition was observed by taking the TLC of reaction mixture. Schiff bases were separated by rotary evaporation of reaction mixture and then recrystallized from ethanol and ethyl acetate. In this way series of Schiff bases 3a-e were synthesized and purified. Deacetylation of above synthesized Schiff bases was done by hydrolysis of Schiff bases (3a-e) in aqueous solution 20 ml catalyzed by conc. HCl (4ml). On completion of reaction deacetylated Schiff bases were separated, purified and dried. Deacetylated Schiff bases (0.01mol) were condensed with perylene dianhydride (0.01mol) in o quinoline at temperature 165 C catalyzed by zinc acetate Zn(CH3COO)2 (0.5g). Reaction mixture was stirred for 24h at this temperature, until completition of reaction was observed by TLC (pure dichloromethane). On cooling the reaction mixture, products were separated which were then filtered and dried in oven at 80oC. In this way a series of perylene Schiff base diimides 13a-e were synthesized [86].

Scheme 2.5 Synthesis of perylene Schiff base diimide dyes 13a-e Syntheses of PDA dyes (13a-e) have been achieved by two step procedure involving the synthesis of Schiff bases and their condensation with perylene dianhydride. Schiff 62

bases (11a-e) were synthesized by reacting p-amino acetanilide with different aldehydes (10a-e) in ethanol at reflux temperature for 10-20h continuous heating and stirring. Synthesized intermediates aqueous solution were heated at 100oC for 2h in acidic conditions and removed the acetyl protecting group and obtained the 11a-e Schiff bases. These Schiff bases were reacted with perylene dianhydride in quinoline at 165oC for 24h continuous heating and stirring. This reaction was conducted in quinoline which is high boiling solvent and more heat can be applied to accomplish the reaction. Zinc acetate was used to catalyze the reaction which speeds up the rate of reaction by making a loose complex with oxygen atom of dianhydride due to deficiency of electrons at zinc. UV, FTIR, 1H-NMR and 13C-NMR studies were done to elucidate the structures of newly synthesized PDA dyes. The U.V/visible absorption spectra of the PDA dyes (1×10−7 M) were taken at room temperature in dimethyl sulfoxide (DMSO) (Figure 2.13) and the selected spectral data are summarized in Table 2.19.

Figure 2.13 UV-Visible spectra of perylene Schiff base diimide dyes 13a-e in DMSO Table 2.19 Absorption maxima of perylene Schiff base diimide dyes

Compounds λmax (Absorption) λem (Emission)

13a 469 537

13b 520 550

13c 492 525

13d 526 Nil

13e 523 533

Three absorption maxima (λmax), bands in UV-visible spectra of PDA dyes were observed during electronic transition of dye molecules in DMSO, at 430-450, 470-490 and 510-530 nm. The λmax for all the dyes was the result of π-π* transitions of the molecules indicative of the existence of alkenic and imine linkages present in dyes. The λmax in range 510-530 nm is due to π-π* transitions of imine linkages C=N as well as conjugated C=C bonds of perylene ring. For dyes (13a-e) effect of substituents is not high, depicted from their λmax which is not too much different from one dye to another. This phenomenon was observed due to the difference in the conjugational or migrating ability of the electron lone pairs on nitrogen atoms and, the azohydrazo tautomerism of azo dyes as evidenced from their structures (Scheme 2.5). This also indicated that the electronic behavior of the nitrogen atoms of azo group was to some extent different in different derivatives and in solvents of different polarities. Fluorescence study of compounds (13a-e) was conducted by preparing their dilute solution in DMSO (1x 10-7M). All the compounds exhibited strong fluorescence above 500 nm except 13d which might be attributed to aggregation of flat molecules of this dye and electron withdrawing groups attached to this molecule. Highest fluorescence was observed for dye 13a and 13b at wavelength 537 and 550 nm respectively due to the highly conjugated systems like pyrene and fluoeronone present in these compounds (Figure 2.14).

Figure 2.14 Fluorescence spectrum of compounds (13a-e) The FTIR spectra of rylene dyes (13a-e) exhibited absorption bands due to O-H, Ar-H, C=O of diimides, C=C and C=N, stretching and bending vibrations at 3410-3455, 3080-3120, 16801690, 1585-1630, 1430-1445, and 1230-1250 cm-1 respectively. In particular the peak observed at 1100- 1150 cm-1 was as a result of carbon oxygen single bond stretching vibrations. The absorption bands at 1585-1630 cm-1 and 770-810 cm-1 depicted the presence of C=C stretching and bending vibrations respectively. Schiff base linkage was confirmed by absorptions at 14301445 cm-1. Stretching vibrations absorptions in between 3080-3120 cm-1 were because of C=C-H bonds of aromatic rings present in dye molecules (13a-e).

1H-NMR and 13C-NMR studies proved the synthesis of rylene dyes (13a-e). Dye 13c showed characteristic peak for imine functionality at 8.90 ppm and a pair of doublet at 7.36 and 7.39 ppm due to phenylene ring sandwiched between nitrogen atoms. A pair doublet peak patteren at δ 8.14 and 8.18 ppm was due to two chemically and magnetically non equivalent protons of naphthalene unit of perylene ring and doublet peak and pair of triplets at 7.81, 7.71 and 7.53 ppm due to phenyl 13 ring attached with NO2 group. C-NMR spectrum of 13c showed peak for carbonyl group of imide functionality at 162.4 ppm and imine carbon showed signal at 158.7 ppm which were the distinguishing signals for these dyes. Fifteen signals in the range 121-151 ppm were due to aromatic carbon nuclei in the molecules (Figure 2.15 and 2.16).

Figure 2.15 1H-NMR spectrum of compound 13c

Figure 2.16 13C-NMR spectrum of compound 13c 2.4.1 Electrochemical Properties

The electrochemical characterization of all compounds was made in detail using cyclic voltammetry in aqueous solution containing 0.1 M TBAPF6 as a supporting electrolyte (Figure 2.17). All dyes exhibited irreversible oxidation peaks in their voltammograms and oxidation onset potential were determined from their CV curves. Redox potential (Eox) of dyes (13a-e) were different from each other and were in the range 0.229 to -0.590 V as shown below in the Table 2.20. Redox potential was highest for dye 13e and lowest for 13a. Eox were used to determine the energy levels of highest occupied molecular orbital (HOMO) by following the empirical Bredas equations (Table 2.20).

Figure 2.17 Cyclic Voltammogram of perylene Schiff base diimide azo dyes (13a-e) in DMSO LUMO energy levels were in -4.21 to -5.20 eV, and highest energy LUMO levels seen for 13a dye bearing pyrene moiety and lowest were in 13e bearing dimethyl amino group at phenylene ring at imide position of perylene ring. These results were in accordance with the general phenomenon observed electrochemical studies of compounds that the electron donating groups decrease the energy gap between HOMO and LUMO levels and electron withdrawing groups increase this gap of energy. HOMO energy levels in the dyes have energy range -6.85 to 7.57 eV and lowest HOMO levels are present in 13e which and highest for 13a because in 13a there is more availability of electrons due highly rich pyrene ring which increases the energy of HOMO levels and thereby decreases the energy gap. Optical band gap energies were calculated by standard procedure and were in the range 2.37 to 2.64 eV. For 13e lowest highest energy gap is observed which is due dimethyl amino group at Schiff base condensed along molecular axis, but comparison cannot be made as these compounds have diverse Schiff bases (Table 2.20) [88].

Table 2.20 Half wave potential (E1/2) and LUMO energy levels of perylene Schiff base diimide azo dyes (13a-e) Compounds E1/2 (V) Eg (V) HOMO (eV) LUMO (eV) 13a -0.590 2.64 -6.85 -4.21 13b -0.135 2.38 -7.04 -4.66 13c +0.360 2.52 -7.68 -5.16 13d +0.229 2.37 -7.37 -5.02 13e +0.400 2.35 -7.57 -5.20

Chapter 3 EXPERIMENTAL 3.1 Materials Tetrachloro perylene dianhydride was obtained from Honest Joy Holdings limited China. Sodium nitrite was obtained from BDH. Hydrochloric acid and phenol were purchased from Merck. Potassium carbonate was purchased from Daejing Korea. Solvents such as ethanol, ethyl acetate, DMF, and methanol were common laboratory grade chemicals and were purified. Perylene tetracarboxylic dianhydride was purchased from Sigma-Aldrich. Hydrochloric acid, decanol, 2- octanol, hexanol, n-butanol, propanol, ethanol, cyclopentanol, cyclohexanol, ethyl acetate, DMF quinoline and methanol were obtained from E. Merck. Potassium carbonate was purchased from Daejing. 3.2 Purification of solvents: Standard methods and procedures were followed for the purification and drying of solvents. The dried solvents were stored over type 4A° molecular sieves. A brief account of the purification procedure is given below. a) Acetone Calcium chloride anhydrous was added to flask having acetone and refluxed for 3-5 hours. Pure acetone was distilled at 56 °C. b) Chloroform Chloroform was pre-dried over anhydrous calcium chloride for 4 hours and distilled at 65-66 °C. c) Dichloromethane It was dried by same procedure used for chloroform and distilled at 39-40 °C. d) Ethyl Acetate It was dried upon stirring on anhydrous calcium hydride and distilled at 77 °C. e) Ethanol Ethanol was refluxed over activated calcium oxide for 4-6 hours followed by distillation at 7778°C. f) Methanol Calcium oxide was introduced into a round bottom flask containing methanol. It was refluxed for 4 hours and distilled at 64 C.

f) Tetrahydrofuran and Diethyl ether Both solvents were dried upon reflux on sodium wire using benzophenone as an indicator, followed by distillation at 66 °C and 34-36 °C respectively, when colour changes to violet blue. THF was distilled freshly each time before use. g) Acetonitrile The solvent was dried upon molecular sieves (4Å) by standing overnight and fractionally distilled at 80-81°C. 3.3 Instrumentation Melting points were determined using digital Gallenkamp (Sanyo) model MPD BM 3.5 with digital thermometer and are uncorrected. Infrared spectra were recorded using a Shimadzu IR 460 as KBr pellets and FTX 3000 MX spectrophotometer using the ATR method. 1H NMR and 13C NMR spectra were obtained using a Bruker Avence (300 MHz) and (400 MHz) spectrophotometers respectively in CDCl3, DMSO-d6, CD3OD-d4 solution using TMS as an internal reference. Chemical shift are given in δ-scale (ppm). Abbreviations s, d, dd, t, at, m have been used for singlet, doublet, double doublet, triplet, apparent triplet, multiplet respectively. Elemental analyses were performed on CHNS 932 LECO instrument. UV-Vis spectra were taken using a CECIL-7400

UV/Visible Spectrophotometer and fluorescence spectra were recorded using the Hitachi FL solutions 7000 fluorescence spectrophotometer. Cyclic voltammetry was performed on CH-800 C potentiostate using 0.1M TBAPF6 as internal reference in DMSO and DCM (purged with argon for 10 minutes) on glassy carbon and platinum (0.2 mm diameter) as working electrodes versus Ag/AgCl reference electrode and platinum wire as counter electrode at room temperature. 3.4 Chromatographic Techniques 3.4.1 Thin Layer Chromatography (TLC) The progress of reactions was monitored through thin layer chromatography by using precoated silica gel aluminum sheets 2.0 x 5.0 cm (layer thickness 0.2 mm, HF254, Reidal-de-Haen from Merck). Chromatograms were detected by using ultraviolet light (254-360 nm). For development of chromatograms different solvent systems were used: n-Hexane: Ethyl acetate (4:1) n- Hexane: DCM (6:1) n-Hexane: Ethyl acetate (9:1) DCM: Methanol (10:1)

3.5 Experimental Procedures 3.5.1 General procedure for synthesis of phenolic azo dyes (3a-j). Suitably substituted anilines (1a-j) (0.01mol) were dissolved in 20 ml water and 3.5 ml o concentrated HCl, with stirring maintaining the temperature at 0-5 C. A solution of NaNO2 (0.01 mol) in 10 ml water was added promptly to solution of aniline with continuous and vigorous stirring. Stirring was further continued for 1h maintaining the temperature in the same range. After 1h the reaction mixture was checked for the completeness of reaction on a paper chromatogram using water as mobile base. The dried chromatogram was sprayed with solution of p-N,N-dimethyl aminobenzaldehyde in ethanol as spraying agent. On completion of reaction the diazonium salts (2a-j) were kept in a freezer. Phenol (0.01mol) was dissolved in water (15ml) and K2CO3 (2g), kept in the ice bath at temperature 0-5 oC with stirring. The diazo solution was added drop wise to the stirred solution of phenol during 30 minutes, maintaining the pH above 8. The progress of reaction was monitored by paper chromatography using H-acid solution in alkaline media. On completion the solids were filtered, dried in oven at 70oC, for 3hs to afford the 4-hydroxyazobenzene derivatives (3a-j) in 87-90% yields. In case where regioisomeric products were obtained; column chromatography was used for separation. 3.5.2 General procedure for synthesis of perylene dianhydride azo hybrid Dyes (5a-j) Tetrachloroperylene dianhydride 0.001mol (0.53g) was taken in 250ml round bottomed flask containing 20ml DMF placed on hot plate having oil bath. Started stirring and heating, and added 4-hydroxy azobenzene derivatives 0.004 mol (3a-j), then added 2.0g of K2CO3. Kept the temperature at 110oC for 3hours.After this TLC of reaction mixture was taken in 4:1 ethyl acetate: pet ether. From TLC it was observed that the reaction had been completed in 3hours. Filtered the reaction mixture and obtained residue which was dissolved in 30ml of water. Acidified the media with conc. HCl, (1ml), precipitation occurred immediately, filtered and dried the product. Yield was 90-95% in different dyes. In this way a series of dyes 5a-j were synthesized.

1,6,7,12-tetra-(4’-sulfophenylazophenoxy) perylene dianhydride (5a) o 1 Yellow crystals, m.p> 350 C. H-NMR (300MHz, D2O) δ (ppm): 7.79 (d, 2H, J= 8.4Hz), 7.59 (d, 13 2H, J= 7.8Hz), 7.54 (d, 2H, J= 8.4Hz), 6.62 (d, 2H, J= 7.8Hz), 7.73 (s, 4H). C-NMR (75MHz, D2O) δ (ppm): 171.70, 153.16, 147.77, 144.17, 127.53, 127.47, 126.41, 126.36, 122.25, 118.11 -1 and 95.73. FTIR (Neat, cm ) νmax: 3050, 1820, 1760, 1620 1580, 1547, 1521, 1457, 1417, 1357, 1267, 1171, 1088, 1011, 963, 907,878, 830, 817, 791, 668, 552 cm-1. 1,6,7,12-tetra-(4’-carboxyphenylazophenoxy) perylene dianhydride (5b)

o 1 Yellowish orange crystals, m.p> 350 C. H-NMR (300MHz, D2O) δ (ppm): 8.09 (d, 2H, J= 8.4Hz), 7.81(d, 2H, J= 7.5Hz), 7.76 (d, 2H, J= 8.4Hz), 6.65 (d, 2H,, J= 7.5Hz), 7.74 (s, 4H), 11.0 13 (br singlet 1H) ppm. C-NMR (75MHz, D2O) δ (ppm): 172.21, 168.95, 158.59, 155.40, 155.09, 150.13, 149.73, 131.71 128.89, 122.99, 122.83, 120.04, 119.85, 117.66 and 98.14. FTIR (Neat, -1 cm ) νmax: 3050, 1820, 1760, 1620 1580, 1584, 1549, 1446, 1410, 1386, 1355, 1271, 1212, 1169, 1014, 962, 905, 877, 828, 795, 750 cm-1. 1,6,7,12-tetra-(2’-carboxyphenylazophenoxy) perylene dianhydride (5c) o 1 Yellowish orange crystals, m.p> 350 C. H-NMR (300MHz, D2O) δ (ppm): 8.05 (m, 1H), 7.95(m, 1H=R1), 7.65-7.80 (m, 2H) 7.76 (d, 2H, J= 8.4Hz), 6.67 (d, 2H=R4 J= 7.4Hz), 7.73 (s, 13 4H), 11.0 (br singlet 1H) ppm. C-NMR (75MHz, D2O) δ (ppm): 170.53, 167.27, 158.59, 155.40, 150.73, 150.13, 149.73, 134.11, 131.71, 129.28, 128.82, 122.99, 122.83, 120.05, 120.04, 119.85, -1 119.18, 117.68, and 111.14. FTIR (Neat, cm ) νmax: 3050, 1820, 1760, 1580, 1584, 1549, 1446, 1410, 1386, 1355, 1271, 1212, 1169, 1014, 962, 905, 877, 828, 795, 750 cm-1. 1,6,7,12-tetra-(4’-nitrophenylazophenoxy) perylene dianhydride (5d)

o 1 Yellowish brown crystals, m.p> 350 C. H-NMR (300MHz, D2O) δ (ppm): 8.20 (d, 2H, J= 8.1Hz) 13 7.98 (d, 2H, J= 7.9Hz), 7.76 (d, 2H, J= 8.1Hz), 6.65 (d, 2H=R4, J= 7.9Hz), 7.74 (s, 4H), ppm. C- NMR (75MHz, D2O) δ (ppm): 158.59, 156.85, 155.40, 150.13, 149.73, 148.81, 125.32, 122.99, -1 122.83, 120.24, 120.04, 119.85, 119.18, and 111.14. FTIR (Neat, cm ) νmax: 3050, 1820, 1760, 1620 1580, 1584, 1549, 1446, 1410, 1386, 1355, 1271, 1212, 1169, 1014, 962, 905, 877, 828, 795, 750 cm-1. 1,6,7,12-tetra-(3’-nitrophenylazophenoxy) perylene dianhydride (5e) o 1 Yellowish orange crystals, m.p> 350 C. H-NMR (300MHz, D2O) δ (ppm): 8.24 (m, 1H=R1), 8.48 (s, 1H), 7.72 (m, 1H), 8.21 (m, 1H), 7.76 (d, 2H, J= 7.9Hz,), 6.65 (d, 2H,, J= 7.9Hz), 7.75 (s, 13 4H) ppm. C-NMR (75MHz, D2O) δ (ppm): 171.52, 158.59, 155.40, 150.13, 149.73, 147.61,

-1 131.71, 129.83, 128.78, 123.79, 120.04, 119.85, 114.43, and 96.14. FTIR (Neat, cm ) νmax: 3050, 1820, 1760, 1620 1580, 1584, 1549, 1446, 1410, 1386, 1355, 1271, 1212, 1169, 1014, 962, 905, 877, 828, 795, 750 cm-1. 1,6,7,12-tetra-(2’-nitrophenylazophenoxy) perylene dianhydride (5f) o 1 Yellow crystals, m.p> 350 C. H-NMR (300MHz, D2O) δ (ppm): 7.72- 8.39 (m, 4H), 7.76 (d, 2H), 13 6.67 (d, 2H, J= 7.8Hz), 7.72 (s, 4H). C-NMR (75MHz, D2O) δ (ppm): 170.23, 158.59, 155.40, 150.13, 149.73, 145.85, 138.67, 134.00, 131.71, 126.77, 125.31, 122.99, 122.83, 120.04, 119.85 and 95.53. FTIR (Neat) 3050, 1820, 1760, 1620 1580, 1547, 1521, 1457, 1417, 1357, 1267, 1171, 1088, 1011, 963, 907,878, 830, 817, 791, 668, 552 cm-1.

1,6,7,12-tetra-(4’-metylyphenylazophenoxy) perylene dianhydride (5g) o 1 Yellowish orange crystals, m.p > 350 C. H-NMR (300MHz, D2O) δ (ppm): 2.35 (s, 3H), 7.26 (d, 2H, J= 7.9Hz), 7.81(d, 2H, J= 7.9Hz), 7.78 (d, 2H, J= 7.8Hz), 6.65 (d, 2H, J= 7.8Hz), 7.73 (s, 13 4H) ppm. C-NMR (75MHz, D2O) δ (ppm): 171.56, 158.59, 155.40, 150.13, 149.36, 131.71, -1 129.72, 122.99, 122.83, 122.58, 120.04, 118.62, 111.14 and 21.13. FTIR (Neat, cm ) νmax: 3050, 1820, 1760, 1620 1580, 1584, 1549, 1446, 1410, 1386, 1355, 1271, 1212, 1169, 1014, 962, 905, 877, 828, 795, 750 cm-1. 1,6,7,12-tetra-(3’-methoxyphenylazophenoxy) perylene dianhydride (5h) o 1 Yellowish orange crystals, m.p> 350 C. H-NMR (300MHz, D2O) δ (ppm): 3.9 (s, 1H), 6.977.82(m, 4H), 7.65-7.80 (m, 2H), 7.76 (d, 2H J= 7.75Hz), 6.67 (d, 2H J= 7.75Hz), 7.73 (s, 4H) 13 ppm. C-NMR (75MHz, D2O) δ (ppm): 170.1, 160.38, 158.74, 152.48, 150.16, 149.73, 131.54, 129.81, 121.68, 122.96, 120.04, 119.73, 116.74, 114.02, 106.29, 105.56 and 56.04. FTIR (Neat, -1 cm ) νmax: 3050, 1820, 1760, 1620 1580, 1584, 1549, 1446, 1410, 1386, 1355, 1271, 1212, 1169, 1014, 962, 905, 877, 828, 795, 750 cm-1. 1,6,7,12-tetra-(4’-methoxyphenylazophenoxy) perylene dianhydride (5i) o 1 Yellowish brown crystals, m.p> 350 C. H-NMR (300MHz, D2O) δ (ppm): 3.9 (s, 3H), 6.97 (d, 2H, J= 8.1Hz), 7.82 (d, 2H, J= 8.1Hz), 7.76 (d, 2H, J= 7.7Hz), 6.93 (d, 2H, J= 7.7Hz), 7.73 (s, 13 4H) ppm. C-NMR (75MHz, D2O) δ (ppm): 171.24, 162.23, 158.98, 155.40, 151.73, 149.73, -1 147.16, 131.31, 124.40, 123.95, 120.04, 119.76, 104.82, 56.04. FTIR (Neat, cm ) νmax: 3050, 1820, 1760, 1620 1580, 1584, 1549, 1446, 1410, 1386, 1355, 1271, 1212, 1169, 1014, 962, 905, 877, 828, 795, 750 cm-1. 1,6,7,12-tetra-(4’-chlorophenylazophenoxy) perylene dianhydride (5j)

o 1 Yellowish orange crystals, m.p> 350 C. H-NMR (300MHz, D2O) δ (ppm): 7.47 (d, 2H, J= 7.9Hz), 7.86 (d, 2H, J= 7.9Hz), 7.78 (d, 2H, J= 7.73Hz), 6.93 (d, 2H, J= 7.73Hz), 7.74(s, 4H). 13C- NMR (75MHz, D2O) δ (ppm): 171.45, 159.72, 156.37, 151.46, 150.51, 149.37, 135.26, 134.11, -1 129.18, 122.89, , 120.04, 120.55, 119.18 and 98.14. FTIR (Neat, cm ) νmax: 3050, 1820, 1760, 1620 1580, 1584, 1549, 1446, 1410, 1386, 1355, 1271, 1212, 1169, 1014, 962, 905, 877, 828, 795, 750 cm-1. 3.6 General procedure for synthesis of perylene diimide azo hybrid dyes (6a-g) Tetrachloroperylene dianhydride (1) 1 mmol (0.53g) was taken in 100ml round bottomed flask containing 20ml propanoic acid positioned on hot plate having oil bath. Started stirring and heating accompanied by addition of 4-nitro aniline (2) 2 mmol (0.276g), and the reaction mixture was refluxed for 18h.The reaction progress was observed by taking the TLC of reaction mixture in 4:1 pet ether: ethyl acetate. On completion the reaction mixture was poured in to 8 folds ice cooled water, the intermediate (3) was precipitated, filtered and dried in vacuum desiccators. Melting point of synthesized tetrachloroperylenediimides (3) was more than 300°C. To the well stirred solvent DMF 25 ml in 250 ml round bottomed flask, was added tetrachloroperylenediimides (3) 1 mmol (0.770g) and started heating. By maintaining the temperature 110oC, the reaction mixture was added 4-hydroxyazobenzene derivatives (3a-g), 4 mmol and potassium carbonate 2.0g. Continued the stirring and heating for 3h. The reaction progress was monitored by taking the TLC of reaction mixture in 4:1 pet ether: ethyl acetate. On accomplishment of reaction, the reaction mixture was filtered and the residue was dissolved in

30ml of water. On acidification with conc. HCl, (1ml), the precipitation occurred immediately, filtered and dried to afford the products (6a-g) in 90-95%. 1,6,7,12-tetra-(4’-methoxyphenylazophenoxy) perylene-3,4,9,10-(p-nitrophenyl) diimide (6a) o 1 Yellowish orange crystals (79 %),, m.p> 350 C. H-NMR (300MHz, DMSO-d6) δ (ppm): 3.78 (s, 3H), 6.07 (d, 2H, J= 8.7Hz ), 6.949 (d, 4H, J= 8.7Hz), 7.413 (d, 2H, J= 8.7Hz), 7.54 (d, 2H, 13 J= 8.7Hz), 7.95 (s, 4H), 8.55 (d, 2H) ppm. C -NMR (75MHz, DMSO-d6) δ (ppm): 55.7, 114.4, 116.6 120.6, 122.2, 127.7, 134.9, 142.05, 144.3, 146.7, 148.2, 150.4, 151.06, 153.6, 155.7, 157.08, -1 158.6, 161.04, 162.7. FTIR (Neat, cm ) νmax: 3085, 1701, 1634, 1595, 1539, 1449, 1369, 1277, -1 1202, 1147, 1120, 1078, 959, 834, 798, 753 cm . Anal. Calcd. For C84H56N12O16: C, 68.1; H, 3.70; N, 11.35; Found: C, 67.9; H, 3.74; N, 11.20.

1,6,7,12-tetra-(4’-methylphenylazophenoxy) perylene-3,4,9,10-(p-nitrophenyl) diimide (6b) 1 Yellowish orange crystals (87 %), m.p> 350. H-NMR (300MHz, DMSO-d6) δ (ppm): 2.38 (s, 3H), 6.725 (d, 2H, J= 8.7Hz), 6.97(d, 2H, J= 8.6Hz), 7.314 (d, 2H J= 8.7Hz), 7.56 (d, 4H, J= 13 8.7Hz), 7.89 (d, 2H, J= 8.6Hz), 8.53(s, 4H). C -NMR (75MHz, DMSO-d6) δ (ppm): 21.13, 112.9, 116.77, 122.58, 123.40, 123.68, 125.77, 127.01, 130.06, 140.74, 146.88, 150.28, 156.43, 161.54, -1 162.70, 163.78, 165.93. FTIR (Neat, cm ) νmax: 3065, 1690, 1638 1588, 1541, 1448, 1380, 1277, -1 1210, 1160, 1118, 1068, 967, 843, 797, 760 cm . Anal. Calcd. For C84H56N12O12 : C, 73.2; H, 4.0; N, 12.2; Found: C, 73.1; H, 3.93; N, 12.5.

1,6,7,12-tetra-(2’-carboxyphenylazophenoxy) perylene-3,4,9,10-(p-nitrophenyl) diimide (6c) o 1 Yellowish orange crystals (80 %), m.p> 350 C. H-NMR (300MHz, DMSO-d6) δ (ppm): 8.20 (m, 1H), 8.14(m, 1H), 7.65-7.80 (m, 2H) 7.76 (d, 2H, J= 8.7Hz), 6.93 (d, 2H, J= 8.7Hz), 8.09 (d, 13 4H, J= 8.6Hz), 8.5 (s, 4H), 11.0 (br singlet 1H) ppm. C -NMR (75MHz, DMSO-d6) δ (ppm): 111.1, 117.68, 120.04, 122.58, 123.50, 125.49, 127.01, 129.72, 130.06, 140.74, 148.49, 149.67, -1 150.28, 155.64, 156.43, 162.70, 167.27. FTIR (Neat, cm ) νmax: 3052, 1683, 1631, 1580, 1546, -1 1441, 1385, 1265, 1210, 1163, 1025, 963, 862, 790, 762 cm . Anal. Calcd. For C84H48N12O20: C, 65.11; H, 3.10; N, 10.88; Found: C, 64.8; H, 3.2; N, 10.70.

1,6,7,12-tetra-(4’-chlorophenylazophenoxy) perylene-3,4,9,10-(p-nitrophenyl) diimide (6d) o 1 Yellowish orange crystals (85 %), m.p> 350 C. H-NMR (300MHz, DMSO-d6) δ (ppm): 7.47 (d, 2H, J= 8.6Hz), 7.86 (d, 2H, J= 8.6Hz), 7.76 (d, 2H, J= 8.6Hz), 6.93 (d, 2H, J= 8.6Hz), 8.15 (d, 13 2H, J= 8.6Hz), 6.90 (d, 2H, J= 8.6Hz), 8.51 (s, 4H) ppm. C NMR (75 MHz, DMSO-d6) δ (ppm): 112.9, 115.7, 117.1, 121.02, 122.01, 122.89, 123.68, 125.49, 127.48, 129.72, 130.06, 135.93, -1 140.74, 158.5, 161.5, 163.78, 165.93. FTIR (Neat, cm ) νmax: 3048, 1696, 1625, 1583, 1537, -1 1450, 1373, 1212, 1167, 1028, 970, 870, 785, 753 cm . Anal. Calcd. For C80H44N12O12: C, 72.94; H, 3.34; N, 12.76; Found: C, 72.45; H, 3.30; N, 12.5. 1,6,7,12-tetra-(3’-nitrophenylazophenoxy) perylene-3,4,9,10-(p-nitrophenyl) diimide (6e) o 1 Yellowish orange crystals (82%), m.p> 350 C. H-NMR (300MHz, DMSO-d6) δ (ppm): 8.39 (m, 1H), 8.86 (s, 1H), 7.72 (m, 1H) , 8.32 (m, 1H), 7.76 (d, 2H, J= 8.6Hz), 8.10 (d, 2H, J= 8.6Hz),

13 6.82 (d, 2H, J= 8.6Hz), 8.54 (s, 4H) ppm. C -NMR (75MHz, DMSO-d6) δ (ppm): 109.53, 114.4, 117.6, 121.3, 123.8, 125.2, 128.78, 129.83, 130.17, 140.58, 147.14, 149.73, 153.70, 154.1, 159.5, -1 163.6. FTIR (Neat, cm ) νmax: 3057, 1699, 1630, 1580, 1525, 1449, 1358, 1232, 1163, 1048, 995, -1 878, 780, 747 cm . Anal. Calcd. For C80H44N16O20: C, 62.01; H, 2.84; N, 14.47; Found: C, 61.55; H, 2.88; N, 14.25.

1,6,7,12-tetra-(4’-nitrophenylazophenoxy) perylene-3,4,9,10-(p-nitrophenyl) diimide (6f) o 1 Yellowish orange crystals (83 %), m.p> 350 C. H-NMR (300MHz, DMSO-d6) δ (ppm): 8.39 (d, 2H, J= 8.7Hz), 8.19 (d, 2H, J= 8.7Hz ), 7.76 (d, 2H, J= 8.7Hz), 6.95 (d, 2H, J= 8.6Hz), 8.15 13 (d, 2H, J= 8.7Hz), 6.85 (d, 2H, J= 8.7Hz), 8.56(s, 4H) ppm. C -NMR (75MHz, DMSO-d6) δ (ppm): 109.5, 118.9, 120.0, 122.8, 123.9, 125.1, 125.4, 130.1, 140.5, 147.1, 148.8, 149.7, 153.7, -1 156.8, 158.5, 162.62. FTIR (Neat, cm ) νmax: 3069, 1694, 1631, 1584, 1522, 1447, 1368, 1252, -1 1173, 1068, 992, 876, 792, 743 cm . Anal. Calcd. For C80H44N16O20: C, 62.01; H, 2.84; N, 14.47; Found: C, 61.59; H, 2.85; N, 14.23. 1,6,7,12-tetra-(2’-methoxyphenylazophenoxy) perylene-3,4,9,10-(p-nitrophenyl) diimide (6g) o 1 Yellowish orange crystals (85 %), m.p> 350 C. H-NMR (300MHz, DMSO-d6), δ (ppm): 3.80 (s, 1H), 6.90-7.85(m, 4H), 7.65-7.80 (m, 2H), 7.76 (d, 2H, J= 8.6Hz), 6.98 (d, 2H, J= 8.6Hz), 13 8.10 (d, 2H, J= 8.6Hz), 8.54 (s, 4H) ppm. C-NMR (75MHz, DMSO-d6) δ (ppm): 56.79, 109.4, 112.6, 118.90, 120.4, 122.5, 128.8, 133.6, 144.1, 145.3, 147.7, 149.2, 150.4, 151.09, 153.8, 155.6, -1 157.07, 158.9, 161.02, 163.5. FTIR (Neat, cm ) νmax: 3064, 1700, 1634, 1581, 1523, 1441, 1362, -1 1242, 1179, 1065, 995, 866, 782, 755cm . Anal. Calcd. For C84H56N12O16: C, 68.1; H, 3.70; N, 11.35; Found: C, 67.93; H, 3.72; N, 11.22. 3.7 General procedure for synthesis of perylene alkoxy derivatives (7a-h) Tetrachloroperylene dianhydride (4) 0.001 mol (0.53g) was charged in to 250 mL round bottom flask containing 20 ml dry distilled DMF. The flask was placed on oil bath being heated with hot plate. The reaction mixture was stirred continuously during heating. Alcohols (3a-h, 0.004 mol), o followed by 2.0 g of K2CO3 were added in the flask. The mixture was kept at 110 C for 3 hours. The progress of reaction was monitored by performing TLC intermittently in 4:1 ethyl acetate: pet ether solvent. On completion of reaction, the mixture was filtered. The residue was dissolved in 30 mL of water and acidified with concentrated HCl, (1mL). Precipitation occurred immediately. The contents were filtered and dried to obtain final products (7a-h) in 90-95% yield.

1,6,7,12-tetradecyl perylene dianhydride (7a) Yellow crystals, m.p> 350 oC, 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 0.88 (3H, t), 1.26-1.76 13 (14H, m), 4.06 (2H, t), 7.81 (4H, s). C-NMR (75MHz, DMSO-d6) δ (ppm): 155.60, 155.40, 133.13, 123.46, 120.85, 117.94, 109.52, 70.13, 31.65, 29.06, 28.96, 28.71, 26.49, 22.94, 14.02. -1 FTIR (Neat, cm ) νmax : 2975, 1828, 1772, 1620, 1580, 1584, 1549, 1438, 1367, 1110, 940, 905, 855, 810, 785, 722. Anal. Calcd. For C64H88O10 C, 75.41; H, 8.90; Found: C, 74.90; H, 8.55.

1,6,7,12-tetra-1’-methylheptyl perylene dianhydride (7b)

o 1 Yellowish orange crystals, m.p> 350 C, H-NMR (300 MHz, DMSO-d6) δ (ppm): 0.88 (3H, t), 0.96 (3H, d, J= 8.6Hz), 1.26-1.63 (15H, m), 3.70 (1H, sextet), 7.61 (4H, s). 13C-NMR (75MHz,

DMSO-d6) δ (ppm): 155.40, 153.31, 133.65, 124.71, 119.93, 119.62, 110.23, 76.80, 36.03, 31.65, -1 29.32, 25.24, 22.94, 19.26, 14.02. FTIR (Neat, cm ) νmax : 2968, 1825, 1777, 1620, 1580, 1584, 1549, 1429, 1367, 1105, 943, 909, 865, 803, 775, 732. Anal. Calcd. For C56H72O10 C, 74.14; H, 8.22; Found: C, 73.75; H, 8.06.

1,6,7,12-tetrabutyl perylene dianhydride (7c)

o 1 Yellowish orange crystals, m.p> 350 C, H-NMR (300 MHz, DMSO-d6) δ (ppm): 0.9 (3H, t), 13 1.27 (2H, h), 1.62 (2H, p) 4.09 (2H, t), 7.27 (4H, s). C-NMR (75MHz, DMSO-d6) δ (ppm): -1 155.60, 155.40, 133.13, 123.46, 120.85, 117.94, 70.46, 30.51, 19.94, 14.02. FTIR (Neat, cm ) νmax : 2980, 1823, 1770, 1620 1580, 1584, 1549, 1433, 1387, 1155, 968, 915, 850, 810, 780, 725. Anal. Calcd. For C40H40O10 C, 70.37; H, 6.20; O, 23.43; Found: C, 70.13; H, 6.04.

1,6,7,12-tetraethyl perylene dianhydride (7d)

o 1 Yellowish orange crystals, m.p> 350 C, H-NMR (300 MHz, DMSO-d6) δ (ppm): 1.72 (3H, t),

13 4.72 (2H, q), 7.27 (4H, s). C-NMR (75MHz, DMSO-d6) δ (ppm): 156.29, 155.40, 132.88,

-1 123.09, 120.61, 118.38, 109.51, 64.09, 13.83. FTIR (Neat, cm ) νmax : 2925, 2855, 1813, 1775, 1620, 1580, 1584, 1549, 1435, 1382, 1135, 989, 945, 855, 820, 792, 755. Anal. Calcd. For C32H24O10 C, 67.37; H, 4.59; Found: C, 66.97; H, 4.09. 1,6,7,12-tetrapentyl perylene dianhydride (7e) o 1 Yellowish orange crystals, m.p> 350 C, H-NMR (300 MHz, DMSO-d6) δ (ppm): 0.90 (3H, t), 13 1.27-1.33 (24H, m), 4.06 (2H, t), 7.27 (4H, s). C-NMR (75MHz, DMSO-d6) δ (ppm): 155.60, 155.40, 133.13, 123.46, 120.85, 117.94, 109.52, 70.13, 31.65, 28.71, 26.82, 22.94, 14.02. FTIR -1 (Neat, cm ) νmax : 2905, 1810, 1772, 1620, 1580, 1584, 1549, 1420, 1387, 1115, 982, 915, 875, 850, 795, 740. Anal. Calcd. For C44H48O10 C, 71.72; H, 6.57; Found: C, 71.24; H, 6.25. 1,6,7,12-tetrapropyl perylene dianhydride (7f)

o 1 Yellowish orange crystals, m.p> 350 C, H-NMR (300 MHz, DMSO-d6) δ (ppm): 1.3 (3H, t), 1.90 13 (2H, h) 4.09 (2H, t), 7.27 (4H, s). C-NMR (75MHz, DMSO-d6) δ (ppm): 155.60, 155.40, 133.13, -1 123.46, 120.85, 117.94, 109.52, 71.83, 21.36, 10.62. FTIR (Neat, cm ) νmax: 2925, 1818, 1762, 1620, 1580, 1584, 1549, 1425, 1395, 1145, 972, 915, 870, 825, 765, 753. Anal. Calcd. For C36H32O10 C, 69.22; H, 5.16; Found: C, 68.93; H, 4.89. 1,6,7,12-tetracyclopentyl perylene dianhydride (7g)

o 1 Yellowish orange crystals, m.p> 350 C, H-NMR (300 MHz, DMSO-d6) δ (ppm): 3.71 (1H, p),

13 2.02 (4H, q), 1.71 (4H, t), 7.27 (4H, s). C-NMR (75MHz, DMSO-d6) δ (ppm): 155.40, 152.39, -1 133.68, 125.11, 119.80, 119.62, 110.22, 82.53, 33.39, 24.10. FTIR (Neat, cm ) νmax : 2935, 1815, 1765, 1620, 1580, 1584, 1549 1427, 1385, 1145, 950, 933, 867, 838, 785, 758. Anal. Calcd. For C44H40O10 C, 72.51; H, 5.53; O, 21.95; Found: C, 72.11; H, 5.35.

1,6,7,12-tetracyclohexyl perylene dianhydride (7h)

o 1 Yellowish orange crystals, m.p> 350 C, H-NMR (300 MHz, DMSO-d6) δ (ppm): 3.64 (1H, p), 13 1.95 (4H, q), 1.53 (4H, p), 1.46 (4H, p), 7.27 (4H, s). C-NMR (75MHz, DMSO-d6) δ (ppm): 155.40, 152.39, 133.68, 125.11, 119.80, 119.62, 110.22, 77.90, 30.36, 25.92, 24.59. FTIR (Neat, -1 cm ) νmax : 2950, 1805, 1757, 1620, 1580, 1584, 1549, 1440, 1380, 1160, 962, 905, 877, 828, 795, 750. Anal. Calcd. For C48H48O10 C, 73.45; H, 6.16; Found: C, 73.08; H, 5.90. 3.8 General procedure for synthesis of Perylene Schiff base diimide Dyes (13a-e) To the well stirred solution of 4-aminoacetanilide (1.52 g, 0.001mol) in 25 ml ethanol in 250 ml round bottomed flask was added aromatic aldehyde (2a, 0.001 mol). The reaction mixture was heated at reflux temperature for 12 hour in the presence of glacial acetic acid catalyst (0.5ml). Completeness of reaction was observed by TLC (4:1, pet ether: ethyl acetate). At the completion of reaction, the reaction mixture was rotary evaporated and collected the solid residue which was further purified by recrystallization (50:50 ethyl acetate: ethanol). The intermediate Schiff base was suspended in water 30 ml in 250 ml round bottomed flask and was added conc. HCl (1.5 ml). Started stirring and heating at 90 oC, for 2 h, until reaction was completed as determined from TLC of reaction mixture (4:1, pet ether: ethyl acetate). The reaction mixture was cooled and deprotected Schiff base was separated from water on standing for 1h, which was filtered and dried in oven at 60 oC. In this way other Schiff base were synthesized by treating aldehydes 10be with p- aminoacetanilide (9). Schiff base (11a, 0.002mol) was dissolved in quinoline (20 ml) I 250 ml round bottomed flask and stated stirring. Then perylene dianhydride (4) was added (0.001mol) was added to the above solution and stated heating at 165 oC. Heating was continued for 20 h for the completion of reaction, until t was determined from its TLC (4:1, pet ether: ethyl acetate). On cooling the reaction mixture dye was precipitated from quinoline which filtered, dried and recrystallized from DCM. In this way other dyes was synthesized. 2,9-bis(4-((pyren-1-ylmethylene)amino)phenyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquino line- 1,3,8,10(2H,9H)-tetraone (13a)

o 1 1 Brownish yellow crystals (67 %), m.p> 300 C. H-NMR H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.71 (s, 2H), 8.39 (2H, m), 8.35 (2H, d, J= 8.1Hz), 8.29 (d, 4H, J= 8.6Hz ), 8.26 (d, 4H, J= 8.6Hz), 8.20-8.04 (10H, m), 7.92 (2H, d, J= 8.1Hz), 7.70 (d, 2H,), 7.59(d, 4H, J= 8.5Hz), 7.56 (d, 4H, J= 13 8.5Hz). C-NMR (75MHz, DMSO-d6) δ (ppm): 167.05, 158.3, 133.6, 131.2, 130.9, 129.6, 128.4, 128.3, 126.6, 126.3, 126.1, 125.6, 125.2, 125.1, 124.0, 124.5, 124.1, 122.5, 133.3, 135.4. FTIR -1 -1 (Neat, cm ) νmax: 3057, 1706, 1625, 1583, 1530, 1447, 1262, 1078, 947, 857, 842 cm . Anal. Calcd. For C70H36N4O4 C, 84.32; H, 3.64; N, 5.62; Found: C, 84.13; H, 3.70; N, 5.43.

2,9-bis(4-((9H-fluoren-9-ylidene)amino)phenyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquino line 1,3,8,10(2H,9H)-tetraone (13b)

o 1 Brownish yellow crystals (71 %), m.p> 300 C. H-NMR (300MHz, DMSO-d6) δ (ppm): 9.24 (s, 1H), 8.58 (d, 4H, J= 8.7Hz), 8.16 (d, 4H, J= 8.7Hz), 8.02 (d, 4H, J= 8.6Hz), 7.94 (d, 4H, J= 13 8.6Hz), 7.68 (d, 4H, J= 8.6Hz), 7.57 (d, 4H, J= 8.6Hz). C-NMR (75MHz, DMSO-d6) δ (ppm): 170.31, 162.96, 146.58, 140.00, 138.74, 136.48, 135.62, 134.34, 131.18, 130.58, 129.57, 129.40, -1 128.39, 128.06, 125.60, 124.53, 124.29, 124.01, 122.27. FTIR (Neat, cm ) νmax: 3081, 1682, 1631, -1 1584, 1545, 1501, 1457, 1260, 1087, 952, 868 cm . Anal. Calcd. For C62H32N4O4 C, 83.02; H, 3.60; N, 6.25; Found: C, 82.97; H, 3.66; N, 6.17. 2,9-bis(4-((4- nitrobenzylidene)amino)phenyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline 1,3,8,10(2H,9H)- tetraone (13c) o 1 Yellowish red crystals (75 %), m.p> 300 C. H-NMR (300MHz, DMSO-d6) δ (ppm): 9.82 (dd, 2H), 8.29(d, 4H, J= 8.6Hz), 8.11 (d, 4H, J= 8.6Hz), 8.26 (d, 4H, J= 8.5Hz), 8.00 (d, 4H, J= 8.5Hz), 13 7.60 (d, 4H, J= 8.4Hz), 7.54 (d, 4H, J= 8.4Hz). C-NMR (75MHz, DMSO-d6) δ (ppm): 177.02, 162.96, 151.75, 149.88, 140.00, 139.97, 133.99, 131.49, 130.58, 128.67, 128.06, 125.64, 125.60, -1 124.60, 124.53 and 124.29. FTIR (Neat, cm ) νmax: 3061, 1701, 1617, 1590, 1531, 1440, 1267, -1 1171, 1088, 925, 863, 830, 817 cm . Anal. Calcd. For C50H26N6O8 C, 71.60; H, 3.12; N, 10.02; Found: C, 71.45; H, 3.18; N, 10.00. 2,9-bis(4-((2-hydroxy-6-nitrobenzylidene)amino)phenyl)anthra[2,1,9-def:6,5,10-d'e'f'] diisoquinoline-1,3,8,10 (2H, 9H)-tetraone (13d)

o 1 Yellowish red crystals (72 %), m.p> 300 C. H-NMR (300MHz, DMSO-d6) δ (ppm): 9.05 (dd, 2H), 8.17 (d, 4H, J= 8.7Hz), 8.15 (d, 4H, J= 8.7Hz), 7.94 (d, 2H, J= 8.5Hz), 8.20 (t, 2H), 8.26 (t, 13 2H), 7.58 (d, 4H, J= 8.6Hz), 7.50 (d, 4H, J= 8.6Hz). C-NMR (75MHz, DMSO-d6) δ (ppm): 168.52, 162.96, 151.75, 149.88, 140.00, 139.97, 133.99, 131.49, 130.58, 128.67, 125.60, 124.60, -1 124.53, 124.29. FTIR (Neat, cm ) νmax: 3455, 3045, 1698, 1618 1585, 1537, 1521, 1457, 1417, -1 1267, 1171, 1080, 907, 870, 828, 810, 780 cm . Anal. Calcd. For C50H26N6O10 C, 68.97; H, 3.01; N, 9.65; Found: C, 68.88; H, 3.07; N, 9.56. 2,9-bis(4-((4-(dimethylamino)benzylidene)amino)phenyl)anthra[2,1,9-def:6,5,10-d'e'f'] diisoquinoline-1,3,8,10(2H,9H)-tetraone (13e)

o 1 Yellowish red crystals (70 %), m.p> 300 C. H-NMR (300MHz, DMSO-d6) δ (ppm): 9.21 (dd, 1H), 8.27 (d, 4H, J= 8.6Hz), 8.11 (d, 4H, J= 8.7Hz), 7.65 (d, 4H, J= 8.6Hz), 7.53 (d, 4H J= 8.6Hz), 7.56 (d, 4H, J= 8.4Hz), 6.68 (d, 4H, J= 8.4Hz), 2.91 (s, 12H). 13C-NMR (75MHz,

DMSO-d6) δ (ppm): 171.02, 162.96, 155.51, 148.84, 140.28, 133.63, 131.23, 130.58, 128.67,

-1 128.06, 125.64, 125.60, 124.53, 124.29, 122.95, 111.56, and 41.91. FTIR (Neat, cm ) νmax: 3041, 2923, 1690, 1628 1582, 1541, 1511, 1457, 1262, 1088, 1011, 878, 830, 817, 791 cm-1.

Anal. Calcd. For C54H38N6O4 C, 77.68; H, 4.59; N, 10.07; Found: C, 77.53; H, 4.64; N, 10.03.

PART II

Chapter 4 INTRODUCTION

4.1 Azo Dyes Azo dyes are amongst the most versatile classes of dyes [89, 90] which are characterized by the presence of one or more azo linkages (N=N). Monoazo dyes have only one N=N double bond, while diazo and triazo dyes possess two and three azo groups correspondingly. The azo groups are mostly associated with benzene and naphthalene rings, but can also be connected to aromatic heterocycles or enolizable aliphatic groups. These side groups are compulsory for conveying the color of the dye, with many changed shades and passions being possible. A common example of an azo dye is shown in Figure 1.1.

Figure 4.1 Structure of a typical azo dye. The structure of azo dyes have been intensely studied and many spectral data analyses have already been reported [90, 91]. The dyes have been most extensively used in areas such as dying textile 77

fibers, biomedical studies, advanced uses in organic synthesis and high technology areas like lasers, liquid crystalline demonstrations, electro-optical maneuvers and ink-jet printer [9195]. Organic dyes have also been reported as effective corrosion inhibitors of mild steel in different media [96-106]. The basic reaction behind azo dyes was discovered in 1858 by P. Griess after the discovery of aniline in 1856 and coupling reaction was discovered in 1864. Diazotization of primary aromatic amine was achieved by addition of aqueous solution of NaNO2 to a solution of amine hydrochloride in presence of excess of HCl at temperature below 5oC (scheme 1.1).

Scheme 4.1 Synthesis of diazonium Salt 4.2 Classification of Azo Dyes Azo dyes are basically characterized by the presence of different kinds of aromatic moieties either they are carbocyclic aromatic systems or heterocyclic aromatic systems. So the two basic classes of Azo dyes are carbocyclic azo dyes and heterocyclic azo dyes, but the more useful classification of azo dyes is due to their application properties. According to the application characteristics classes of azo dyes are direct dyes, acid dyes, reactive dyes, disperse dyes, metal complex dyes, mordant dyes and formazan dyes. 4.2.1 Direct Dyes This is an important class of dyes for the dyeing of paper. Direct dyes are also called substantive dyes [107, 108] because they tend to have a high affinity for cellulose fibers due to their linear molecular structure and a system of conjugated double bonds and usually also exhibit good wet fastness properties with the addition of a fixative. They are applied from neutral or slightly alkaline baths containing additional electrolyte on cotton, rayon, paper and nylon. These may be anionic direct dyes or cationic direct dyes depending upon the functional groups present in these dyes (Figure 1.2 and 1.3).

Figure 4.2 Examples of anionic direct azo dyes (1, 2 and 3)

Figure 4.3 Examples of cationic direct azo dyes (4 and 5)

4.2.2 Acid Dyes Acid dyes differ from the direct dyes in that they have smaller molecules and are less substantive due to the lack of a conjugated double-bond system. They are used to dye polyamide or wool, where they produce good dyeing properties owing to their small molecule sizes and good solubility. The acid dyes have no affinity to vegetable fibers. Although they penetrate well into the capillaries of the fibers, no fixed bond is formed and there is virtually no formation of a charge- transfer complex. Like direct dyes these may be cationic or anionic depending upon different functionalities present within the molecules (Figure 1.4 and 1.5) [109, 110].

Figure 4.4 Examples of anionic acid dyes (6, 7 and 8)

Figure 4.5 Examples of cationic acid dyes (9 and 10) 4.2.3 Reactive Dyes Reactive dyes are the newest class of dyes for cellulose fibers. ICI introduced the first group of reactive dyes for cellulose fibers (with 2,4-dichloro-1,3,5-triazine anchor) in 1956. In reactive dyes, a chromophore is combined with one or more functional groups, the so-called anchors that can react with cellulose (Figure 1.6) [111].

Figure 4.6 Reactive dyes based on diazine and vinylsulphone para ester

4.2.4 Disperse Dyes Disperse dyes are colorants with low water solubility that, in their spread colloidal form, are appropriate for dyeing and printing hydrophobic threads (fibers) and fabrics. Disperse dyes consist of very small molecules and therefore are ideal for dyeing the dense fibers like polyesters. Industrially applied disperse dyes are based on numerous chromophore systems which include approximately 60% azo dyes (Figure 1.7) and 25% anthraquinone dyes with the remainder distributed among quinophthalone, methine, naphthalimide, naphthoquinone, and nitro dyes [112, 113].

Figure 4.7 Typical examples of disperse azo dyes 4.2.5 Metal Complex Dyes Metal complex dyes are very versatile in terms of applications. Virtually all substrates, apart from a few synthetic fibers, can be dyed and printed with this class of dyes. Countless shades from greenish yellow to deep black can be generated, depending upon the metal, the dye ligand, and the combination of dye ligands in mixed complex dyes (Figure 1.8) [114].

Figure 4.8 Metal complex Azo Dyes.

4.2.6 Mordant Dyes In these dyes mordanted fiber is used which is capable of producing of producing insoluble colored complexes (lakes) with certain azo and anthraquinone derivatives. Alizarin the bestknown example of mordant dyes is isolated from the root of the Madder plant, but has now been replaced by the synthetic product. Mordant azo dyes must contain hydroxyl or carboxyl groups in the position ortho to the azo group on one or both of the aromatic nuclei. The shade of the dyeing depends on the type of metallic mordant used (Figure 1.9) [115].

Figure 4.9 Typical examples of Mordant Azo Dyes 4.2.7 Formazan Dyes Formazan dyes are closely related to azo dyes and are derived from the following basic structure as shown in Figure 1.10.

Figure 4.10 Basic structure of formazan azo dyes Formazans unsubstituted in the 1 and 5-positions and 1, 5-dialkyl-substituted formazans are unknown. Aryl or heteroaryl groups are the most common 1, 5-substituents (Figure 1.11). The 3- or meso position can be occupied by a variety of substituents (e.g., aryl, heteroaryl, H, OH, SR, halogen, NO2, CN, and alkyl) [116].

Figure 4.11 1, 5- substituted examples of formazan azo dyes

4.3 Derivatives of Azo Dyes A lot of derivatives of azo dyes have been synthesized and are currently being synthesized depending upon the choice of applications keeping in view the availability of reagents, ease of

handling, facile in synthesis, ease in applications and environmental issues. A large share of derivatives in azo dyes comes from reactive dyes and acid dyes. 4.3.1 Reactive Dyes Reactive dyes are colored compounds which contain one or two groups capable of forming covalent bonds between a carbon or phosphorus atom of the dye ion or molecule and an oxygen, nitrogen, sulphur atoms of a hydroxyl, an amino or a mercapto group correspondingly, of the substrate. Such covalent bonds are made with the amino, hydroxyl groups of cellulosic threads (fibers), with the amino, hydroxyl and mercapto groups of protein fibers and with the amino groups of polyamide. Reactive dyes are the only textile colorants which establish a covalent relationship with different substrates having nucleophilic functionalities in their structures. They are used for the dyeing and printing of cellulose, wool, silk and to a lesser extent polyamide fibers. Brilliant shades, excellent wet fastness of dyeing and simple dyeing operations have contributed a lot to a rapid increase in demand of reactive dyes. However, additional properties have been demanded by dye works and apparel manufactures [117-120], with the growth in the usage of reactive dyes, particularly high fixation in exhaustion dyeing and high fastness to chlorine perspiration, light and wash fastness in the presence of peroxides [121-123]. Major commercial importance in reactive dyeing is of hot brand reactive dyes particularly in printing [124, 125]. Several new reactive systems as well as intermediates have been developed from time to time and can be viewed in the form of derivatives of substituted ethylamine, ethylamides, pyridazine, phthalazine and quinoxaline (Figure 1.12).

Figure 4.12 Different reactive functionalities in reactive dyes 4.3.2 Acid Dyes Acid dyes are characterized by the presence of acidic functional groups within the dye molecules which may be carboxylic, sulfonic or phosphoric group. Acid dyes exist mostly in metal complex forms. Three metals of prime importance in azo dyes are copper, chromium and cobalt. The most important copper dyes are the 1:1 planar copper (II) azo dye complexes (Figure 1.13). In contrast, chromium (III) and cobalt (III) form 2:1 dye: metal complexes that have nonplanar structures. So there are two types of metal complex acid dyes.

Figure 4.13 1:1 and 2:1 metal complex acid dyes

a) 1:1 Metal-Complex Dyes In these complexes the mole ratio between dye ligands and metal atoms is 1:1. Among these dyes primarily the 1:1 chromium complexes containing sulfonic acid groups have achieved commercial importance. They are applied from a strongly acid bath, which imposes certain limits on their range of applications. The 1:1 metal complexes are not suitable for polyamide, which is partially decomposed under the dyeing conditions for these products. Their main area of application is in the dyeing of wool, but they are also suitable for leather dyeing [126]. b) 1:2 Metal-Complex Dyes 1:2 metal-complex acid dyes exhibit anionic character. Those which have gained commercial importance are primarily the ones that are free of sulfonic acid groups and for which adequate water solubility is provided by nonionic, hydrophilic substituents, such as methyl sulfone or sulfonamide groups [127]. The introduction of 1:2 metal-complex dyes which are applied from a neutral to weakly acid bath, represented a significant technical advance over the strong-aciddyeing 1:1 chrome complex dyes. It has led to better protection of the fiber material, simplification of the dyeing process, and improvement of the fastness properties. 4.3.2.1 Copper Complexes Synthesis of copper complexes of tridentate metallizable azo and azo methine dyes is easily carried out in aqueous medium with copper salts at pH 4.7 in the presence of buffering agents such as sodium acetate or amines. Sparingly water soluble precursors can be metallized in alkaline medium at up to pH 10 by using an alkali-soluble copper tetrammine solution as coppering reagent, which is available by treating copper sulfate or chloride with an excess of ammonia or alkanol amines [128, 129]. Three other approaches to copper complexes are also applicable, all of which do not start from o,o-Dihydroxyazo compounds. These are valuable and convenient methods in those cases where o,o-Dihydroxyazo compounds are difficult to prepare from diazotized o- Aminophenols (Figure 1.14) [130-132].

Figure 4.14 o,o-dihydroxyazo copper complex acid dyes 4.3.2.2 Chromium Complexes Chromium complexes of tridentate azo dyes are the most important class of metal-complex dyes. This is due to the reluctance of hexa coordinated chromium (III) complexes to exchange ligands, which, however, complicates the preparation of chromium complex dyes from hexa aqua chromium (III) salts, and makes it possible to prepare triaqua 1:1 chromium complex dyes. Generally, 1:1 chromium complexes can be made in acid medium below pH 4, whereas 1:2 chromium complexes are prepared at higher pH in weakly acid to alkaline medium. The stability of the chrome dyes parallels the pH conditions for production. The 1:1 chromium complex dyes are only stable in the presence of mineral acids, and 1:2 chromium complex dyes are unstable at 84

high acidity and disproportionate into the corresponding 1:1 chromium complex and the metalfree dye (Figure 1.15) [133-135].

Figure 4.15 1:2 and 1:1 chromium complex dyes 4.3.2.3 Cobalt Complexes Preparation of 1:2 cobalt complexes does not require such high reaction temperatures as the corresponding 1:2 chromium complexes, since the aqua cobalt complexes are less inert than those of chromium. The usual method is the reaction of Co (II) salts in alkaline medium at about 60°C, which leads rapidly to the diamagnetic 1:2 Co (III) complexes. Atmospheric oxygen serves as oxidant. Co (III) exhibits a greater tendency to form complexes with nitrogen-donor ligands than with oxygen-donor ligands (Figure 1.16) [136-138].

Figure 4.16 1:2 cobalt complex dyes 4.4 Applications of Azo Dyes 4.4.1 Dosimetric Indicators Indicators are the organic substances, the presence of very small amount of which indicates the termination of a chemical reaction by a change of colour. Indicators are of various types, e.g., acid- base indicators, redox indicators, adsorption indicator, etc. Acid-base indicators are the organic substances, which have one colour in acid solution while different colour in alkaline solution. Azo dyes are widely used as indicators in acid base and complexometric titrations (Figure 1.17) [139- 142].

Figure 4.17 Examples of Azo dyes used as indicators 4.4.2 Dyeing of Protein, Polyester and Cellulosic fibers Dyes used for fabric such as cotton, wool, and silk are complex organic molecules that contain a chromophore group, which is a part of conjugated system within the molecule. These molecules can absorb certain wavelengths of visible light and reflect the remaining light and, thus, give a fabric its color. Not only do the dyes have polar or ionic groups, but fabrics such as cotton and wool also contain polar groups such as –OH (hydroxyl) and –NH (amide) which help in dye attachment to the fabric [143-145]. Sometimes chemical bonds are formed between the dye and the fabric molecules which hold the two together. Another process involves the use of a mordant, which serves as sort of an intermediary that bonds the dye and the fabric. If the dye molecules attach firmly to the fabric, the color will be "fast", that is, it does not run when wet or washed, after the initial rinsing of excess dye (Figure 1.18) [146, 147]. Azo dyes have been extensively and excessively used for dyeing cotton.

Figure 4.18 Examples of Azo dyes used for dyeing of protein, polyester and cellulosic fibers 4.4.3 Food Colorants Azo dyes are much more stable than most of the natural food dyes. Azo dyes are stable in the whole pH range of foods, are heat stable and do not fade when exposed to light or oxygen. This makes azo dyes applicable in nearly all foods. The only disadvantage is that azo dyes are not soluble in oil or fat. Only when azo dyes are coupled to a fat soluble molecule, or when they are dispersed as very fine particles, oils can be colored (Figure 1.19) [148-151].

Figure 4.19 Examples of azo dyes food colorants

4.4.4 Cosmetic Colorants Coloration of cosmetic products, especially decorative cosmetics, has been performed over a long periods of time. Azo dyes are being used especially due to attractive shades and colors (Figure 1.20) [151-153] in cosmetics.

Figure 4.20 Examples of azo dyes used in cosmetics 4.4.5 Staining of Biological Tissues Histological staining involves the use of dyes to highlight specific intra- or extracellular elements within tissue. A vast array of dyes and associated staining protocols exist in use. Each dye is targeted toward different cellular structures. The response to a given protocol can vary among samples. Many protocols are up to 100 years old and were developed using partially characterized textile dyes. As a result, the detailed mechanism underlying many popular staining techniques is unclear (Figure 1.21) [154-156]. Azo dyes are being used for staining purpose.

Figure 4.21 Examples of Azo dyes used for staining

4.4.6 Solar Cell Sensitizers Azo dyes and pigments have attracted considerable attention as photosensitizes in dye-sensitized solar cells DSSCs in recent times, since they represent not only the largest chemical class in the colour index, but also the largest class of the dyes used in the industry and the analytical chemistry [157-160]. Azo dyes economically superior to organometallic dyes because they are cheap and color variants. Oxidation potential measurements for used azo dyes ensured an energetically permissible and thermodynamically favorable charge transfer throughout the continuous cycle of photo-electric conversion. The performance of dye-sensitized solar cells based on azo dyes has been studied. The results illustrate that the dye containing acetic acid and sulfonic acid as the acceptor group gave the maximum conversion efficiency, 3.52 and 3.17 in the presence and absence of anti-aggregation agent, respectively (Figure 1.22) [161,162].

Figure 4.22 Typical Example of Azo dyes used in DSSC 4.4.7 Medicinal Potential Azo dyes exhibit a variety of interesting biological activities such as antibiotic, antifungal and anti- HIV. One of the positive pharmaceutical application of azo dyes and their specific azo reduction in vivo is polymeric azo compound for site specific drug delivery in the colon diseases such as colitis and irritable bowel syndrome [163]. Azo Schiff bases exhibited antibacterial activity against Bacillus subtilis (bacterium responsible for causing ropiness in spoiled bread dough) and antifungal against several fungi, including Candida albicans, Cryptococus neoformans, Tricophyton mentagrophytes [164]. Using of azo dyes as pro-drug can be exemplified by low-molecular or polymeric (immobilized on polyethylene glycol matrix (Figure 1.23) 5-aminosalicylic acid derivatives that exhibit anti-inflammatory and cytoprotective potency [165-170]. Some of azo dyes are being used as anti HIV (Figure 1.24). 88

Figure 4.23 Azo derivative of 5-aminosalicylic acid immobilized on polyethylene glycol

Figure 4.24 Bisazo compound with anti-HIV activity

4.5 Summary of Applications and Derivatization of Azo Dyes Azo dyes constitute 60-70% of all the synthetic dyes being used as commercial colorants. Azo compounds are continuously gaining attention in scientific research because of variety of derivatives obtained by the slight modifications of dye intermediate structures. Excellent thermal and optical properties of azo dyes have made them a field of diverse applications such as optical recording medium, toner, ink-jet printing, and oil-soluble light fast dyes. Biocidal treatment of textile materials is being done by azo dyes of suitable structures carrying some bioactive templates which exhibit physical interactions and chemical bonding with molecules of fibrous materials.

Medicinal importance of azo compounds is also well documented because of their use as antineoplastics, antidiabetics, antiseptics, antibacterial and antitumor. In a number of biological reactions such as inhibition of DNA, RNA and protein synthesis, carcinogenesis and nitrogen fixation, the involvement of azo dyes is known.

Diazonium salts undergo a variety of reactions with different substrates under different conditions to produce different molecules [171-174]. A summary of these reactions is shown in Scheme 1.2.

Scheme 4.2 Summary of reactions exhibited by diazonium salts

Objective and Plan of Research It was revealed from the detailed literature survey that azo dyes are being used in diverse fields owing to their properties associated with their brilliant colors or away from their colors. They are being employed in heavy duty applications as functional dyes. A lot of development has been in this regard, but there is lack of advancement in certain properties to meet the requirement for application of azo dyes. In case of textile and leather field, a large quantity of dye is wasted during application on fibers which not only causes loss to industrialist but also creates environmental problems. In case of solar cell applications of azo dyes, thermal and photo chemical stability of dyes is less which affects on the efficiency of solar cells. Detection probes based on azo dyes are less in number which can be used satisfactorily for detection of ions in different media. So keeping in view the deficiencies and availability of raw materials planning was made to develop certain dyes devoid of major demerits to meet the application standards of different categories. In this regard following efforts were made: • To synthesize the different bis anilines and use them as bridges in reactive dyes for a multiplicity of reactive groups so that they may impart their role in exhaustion and fixation of dyes on fibers. • To devise the new process for the synthesis of heterocyclic azo dyes. • To synthesize the Calix azo dyes, having ligand capacity to bind with different metal ions at the four sides of calix.

Chapter 5 RESULTS AND DISCUSSION

5.1 Synthetic Pathway to Reactive Azo Dyes (9a-j) The synthesis of bisazomonochlorotriazine (MCT) reactive dyes was accomplished following a two-step procedure. It is crucial to note that only a single type of bisazomonochlorotriazine (MCT) reactive dyes were synthesized having a common coupler, bisazo component and reactive system but different bridging bis anilines. Numerous literature reports are available in recent years to supplement this paradigm shift in azo dyes utilization. In order to create novelty and to increase the fixation of reactive dyes on fibers bis anilines were used as bridging groups. Thus substituted anilines were treated with a dilute solution of formaldehyde, at low temperature in order to avoid polymerization. Conc. HCl was used to protonate the NH2 group in differently substituted anilines, to peter out the nucleophilicity of NH2 group. Short duration of time (2h) was required for the completion of the reaction. Neutralization of reaction mixture was achieved with 10% NaOH solution which resulted in the separation of product, by snatching the proton and making the product insoluble in aqueous medium. The synthetic route to novel bis anilines (1k-t) has been sketched in Scheme 5.1.

Scheme 5.1: Synthesis of 5, 5'-methylenebis (3-aminoanilines) linkers Synthesis of dyes bisazomonochlorotriazine (MCT) reactive dyes (9a-j) has been conducted in accordance with the Scheme 2.2. The rational for the selection of these dyes for synthesis, is to acquire various scaffolds of this nature by derivatization which will help in the future structure activity relationship (SAR) study of these compounds. Here reactive dyes (9a-j) have been synthesized from 1-amino-8-naphthol-3,6-disulphonic acid (H-acid) coupler and 4,4’-diamino diphenylamine-2- sulfonic acid (FC-acid) as bisazo component. Accordingly, FC-acid (2) was tetra azotized at low temperature 0-5oC in order to stabilize the azo compound. Coupling of tetrazo F.C acid with H-acid (3) was achieved to afford (4) in alkaline medium to accomplish the coupling at position ortho to the hydroxyl group of H-acid at 0-5oC and the coupling was completed in 3h. Coupling at ortho position of hydroxyl group occurs at alkaline pH and in acidic medium coupling ortho to amino group takes place. Cyanuration of dye (4) was achieved by addition of dye to cyanuric chloride solution in an ice bath at pH 7 in 1:2 molar ratio followed by filtration, and drying of the dye at 70oC. Low temperature and neutral pH was maintained throughout condensation with cyanuric chloride for the replacement of only one chloro

group with the amino group of H-acid coupling component of dye 4 to afford intermediate 5. When pH and temperature conditions are varied then chances of substitution at second and third chloro groups are also enhanced. Bis anilines (1k-t) were added to solution of dye at pH 7 at room temperature in molar ratio 1:1 to furnish dyes (9a-j). Different bis anilines were used as bridging component for dyes, which did not affect the λmax too much but increased the substantivity of dye with fiber (Scheme 5.2).

Scheme 5.2 Synthetic route to bisazo MCT reactive dyes (9a-j)

5.2 Spectral Properties of Reactive Dyes (9a-j) The absorption maxima (λmax) of the dyes (9a-j) were recorded in water and are shown in Figure 5.1. These dyes showed two absorption maxima, one in the UV range due to π-π* transition of the C=C present in the aromatic moiety common in all dyes and other in the visible region, and is due to π-π* transition of azo linkage N=N of dyes. All dyes have same chromophoric functionalities but difference is of bridging groups. These bridging groups affect the λmax of dyes, but effect is not too high as these are not directly attached to chromophoric groups of dyes. The values of log ε (molar extinction coefficient) are summarized in Table 5.3 and are in the range 3.54-3.97, due to the high absorption intensity of the dyes. Intermediate dye (4) has λmax 626 nm which is without bridging anilines. Dyes 9a-c have λmax at 599, 608 and 604 nm respectively as these contain bridged anilines having a carboxylic function at o, m and p-positions to NH groups. There was a shift of 18-27 nm in λmax because of different electron donating and withdrawing groups through resonance and inductive effects. Dye 9b has higher λmax than 9a and 9c because of carboxylic group at m- position where only inductive effect operates. Dyes 9d and 9e have λmax 640 and 634 nm while 9f and 9g have λmax values 609 and 615 nms respectively. Here the introduction of auxochromes like hydroxyl group in bridging anilines produced bathochromic effect in 9d and 9e and caused shift of 8-14 nm λmax from 5. Dyes 9f and 9g carried bridging anilines containing both electron donating as well electron withdrawing groups which cancel the effect of each other and their values were close to the original dye 5. Dyes 9h and 9j have λmax lower than original dye 5 due to electron withdrawing groups NO2 and Cl at m-position to NH and their values were 601 and 620 nm respectively. The λmax of dye 9i was larger than dye 5 and was 638 nm due to methoxy group at ortho to NH. From the U.V analysis of dyes, it was observed that the dyes containing electron withdrawing groups in bridging anilines had are lower λmax while those containing electron donating groups had higher wavelength of maximum absorption than original dye without bridging groups.

Figure 5.1 Combined UV-Visible spectrum of MCT dyes (9a-j)

Table 5.1 Characterization data of dyes (9a-j)

Mol. C% H% Cal N% S% Rf* Dye Molecular Formula Yield% Wt Cal/Found /Found Cal/Found Cal/Found Value 9a C53Cl2H34N15O21S5 1447 75.32 43.95/43.35 2.34/2.30 14.51/14.35 11.05/10.98 0.55

9b C53Cl2H34N15O21S5 1447 76.11 43.95/43.10 2.34/2.26 14.51/14.32 11.05/10.95 0.54

9c C53Cl2H34N15O21S5 1447 75.65 43.95/43.68 2.34/2.28 14.51/14.38 11.05/10.80 0.55

9d C51Cl2H34N15O19S5 1391 81.15 43.99/43.55 2.44/2.40 15.09/15.00 11.50/10.85 0.47

9e C51Cl2H34N15O19S5 1391 80.72 43.99/42.95 2.44/2.43 15.09/14.93 11.50/10.97 0.49

9f C51Cl2H32N17O23S5 1481 83.22 41.32/40.53 2.16/2.05 16.07/15.90 10.80/11.00 0.45

9g C51Cl4H32N15O19S5 1460 85.71 41.91/40.70 2.19/2.15 14.38/14.05 10.95/10.91 0.41

9h C51Cl2H32N17O21S5 1449 78.77 42.23/42.06 2.20/2.12 16.42/16.22 11.04/10.97 0.43

9i C51Cl2H36N15O19S5 1393 84.57 43.93/42.33 2.58/2.56 15.07/14.99 11.48/11.30 0.43

9j C51Cl4H32N15O17S5 1428 80.43 42.85/41.35 2.24/2.18 14.71/14.50 11.20/11.15 0.39

*Toluene: Ethyl acetate (7.5: 2.5 v/v), Silica gel-G F254 TLC plate.

From the FTIR spectra of bis anilines (1k-t), appearance of CH2 stretching and bending vibrations is evidenced which are absent in the respective substituted anilines. The infrared spectra of the synthesized bisazo MCT reactive dyes showed absorption peaks due to O-H,N-H, Ar-H, C-H, C=O, C=C, C=N, NO2 , SO3H, N=N and C-Cl stretching vibrations at 3400-3500, 3160- 3000, 2929, 1700-1760, 1660,1590, 1502-1520, 1070, 723, and 672 cm-1 as depicted from their FTIR spectra. Specifically speaking, using FTIR spectrum of 9a-c reactive dyes, a broad band was observed in the range 3000-3500 cm-1 which was due to H-bonding of OH and COOH groups present in the bridging groups. This broad band was masking the peaks of N-H functionality. A peak was observed in the range 1710-1750 cm-1 which was due to the C=O functionality of dyes. The absorption bands at 1660, 1590, 1502 and 750 cm-1 depicted the presence of C=C stretching and bending vibrations of aromatic moieties, respectively, C-Cl peak was observed in all dyes at 672-700 cm-1 which confirmed the triazine ring system in dyes. Azo linkage is inveterated by the peaks in the range 1502-1520 cm-1. Dyes 9d and 9e have bridging anilines containing OH groups at o- and p-position to N-H group of diamines, they show a broad peak at 3448 cm-1which is due to OH and N-H stretching vibrations. Similarly 9i showed a peak at 3410 cm-1due to N-H stretching vibrations. This also shows a prominent peak at 1120 cm-1 due to C-O-C stretching vibrations, as it contains methoxy group at o-position to N-H. Reactive dyes 9d, 9e and 9i showed the remaining peaks similar to dyes 9a-c due to common moieties in all dyes. Dyes 9f and 9g had bridging groups containing NO2 and Cl, -1 p-position to NH group, so a peak was observed for NO2 group at 1550 cm and a peak for C-Cl was observed at 710 cm-1 in addition to C-Cl absorption of the triazine ring system. The dye 9h -1 had NO2 at m-position to NH, this also showed peak for NO2 at 1530 cm . The compound 9i had Cl at p-position to NH, so here another peak at 700 cm-1 was observed due to Cl attached to benzene ring. Peak for C-Cl in the triazine ring system was at lower wave number than Cl attached to benzene ring due to the fact that in the latter case bond was stronger than previous one, so peak 96

appeared at higher wave number. An absorption band at 2900-2950 cm-1 was common in all dyes (Figure 5.2).

Figure 5.2 FTIR spectrum of synthesized reactive azo dye 9d. 1 The H-NMR spectrum of dyes 9a-c showed signals down field at 11.15-11.62 ppm due to COOH groups present in the bridging diamines and in the aromatic region of the TMS scale in between δ7.60-8.3 ppm due to 19 aromatic protons. A broad singlet is observed at 5.0-5.10 ppm because of 2-OH groups attached to naphthalene ring. At range 1.90-2.10 ppm sharp singlet was observed due to bridging CH2 group present in bis-anilines. All these dyes 9a-j are compounds of a series where the difference arises in case of bridging groups and chromophores, coupling component and reactive systems were same. Here peak positions and intensity was varied for OH and bridging methylene protons. In case of dyes 9f-h and 9j which contained NO2 and Cl at m- and p-position to N-H group of bis anilines, methylene as well as N-H peaks shifted to downfield. The dyes 9d, 9e and 9i have OH and OCH3 groups at o and p- positions to NH groups, their signals shifted upfield and extra peaks were observed at 5.3-5.40 and 3.9 ppm for 1H and 3H attached to oxygen atoms and benzene rings. 5.3 Dyeing Properties of Reactive Dyes (9a-j) All the dyes (9a-j) were applied at 2.0 % depth (Figures 5.3 and 5.4) on cotton fibers according to the usual procedure [175] in the dye bath containing materials (listed in Table 5.2)

Figure 5.3 Samples of dyes applied on cotton cloth pieces before washing treatments for wash fastness.

Figure 5.4 Samples of dyes applied on cotton cloth pieces after washing treatments for wash fastness. Table 5.2 Dye-bath containing materials Materials Cotton Fabric 0.5g Amount of Dye 25mg Sodium Sulfate 1ml (25% W/V) Sodium Carbonate 1ml (10% W/V) Sodium Chloride 1ml (10% W/V) EDTA 1ml (10% W/V) pH 8.5-9.0 Dyeing Time 1h Dyeing Temperature 80 oC Total Volume 20 ml

5.3.1 Exhaustion and Fixation Study of Reactive Dyes (9a-j) Exhaustion and fixation values are determined by the application of dyes at 2% dyeing on cotton fibers. Exhaustion and fixation values are shown in Table 5.3 [176]. Table 5.3 Exhaustion and fixation data of reactive dyes (9a-j) Shade on λmax (nm) Log ε Dyes % Exhaustion % Fixation Fiber in H2O 9a Sky Blue 599 3.63 65.77 91.54 9b Sky Blue 608 3.54 63.53 92.35 9c Sky Blue 604 3.67 64.36 91.98 9d Sky Blue 643 3.65 72.35 81.23 9e Sky Blue 635 3.72 74.26 80.18 9f Sky Blue 609 3.49 71.28 82.23 9g Sky Blue 615 3.85 75.25 79.63 9h Sky Blue 601 3.97 73.18 81.25 9i Sky Blue 638 3.95 73.22 81.15 9j Sky Blue 620 3.82 75.55 79.49

Exhaustion refers to the degree of dye transfer from dye bath to fiber, usually expressed as percentage of the amount of dye originally placed in the dye bath to the dye adsorbed in capillaries of fibers. For economic and environmental reasons a high degree of exhaustion and fixation is required. Fixation of dye deals with the amount of dye fixed to the fiber of textile materials. The driving force for exhaustion is a concentration of dye in two phases and for fixation is the physical 98

as well as chemical interaction. In order to get a high degree of exhaustion auxiliary chemicals like NaCl, Na2SO4 and EDTA were added in the dye bath to improve exhaustion. Salts NaCl and Na2SO4 open the grains of cotton fibers and enhance the dye absorption and exhaustion of dyes. EDTA does so by trapping the calcium, magnesium and zinc ions, and prevents the dye precipitation. Triazine has been used as a reactive component to interact with cellulose fibers. Hydroxyl of cotton fibers (cellulose) in alkaline pH interacts with electron deficient carbon attached to chloro group and high temperature required to replace the third group and to establish covalent bonding. The percentage exhaustion [177] and percentage fixation of 2% dyeing on cotton ranges from 65- 75% and 75-92 % respectively as it is represented in the Table 5.3. All the dyes havd good exhaustion and fixation values which was expected due to the rapid diffusion of the dye molecule within the fabric under dyeing condition and physical as well as chemical interactions of polar groups present in disazo, coupler and bridging components. Reactive component establishes covalent linkages with fiber. Dyes 9a-b and 9j have high exhaustion and fixation values owing to the presence of carboxylic groups in the bridging anilines. Good exhaustion and fixation values of dyes are in accordance with the structure of dyes bearing polar groups which establish physical and chemical interactions with fibers. 5.3.2 Fastness Properties of Reactive Dyes (9a-j) Fastness properties of dyes were assessed after application of 2% dye with respect to cotton fibers as represented in Table 5.4. These were light fastness, wash fastness and rubbing fastness which provided the clear picture regarding quality of dye.

Table 5.4 Fastness properties data of the reactive dyes (9a-j) Dyes Light Fastness Wash Fastness Rubbing Fastness Dry Wet 9a 6-7 4-5 4 3

9b 6-7 4-5 4 3

9c 6-7 4 5 4

9d 5-6 4-5 4-5 3-4

9e 5-6 4-5 4-5 3-4

9f 5-6 3-4 4-5 3-4

9g 5-6 4 4 3-4

9h 5-6 4 4-5 3-4

9i 5-6 5 4-5 3-4

9j 5-6 4-5 4-5 3-4

Light fastness is the degree to which a dye resists fading due to light exposure. Different dyes have different degrees of resistance to fading by light. Light fastness of all dyes was high in the range 4-5. These dyes have little susceptibility to light damage, simply because their strong colors are indications that they absorb the wavelengths that they don’t reflect back. Light is absorbed by pigmented compounds may serve to degrade them. Wash fastness is the resistance offered by dyed fibers to retain color when washed by soaps and detergents. In the test, change in color of the textile and also staining of color on the adjacent fabric are assessed [178]. Wash fastness of dyes was in the range 5-6. Color Fastness to rubbing is a main test which is always required for every colored fabric either it is printed or dyed. Rubbing fastness was designed to determine the degree of color which may transfer from the surface of a colored fabric to a specified test cloth for rubbing. Rubbing fastness of all dyes was very high 4-5. Rubbing fastness is an indicator for other improved properties like wash fastness, substantively and durability in use. It was obvious from rubbing fastness value that all these dyes have high washing fastness and fixation on the cotton fibers.

5.4 Synthetic Pathway to Reactive Azo Dyes (10a-h)

The reaction sequences employed for the synthesis of the target compounds (10a-h) are illustrated in Scheme 5.3. Accordingly, 4-nitro-2-aminophenol (4-NAP) (2) was diazotized by treating with sodium nitrite in the presence of HCl at low temperature 0-5oC. The control of temperature and pH was essential; high temperature converts the azo group into N2 gas and OH group is introduced which not only decreases the yield but also affect the as quality of dye. Coupling of diazo of 4- NAP (7) with H-acid (3) in alkaline medium resulted the coupling at a position ortho to the hydroxyl group of H-acid. The dyes were salted out by the addition of 10% sodium chloride solution to the reaction mixture. Cyanuration of dye (8) was achieved by addition of dye to cyanuric chloride solution in ice bath under neutral conditions at molar ratio 1:1. Filtered, separated and dried the dye in oven at 50oC. Separately synthesized bis anilines (1k-r, Figure 5.5) were added to solution of dye at pH 4-5 at room temperature in molar ratio 1:2 to furnish compounds 10a-h. The diverse bis anilines used as bridging components for dyes did not affect the λmax but increased the substantivity of dye with fiber. Characterization data of dyes 10a-h are given in Table 5.5.

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Figure 5.5 Structures of methylene bisanilines (1k-r) used as linker

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Scheme 5.3 Synthetic route to monosazo MCT reactive Dyes (10a-h) Table 5.5: Characterization data of reactive dyes (10a-h) Dye Molecular Mol. Yield C% H% N% S% Rf* Formula Wt (%) Cal/Found Cal/Found Cal/Found Cal/Found Value

10a C51Cl6H30N16O20S4 1523 76 40.09/40.05 1.98/1.95 14.67/14.60 8.97/9.02 0.52

10b C51H32Cl2N18O26S4 1476 77 40.51/40.43 2.13/2.10 16.67/16.53 8.48/8.52 0.56

10c C53H34Cl2N16O24S4 1510 76 43.07/43.02 2.32/2.25 15.16/15.09 8.68/8.73 0.48

10d C51H32Cl2N18O24S4 1478 81 41.39/41.33 2.18/2.10 17.03/17.00 8.66/8.69 0.46

10e C53H38Cl2N16O22S4 1420 79 43.90/43.81 2.64/2.51 15.45/15.40 8.84/8.86 0.50

10f C53H34Cl2N16O24S4 1476 84 43.07/43.01 2.32/2.29 15.16/15.10 8.68/8.70 0.43

10g C51H32Cl4N16O20S4 1478 84 41.99/41.80 2.21/2.11 15.36/15.32 8.79/8.83. 0.45

10h C51H34Cl2N16O22S4 1487 81 43.08/43.03 2.41/2.35 15.76/15.56 9.02/9.10 0.44 Toluene: Ethyl acetate (7.5: 2.5 v/v), Silica gel-G F254 TLC plate. 5.5 Spectral Properties of Reactive Azo Dyes (10a-h) The absorption maxima (λmax) of the reactive azo dyes (10a-h) were taken in water and are presented in Table 5.6. The λmax values are directly related to the nature, electronic power and position of the substituents on the naphthyl ring of the coupler moiety as well as in the bridging anilines [179]. Dyes 10a and 10h exhibited two absorption maxima, one in the UV range due to π- π* transitions of aromatic moieties present in all dyes and other in the visible range at 557-570 nm. The absorption band which was present in the visible region was due to π-π* transition of azo linkage N=N of dyes conjugated with aromatic nuclei and was responsible for the reddish violet color of 10a-h dyes (Figure 5.6). All dyes had same chromophoric functionalities, but the difference was of bis-anilines used as bridging groups. These bridging groups affect the λmax of dyes very but the effect is not too high as these are not directly attached to chromophoric groups

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of dyes and these effects cannot be explained in regular way due to a diverse variety of groups in the bis anilines [180].

Figure 5.6 Combined UV-Visible Spectrum of MCT reactive dyes (10a-h) The values of log ε (molar extinction coefficient) are summarized in Table 5.6 and for dyes (10a- h) were in the range of 5.4 to 6.7. From the U.V analysis of dyes, it was gathered that the dyes containing electron withdrawing groups cause blue shift in absorption λmax while electron donating group produced red shift in absorption maximum. The infrared spectra of the synthesized Monoazo MCT reactive dyes based on H- acid showed absorption bands due to O-H, N-H, Ar-H, C-H, C=O, C=C, C=N, NO2 , SO3H, N=N and C-Cl is stretching vibrations at 3422-3526, 3048-3078, 2843-2965, 1735-1745, 1622-1663, 1565-1590, 1539-1505, 1427-1481, 1120-1070, 672-690 cm-1 respectively as depicted from their FTIR spectra [181, 182]. Specifically speaking, using FTIR spectrum of 10c and 10f a broad band was observed in the range 3200-3526 cm-1 which was due to H-bonding of OH and COOH groups present in the bridging anilines. This broadband masked the peaks of N-H functionality due to its broadness and provided conclusive evidence regarding carboxylic group in dye molecules. A peak was observed in the range 1735-1745 cm-1 which was due to the C=O functionality of dyes. The absorption bands at 1622-1663, 1565-1590, and 1539-1505 cm-1 depicted the presence of C=C stretching vibrations and bending vibrations at 770-723 cm-1 due to aromatic rings. The peak for C-Cl bond was observed in all dyes at 672-690 cm-1 which confirmed the triazine ring system in dyes. Azo linkage is inveterated by the peaks in the range 1539-1505 cm-1. Dyes 10b and 10h had bridging anilines containing OH groups at o- and p-position to NH group of diamines, they show a broad peak at 3422-3526 cm-1which is due to OH and N-H stretching vibrations. Similarly reactive dye 10e showed a peak at 3465 cm-1due to N-H stretching vibrations. This also showed a prominent peak at 1055 cm-1 due to ether stretching vibrations, as it contained methoxy group at o-position to N-H. The dyes 10b and 10d had NO2 peaks at mposition to NH group so a peak at 1550-1560 cm-1 was present in their IR spectra. The compounds 10a and 10g had bridging groups containing chloro groups at o, p and m-position to NH group, so C-Cl was observed at 710-720 cm-1 in addition to C-Cl absorption of the triazine system. An absorption band at 2843-2965 cm-1 was common in all dyes and was due to a methylene group symmetric and asymmetric stretching vibrations [183].

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The 1H-NMR spectrum of dye 10c showed down field signal at 12.74 ppm due to COOH groups present in the bridging diamines and in the aromatic region of the TMS scale in between δ7.158.10 ppm due to eighteen aromatic protons. Broad singlets were observed at 9.40-9.46, 8.61-8.75 and 8.30-8.38 ppm because of O-H and NH groups attached to naphthalene ring and benzene ring, and these peaks are common in all dyes. At range 1.785-2.05 ppm sharp singlet was observed due to bridging CH2 group present in bis-anilines. All these dyes 10a-h are compounds of a series where the difference occurred in case of bridging groups while chromophores, coupling component and reactive systems were same. Here peak positions and intensity was varied for O-H and bridging methylene protons. In case of compounds 10a, 10b, 10c and 10d which contained NO2 and Cl at o, m- and p-position to NH group of bis-anilines, methylene as well as N-H peaks shifted downfield but for dyes 10e and 10h which had OCH3 and OH groups at o position to N-H moiety, their signal shifted upfield and extra peak for 3H in 10e was seen at 4.07 ppm. 13C-NMR spectra of dyes (10a- h) showed signals for different aliphatic carbons at 4050 and for aromatic carbons in the range 109-160 ppm which confirmed the synthesis of these compounds. 5.6 Dyeing properties of Reactive Azo Dyes (10a-h) All the dyes (10a-h) were applied at 2.0 % depth on cotton fibers according to the usual procedure in the dye bath containing materials to achieve dominant and attractive shades of targeted dyes. 5.6.1 Exhaustion and fixation study of Reactive Azo Dyes (10a-h) Exhaustion and fixation study was made by the application of dyes at 2% dyeing on cotton fibers. Exhaustion and fixation values are shown in Table 5.6.

Table 5.6 Exhaustion and fixation data of reactive dyes (10a-h) Dyes Shade on Fiber λmax (nm) Log ε % Exhaustion % Fixation

in H2O 10a Reddish violet 564 6.704 62.17 70.23 10b Reddish violet 567 6.443 72.33 87.53 10c Reddish violet 560 6.373 76.67 92.88 10d Reddish violet 557 6.043 63.45 81.23 10e Reddish violet 570 5.410 63.15 76.43 10f Reddish violet 568 5.758 77.53 93.15 10g Reddish violet 560 6.219 65.52 78.37 10h Reddish violet 568 6.042 74.38 83.47

The percentage exhaustion and percentage fixation of 2% dyeing on cotton ranged from 62-77% and 70-93 % for 10a-h, as it is represented in the Table 5.6. Good exhaustion and fixation values had been observed for all the dyes which were expected, owing to the fast diffusion of the dye molecule within the fabric under dyeing condition and physical as well as chemical interactions of

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polar groups present in disazo, coupler and bridging components. A reactive component established covalent linkages with fiber. Dyes 10d and 10f had high exhaustion and fixation values in effect of the presence of carboxylic groups in the bridging anilines. 5.6.2 Fastness properties of Reactive Azo Dyes (10a-h) Fastness study of dyes was made after application of 2% dye with respect to cotton fibers as represented in Table 5.7. Light fastness, wash fastness and rubbing fastness were examined which provided the real image regarding the quality of dye.

Table 5.7 Fastness properties data of reactive dyes (10a-h) Dyes Light Fastness Wash Fastness Rubbing Fastness Dry Wet 10a 5-6 4-5 4 3 10b 6-7 4-5 4 3 10c 6-7 4 5 4 10d 5-6 4-5 4-5 3-4 10e 5-6 4-5 4-5 3-4 10f 6-7 3-4 4-5 3-4 10g 5-6 4 4 3-4 10h 5-6 4 4-5 3-4

Light fastness is related to the resistance offered by the dye to fading on exposure to light. Different dyes exhibited different degrees of confrontation to fading by light. Light fastness of all dyes was high in the range 6-7. These dyes have little susceptibility to light damage, simply because their strong colors are indications that they absorb the wavelengths that they don’t reflect back. Wash fastness is the resistance offered by dyed fibers to retain color when washed by soaps and detergents. In the test, change in color of the textile and also staining of color on the adjacent fabric are assessed. Wash fastness of dyes was in the range 4-5. Color fastness to rubbing is a main test which is always required for every colored fabric either it is printed or dyed. Rubbing fastness was designed to determine the degree of color which may 105

transfer from the surface of a colored fabric to a specified test cloth for rubbing. Rubbing fastness of all dyes was very high 4-5. Rubbing fastness is an indication of other improved properties like wash fastness, substantively and durability in use. It is obvious from rubbing fastness value that all these dyes have high washing fastness and fixation on the cotton fibers. High light fastness, wash fastness and rubbing fastness indicate that dyes have established permanent covalent bond with cotton fibers instead of physical adsorption and this provides the evidence in favour of reactive functioning of dyes. 5.7 Synthetic Pathway to Reactive Azo Dyes (15a-h) The synthesis of bisazo monochloro triazine (MCT) reactive dyes (15a-h) was conducted in accordance with the Scheme 5.4. Here 15a-h reactive dyes were synthesized using 7-amino- 1naphthol-3-sulphonic acid (γ-acid) coupler and 4-aminobenesulfonic (sulfanilic acid) as monoazo component. Accordingly, Sulfanilic acid (11) was diazotized by treating it with Sodium nitrite and HCl at low temperature 0-5oC. Conditions were kept same as discussed above during diazotization. Coupling of tetrazo of sulfanilic acid with γ-acid (12) was achieved to afford (13) in alkaline medium to accomplish the coupling at position ortho to the hydroxyl group of γ-acid at 0-5oC. Addition of diazo was carried out to well stirred solution of γ-acid at pH 8.5-9.0 which was adjusted with a mild base like Na2CO3. The coupling was completed in 3-4 h as determined from TLC and paper chromatography. The dye was salted out by the addition of 7-8% Sodium chloride to dye solution at room temperature. Cyanuration of dye (13) was done by addition of dye to cyanuric chloride solution in an ice bath at pH 7.0-7.5 in 1:1 molar ratio [184, 185]. Cyanurated dye was separated and dried in oven at 50oC. Bis aniline (1k-r) were added to solution of dye at pH 4-5 at room temperature in molar ratio 1:2 to furnish (15a-h) dyes. Different bis anilines were used as bridging component for dyes to enhance the adherence of dye with fiber (Scheme 5.4) without affecting too much on dye shades and hues. The synthesized dyes 15a-h were investigated for elemental analysis and percentages of C, H, N and S in these derivatives provided evidence for prepared compounds (Table 5.8).

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Scheme 5.4 Synthetic route to monoazo MCT reactive dyes (15a-h)

Table 5.8 Characterization data of dyes (15a-h)

Dye Molecular Formula Mol. Yield% C% H% N% S% Rf* Wt Cal/Found Cal/Found Cal/Found Cal/Found Value

15a C53H36Cl2N14O18S4 1401 73 46.94/46.23 2.68/2.54 14.46/14.40 9.46/9.50 0.41

15b C51H34Cl2N16O18S4 1401 76 45.11/44.95 2.52/2.30 16.50/16.35 9.44/9.51 0.53

15c C51H34Cl4N14O16S4 1388 77 44.75/44.20 2.50/2.32 14.32/14.15 9.37/9.40 0.52

15d C53H40Cl2N14O16S4 1358 80 47.93/47.11 3.04/2.99 14.77/14.50 9.66/9.71 0.45

15e C51H34Cl4N14O14S4 1378 79 45.82/45.35 2.56/2.16 14.67/14.45 9.59/9.64 0.48

15f C51H32Cl6N14O14S4 1326 84 43.57/43.10 2.29/2.23 13.95/13.34 9.12/9.20 0.44

15g C51H32Cl6N14O14S4 1354 85 43.57/43.17 2.29/2.20 13.95/13.35 9.12/9.25 0.45

15h C51H36Cl2N14O16S4 1365 80 47.12/47.05 2.79/2.74 15.08/14.95 9.86/9.95 0.56 *Toluene: Ethyl acetate (7.5: 2.5 v/v), Silica gel-G F254 TLC plate. 5.8 Spectral properties of Reactive Azo Dyes (15a-h) The absorption maxima (λmax) of the dyes (15a-h) were taken in water and are presented in Table 5.9. The λmax values were honestly interrelated to the nature, electronic power and location of the substituents on the naphthyl ring of the coupler moiety as well as in the bridging anilines. Dyes (15a-h) exhibited two absorption maxima, one in the UV range due to π-π* transition of C=C present in the aromatic moiety common in all dyes and other in the visible rang at 557 to 570 nm. The absorption band which lies in the visible region is due to π-π* transition of azo linkage N=N of dyes was responsible for the reddish violet color of this series dyes (Figure 5.7). These bridging groups affected the λmax of dyes but the effect was not too high as these were not directly attached to chromophoric groups of dyes and these group effects could not be explained in regular way due 107

to a diverse variety of groups in the bis anilines. Similarly, dyes (15a-h) showed three absorption maxima in the mid UV, far UV and in the visible region of electromagnetic radiation spectrum (Figure 5.7).

Figure 5.7 Combined UV-Visible spectrum of MCT reactive dyes (15a-h) The values of log ε (molar extinction coefficient) are summarized in Table 5.9 for dyes 15a-h and are in the range of 6.0 to 6.4, which showed the high absorption intensity of dyes. From the U.V. visible analysis of dyes, it was investigated that the electron withdrawing groups as well as electron donating groups affect the molar extinction coefficient and λmax of compounds. From the infrared spectra of the synthesized reactive dyes based on γ- acid, sulfanilic acid and cyanuric chloride showed absorption peaks owing to O-H, N-H, Ar-H, CH2, C=O, C=C, C=N, NO2, SO3H, N=N and C-Cl stretching vibrations at 3435-3467, 3032-3072, 2838-2963, 1755, 1625-1653, 1568-1589, 1512-1526, 1433-1465, 1125-1070, 680-689 cm-1. Dyes (15a-h) had OH and N-H groups; they showed a broad band at 3435-3467 cm-1 due to OH and N-H stretching vibrations. All dyes had sulfonic groups which were confirmed by the appearance of absorption bands at 1070-1123 cm-1. Dye 15d exhibited prominent peak at 1055 cm-1 due to C-O-C stretching vibrations, as it carried methoxy group at o-position to N-H. The compound 15b had NO2 peaks at the m- position to the NH group so a peak at 1565 cm-1 was present in their IR spectrum. An absorption band at 2843-2965 cm-1 was common in all dyes and was due to methylene asymmetric stretching vibrations. In case of 1H-NMR spectra of (15a-h), 15a exhibited signal at 13.11 ppm due to COOH groups present in the bridging diamines and the aromatic protons signals in between 6.77-8.08 ppm due to twenty six aromatic protons. Broad singlets were observed at 9.41, 8.40 and 8.33 ppm because of O-H and N-H groups attached to naphthalene ring and benzene ring, and these peaks were common in all dyes (15a-h). At range 1.85-2.10 ppm sharp singlet was observed due to the bridging CH2 group present in the bis-anilines. All these dyes 15a-h are compounds of a series based on sulfanilic acid, γ-acid and cyanuric chloride which were different only in bridging groups. In case of dyes 15b, 15f and 15g, which bore NO2 and Cl at o, m- and p-position to NH of bis-anilines, the signals for methylene as well as N-H groups shifted downfield (Figure 5.8) and in case of compounds 15c and 15e which had OCH3 and OH groups at o positions to N-H groups, their peaks shifted upfield. In dye 15c three protons attached to oxygen were verified from signal at 4.05 ppm

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and the presence of methoxy group was confirmed from spectrum. In 13C-NMR spectra of reactive dyes (15a-h), signals for dye 15c were present in the aliphatic as well as aromatic regions of spectrum at positions 46.6, 99.5, 101.4, 103.75, 116.2, 119.2, 123.8, 126.6, 130.4, 132.0, 133.9, 142.2, 143.3, 152.3, 154.9, 158.8, 170.4 ppm which confirmed the synthesis of dye as depicted from Figure 5.9. In this way other dyes were confirmed for their predicted structures from 13C- NMR spectra.

Figure 5.8 1H-NMR spectrum of reactive dye 15c

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Figure 5.9 13C-NMR spectrum of reactive dye 15c

2.9 Dyeing properties of Reactive Azo Dyes (15a-h) All the dyes 15a-h were applied at 2.0 % depth on cotton fibers according to the usual procedure in the dye bath containing materials to achieve dominant and attractive shades of targeted dyes. 2.9.1 Exhaustion and Fixation Study of Reactive Azo Dyes (15a-h) Exhaustion and fixation study was made by the application of dyes at 2% dyeing on cotton fibers. Exhaustion and fixation values are shown in Table 5.9 [186]. Table 5.9 Exhaustion and fixation data of the dyes 15a-h Dye Shade on Fiber λmax (nm) % Exhaustion % Fixation Log ε in H2O 15a Reddish yellow 502 6.36 75.38 91.93 15b Reddish yellow 496 6.394 66.31 78.24 15c Reddish yellow 496 6.229 69.61 83.68 15d Reddish yellow 492 6.344 68.86 80.19 15e Reddish yellow 498 6.439 63.12 74.18 15f Reddish yellow 492 6.093 62.44 77.75 15g Reddish yellow 495 6.375 62.78 76.69 15h Reddish yellow 499 6.397 74.16 84.29

The percentage exhaustion and percentage fixation of 2% dyeing on cotton ranged from 62-75 and 74-91% for 15a-h as it is represented in the Table 5.9. Moderate exhaustion and fixation values had been observed for all the dyes which were due to the fast diffusion of the dye molecule within the fabric under dyeing condition and the involvement of van der waal’s, as well as electrostatic interactions due to polar and non polar groups present in disazo, coupler and bridging components. The reactive component generated covalent bonding with fiber. Dye 15c had high exhaustion and fixation values due to the presence of carboxylic groups in the bridging anilines [187]. 5.9.2 Fastness properties of Reactive Azo Dyes (15a-h) Fastness study of dyes was made after application of 2% dye with respect to cotton fibers as represented in Table 5.10. Light fastness, wash fastness and rubbing fastness were examined which provided the real image regarding the quality of dye [188].

Table 5.10 Fastness properties data of the dyes 15a-h Dye Light Fastness Wash Fastness Rubbing Fastness

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Dry Wet 15a 6-7 4-5 4-5 3 15b 5-6 4-5 3-4 3 15c 5-6 4 3-4 3 15d 5-6 4-5 4 3-4 15e 5-6 4-5 4 3-4 15f 5 3-4 3-4 3-4 15g 5 3-4 3-4 3-4 15h 5-6 3-4 4 3-4

Light fastness is related to resistance offered by dye to fading on exposure to light. Different dyes exhibited different degrees of confrontation to fading by light. Light fastness of all dyes was high in the range 6-7. These dyes have little susceptibility to light damage, simply because their strong colors are indications that they absorb the wavelengths that they don’t reflect back. Wash fastness is the resistance offered by dyed fibers to retain color when washed by soaps and detergents. In the test, change in color of the textile and also staining of color on the adjacent fabric are assessed. Wash fastness of dyes was in the range 4-5. Color fastness to rubbing is a main test which is always required for every colored fabric either it is printed or dyed [189]. Rubbing fastness of all dyes was very high 4-5. Rubbing fastness provided evidence for the other improved properties of dyes like wash fastness, substantively and durability in use. It was obvious from rubbing fastness value that all these dyes had high washing fastness and fixation on the cotton fibers. High light fastness, wash fastness and rubbing fastness indicated that dyes had established permanent covalent bond with cotton fibers instead of physical adsorption and these properties provided proof in favour of reactive functioning of dyes. 5.10 Synthetic Pathway to Heterocyclic Dyes (19a-i) The synthetic strategy adopted for the synthesis of the intermediate and targeted compounds (19a- i) is depicted in Scheme 5.5. Accordingly, 4-aminoacetanilide (16) was diazotized by treating with sodium nitrite in the presence of acid at a low temperature 0-5oC. The control of temperature and pH was found to be crucial essential as high temperature not only decreases the yield but also affects the quality of dye. The coupling of diazo of 4- aminoacetanilide with different active methylene compounds in alkaline medium resulted in the attachment of diazo to methylene carbon through a nucleophilic attack of active center to furnish the intermediates (17a-d). In the next step, the condensation of intermediate, dyes (17a-d) with separately synthesized various substituted hydrazines (18a-d) was achieved by refluxing the reaction mixture in ethanol at 80oC for five hours to afford the target compounds (19a-i) in high yields.

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Scheme 5.5 Synthetic pathway to heterocyclic direct dyes (19a-i). 5.11 Spectral Properties of Heterocyclic Dyes (19a-i) The absorption maxima (λmax) of the dyes (19a-i) were taken in ethanol and results are presented in the Table 5.11. The absorption maxima (λmax) of dyes depend upon the nature, position and electronic power of the substituents present in the coupler as well as a diazo component of dyes. All dyes exhibited only one absorption maximum in the visible region in the range 387-401 nm due to π-π* transition of azo linkage present in all dyes which imparted them yellow color (Figure 5.10). All dyes have same chromophoric functionalities, but the difference lies in the different hydrazines used for condensation. Different substitution patterns present in hydrazines affect the λmax of dyes, but the effect was small as affecting groups were away from chromophoric groups of dyes. Dye 19i showed orange yellow color which had λmax 401 nm while other dyes 19a-h absorption maxima (λmax) were very close to each other and they were of yellow color.

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Figure 5.10 Combined U.V Visible Spectrum of Heterocyclic azo Dyes (19a-i) The values of log ε (molar extinction coefficient) are summarized in Table 2.11 ranging from 4.10 to 4.60 indicating the high absorption intensity of the dyes. The infrared spectra of the synthesized heterocyclic azo dyes showed general characteristic bands at 3400-3500 and 32553340 cm-1 which indicate the O-H and N-H stretching vibrations due to the presence of these groups in the dyes. The band at 3035-3132 cm-1 and 2858-2950 cm-1depicted the presence of CH stretching vibrations of aromatic as well as aliphatic C-H bonds. Presence of carbonyl groups was evidenced by the band in the range 1675-1725 cm-1. Amide carbonyl showed absorption below 1700 cm-1while carboxylic carbonyl exhibited peaks near 1725 cm-1. The Broad peaks at 3450-3500 cm-1in case of 19b, 19c and 19d were due to intramolecular H-bonding interactions of NH and COOH groups present in dye in close proximity to each other. The absorption bands at 1610-1659, 1570-1590, 1511-1530, 1420-1440 and 1145-1200 cm-1 depicted the presence of aromatic rings, N=N and C- O respectively [190,191]. The presence of the sulfonic groups in dyes 19a and 19h was observed by the appearance of the peaks at 1250-1255 cm-1 in their FTIR spectra. Similarly the peaks for different functional groups in the FTIR spectra of remaining dyes provided evidence in favor of their structure. In case of dye 19f the IR peaks were present at 3303, 3153, 2881, 1696, 1607, 1548, 1515, 1427, 1320, 1176 cm-13303, 3153 due to presence of C=C-H str, C-H str, C=O str, C=C str, N=N str, C-H bend, C-O str vibrations in the molecule (Figure 5.11).

Figure 5.11 FTIR spectrum of heterocyclic dye 19f The 1H-NMR spectrum of all dyes (19a-i) showed a peak at 14.75-14.85 ppm due to amide NH being present in all dyes. Except for dyes 19c and 19g which exhibited one amide peak all others showed two amide peaks the second appearing in the range 11.40-11.47 ppm. Presence of hydroxyl of the carboxyl group in dyes 19b-d and 19i was evidenced by the peaks in region 12.55-13.11 ppm. Sulfonic group in dyes 19a and 19h was confirmed by the broad singlet peak at 8.52-8.55 ppm. A characteristic feature in all dyes was the presence of two symmetric doublets in region 7.53-7.70 ppm due to benzene ring of azo motif. Similarly, other aromatic rings in dyes were confirmed from multiplets at 7.30-7.50 ppm. Two methyl groups were evidenced by singlet at 113

2.20-2.26 ppm and 2.50-2.62 ppm, while dyes 19c and 19g showed three singlets the third methyl resonating at 2.50-2.52 ppm (Figure 5.12). The 13C-NMR spectra of all dyes (19a-i) exhibited signals for imine and carbonyl carbons at 155-157 and 166-167 ppm while the extra peak for carbonyl of carboxyl group was present in 19b-d and 19h at 168.5-170 ppm. The signals at 22-24 and 25-26 ppm were common for methyl groups in all dyes (Figure 5.13).

1 Figure 5.12 H-NMR spectrum of heterocyclic dye 19f in CDCl3

13 Figure 5.13 C-NMR spectrum of heterocyclic dye 19f in CDCl3 5.11.1 Dyeing Properties of Heterocyclic Dyes (19a-i) All the heterocyclic dyes (19a-i) were applied at 2.0% depth on the leather fabric according to the usual procedure to determine the level of fastness displayed by the heterocyclic azo dyes.

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5.11.2 Exhaustion and Fixation Study of Heterocyclic Dyes (19a-i) Exhaustion and fixation study of dyes was made from the absorbance measurements of the original dye bath and the exhausted dye bath. Exhaustion and fixation values are shown in Table 5.11. Table 5.11 Exhaustion and fixation data of heterocyclic azo dyes 19a-i Dye Shade on λmax( nm) Log ε Exhaustion % (C) Fixation % (C)

Leather in H2O 19a Yellow 388 4.54 80.15 93.33 19b Yellow 389.5 4.36 82.55 87.77 19c Yellow 390 4.60 78.19 86.73 19d Yellow 389 4.45 79.45 89.47 19e Yellow 387 4.38 20.15 10.43 19f Yellow 387.5 4.27 21.23 12.38 19g Yellow 391.5 4.32 25.57 13.52 19h Yellow 395.5 4.31 85.59 93.89 19i Orange 401 4.10 86.73 90.25

The percentage exhaustion and percentage fixation of 2% dyeing on leather ranged from 20-86% and 10-93% respectively for dyes 19a-i. Good exhaustion and fixation values have been observed for dyes which have carboxylic and sulfonic acid groups. Dyes 19e-f showed very small values of exhaustion and fixation as compared with other dyes. These dyes have no functional groups which are suitable to develop electrostatic interactions with leather. Dyes 4a and 4h showed high values these parameters due to the fast molecule of the dye molecule within the fabric under dyeing condition and development of physical as well as chemical interactions of polar functional groups present in dye molecules as these contain sulfonic groups. Dyes 19b-d and 19i showed comparatively moderate to good values of exhaustion and fixation, but less than 19a and 19h. It appeared that dyes containing sulfonic groups were more suitable for dyeing leather as compared with carboxylic groups. In figure 5.14 it was shown the application of heterocyclic dyes 19a-i on leather.

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Figure 5.14 Application of Heterocyclic dyes (19a-i) on leather 5.11.3 Fastness Properties of Heterocyclic Dyes (19a-i) Fastness study of dyes was made after application of 2% dye with respect to leather, fabric as represented in Table 5.12. Light fastness, wash fastness and rubbing fastness was examined which provided the real image regarding the quality of dye. Table 5.12 Fastness properties data of heterocyclic azo dyes (19a-i) Dye Light Fastness Wash Fastness Rubbing Fastness Dry Wet 19a 5-6 4-5 5 4 19b 5-6 4-5 4 3 19c 5-6 4-5 5 4 19d 5-6 4-5 4-5 4 19e 4-5 4 4 3 19f 4-5 4 4 3 19g 4 4-5 4 3 19h 5-6 4-5 4-5 3-4 19i 5-6 4-5 4-5 3-4 Light fastness determines the resistance accessible by the dye to fading on exposure to light. Different dyes exhibited different degrees of confrontation to fading by light. Light fastness of 116

approximately all dyes was high in the range 5-6 except for that of 19e-f. These dyes had little propensity to light damage, plainly because of their strong yellow colors which were indications of their absorption of light at wavelengths where they were not reflecting back. In the wash fastness test, change in color of the textile and also staining of color on the adjacent fabric were assessed. Wash fastness of dyes was in the range 4-5. Color fastness to rubbing is a focal test which is always required for every colored fabric either it is printed or dyed [192, 193]. Rubbing fastness was designed to determine the degree of color migration from the surface of a dyed fabric to a specified test cloth for rubbing. Rubbing fastness of all dyes was excellent in the range 3-4. Rubbing fastness was dependent upon the physical interaction of dyes with fabric. It was obvious from rubbing fastness value that all these dyes had high washing fastness and fixation on the cotton fibers. 5.12 Synthesis of Calix [4] resorsoniarene Azo Dyes (23a-f) Synthesis of these azo dyes was achieved in three steps following the Scheme 2.6, which involved the preparation of calixarene, diazotization and coupling to obtain the desired products (23a-f). Calixarene synthesis was done by reacting the formaldehyde with resorcinol in the presence catalytic amount of HCl. This reaction occurred through Friedel Crafts acylation mechanism. Hydrochloric acid protonated the formaldehyde and increased the electrophilicity of carbonyl carbon, and thereby facilitated the attack of nucleophilic carbon ortho to hydroxyl of resorcinol. There were two nucleophilic carbons in resorcinol and both reacted with formaldehyde to produce calix involving four units of each reactant. Diazonium salts of different derivatives of o-amino phenols (22a-f) were prepared separately according to the standard diazotization procedure. Dyes (23a-f) were obtained by coupling of the synthesized diazonium salts with calix [4] resorsoniarene (21) with 22a-f, in basic medium at room temperature. The coupling was achieved in alkaline aqueous solution to increase the nucleophilicity of calix. In this way a series of compounds were prepared [194-198].

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Scheme 5.6 Synthesis of Calix [4] azo dyes (23a-f) 5.13 Characterization of Calix [4] resorsoniarene Azo Dyes (23a-f) Characterization of these calix [4] resorsoniarene azo dyes was made with the help of UV-Vis, FTIR and NMR studies of dyes. Dyes exhibited absorptions in the range 400-500 nm (Figure 5.15) in the visible region of electromagnetic spectrum. These absorption were due to π-π* of electrons which are the part of the conjugated framework of dye molecules. Two absorption maxima were present in dye 23f which may be attributed to two distinct chromophores showing absorptions at different positions. FTIR spectra of these compounds 23a-f provided evidence for the presence different functional groups within dye molecules. Hydroxyl, azo and aromatic motifs are common in all dyes which are being proved from their IR absorptions at specific positions. Hydroxyl group produced broad range absorptions at 3200-3500 cm-1 due to extended hydrogen bonding in dyes. Azo groups in dye molecules were confirmed from their peaks at 1500-1530 cm-1 which were common in all dyes (23a-f). Presence of peaks at 3050-3070, 1535, 1580 and 1610 cm-1 were due to C=C-H, and C=C stretching vibration of aromatic nuclei. Dyes 23c and 23e-f exhibited strong absorptions at 1240- -1 1260 cm due to SO3H group present in these dyes. 1H-NMR and 13C-NMR study of dyes 23a-f confirmed the synthesis of targeted compounds. In case of compound 23a, there were broad peaks in 1H-NMR at 10.14 and 6.35 ppm due to presence OH groups in the azo component and calix coupler respectively. Two mutually coupled protons in the diazo component showed doublet peak at 7.20 and 8.22 ppm. The downfield singlet peak at 8.77 ppm was due to proton ortho to nitro group in the diazo component. One proton peak at 6.97 ppm was because of calix resorsoniarene proton while two protons signals at 3.81 ppm were due

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to methylene protons sandwiched between resorcinol moieties of calix [4] resorsoniarene. 13C- NMR of 23a showed signals at 118.32, 130.90, 138.24 and 155.12 ppm due to calix aromatic nucleus carbons while the signals of carbons at 116.65, 119.55, 126.62, 139.72, 143.74 and 163.30 ppm confirmed the presence of an aromatic nucleus of diazo component. Singlet signal at 31.3 ppm was due to methylene carbons of calix. Similarly, other dyes 23b-f have been confirmed from their 1H-NMR and 13C-NMR spectra.

Figure 5.15 Combined UV Visible spectrum of calix [4] resorsoniarene azo dyes (23a-f)

5.14 Applications of Calix [4] resorsoniarene Azo dyes (23a-f )

These dyes had hydroxyl groups at ortho to azo linkage at four sides of the molecule which were capable to act as ligands. So these dyes were tested for detection of different metal ions like Cu (+2), Fe (+2), Co (+2), Ni (+2) and Cr (+3) in their aqueous solutions. All dyes showed valuable interactions with these metals which were studied with the help of UV visible spectrophotometer. All metal ions on interaction with dyes exhibited bathochromic shifts, but the largest change was seen for Cu (+2) ions in the absorption of visible light and color of the complex from the original dye (Figure 5.16 and 5.17). The color change was clearly observed by naked eye. This metal ion study of calix [4] resorsoniarene azo dyes enable them to be used as sequestrants for metal ions in their aqueous and alcoholic solutions [199, 200].

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Figure 5.16 Most probable metal ion interaction mechanism of calix [4] resorsoniarene azo dyes (23a-f)

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Figure 5.17 Study of different metal ion interactions with Calix [4] resorsoniarene azo dyes (23a- f)

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Chapter 6 EXPERIMENTAL

6.1 Materials 1-Amino-8-hydroxy-naphthalene-3,6-disufonic acid (H-acid), 4'4-diamino diphenylamine- 2sulfonic acid (F.C-acid), 7-Amino-1-hydroxy naphthalene-3-sulfonic acid (γ- acid), 4Aminobenzene sulfonic acid, 4-Nitro-2-aminophenol, 4-Chloro-2-aminophenol, 2- Aminobenzoic acid, 3-Aminobenzoic acid, 4-Aminobenzoic acid, 3-Chloroaniline, 2- Aminophenol, 4Aminophenol, 3-Nitroaniline, 2-Methoxyaniline, 4-N,N- Dimethylaminobenzaldehyde, resorcinol, 1-amino-2-naphthol-6-nitro-4-sulfonic acid, 1-Amino- 2-naphthol-4-sulfonic acid, 3amino-4-benzene sulfonic acid, sodium nitrite, sodium hydroxide, sodium carbonate, formaldehyde were obtained from Daejing Korea. 4-Aminoacetanilide, acetoacetic acid, ethyl acetoacetate, acetyl acetone, acetoacetanilide, phenyl hydrazine, 4- hydrazinylbenzenesulfonic acid, 4-hydrazinylbenzoic acid, 2-hydrazinylbenzoic acid were purchased from Sigma Aldrich. Hydrochloric acid was purchased from Merck. Solvents such as ethanol, ethyl acetate, dichloromethane, DMSO, DMF, acetone, chloroform, and methanol were common laboratory grade chemicals and were purified before use. 6.2 Purification of Solvents Standard methods and procedures were followed for the purification and drying of solvents. The dried solvents were stored over type 4A° molecular sieves. Same solvents were used as mentioned at page 55-56. 6.3 Instrumentation Melting points were determined using digital Gallenkamp (Sanyo) model MPD BM 3.5 with digital thermometer and are uncorrected. Infrared spectra were recorded using a Shimadzu IR 460 as KBr pellets and FTX 3000 MX spectrophotometer using ATR method. 1H NMR and 13C NMR spectra were obtained using a Bruker Avence (300 MHz,) and (400 MHz,) spectrophotometers in CDCl3, DMSO-d6, CD3OD-d4 solution using TMS as an internal reference. Chemical shift are given in δ- scale (ppm). Abbreviations s, d, dd, t, at, m have been used for singlet, doublet, double doublet, triplet, apparent triplet, multiplet respectively. Elemental analyses were performed on CHNS 932 LECO instrument. UV-Vis spectra were taken by CECIL-7400 UV/Visible Spectrophotometer.

6.4 Chromatographic Techniques 6.4.1 Thin Layer Chromatography (TLC) Same procedure and mobile phases were used to monitor the progress of reactions as discussed at pages 55-56. 6.5 Experimental Procedures 6.5.1 (a) General Procedure for the Synthesis of Bisanilines (1k-t) Suitably substituted aniline (0.01mol) was dissolved in water (12.5 ml) and 36.5% hydrochloric acid (2.5 ml) at 50°C. The reaction mixture was then reacted with 3% aqueous formaldehyde (3.5 ml) solution at 60°C with stirring for 1 hour and neutralized with 10% sodium hydroxide solution. Precipitates obtained, were filtered, washed with hot water, dried and recrystallized from acetic acid. By adopting the above procedure bis anilines (1k-t) were synthesized. 5, 5'-Methylenebis (2-aminobenzoic acid) (1k) Yellowish orange solid (80%) m.p. 143-145°C.Rf = 0.75 (EtOAc: Pet Ether, 4: 1 v/v). FTIR (KBr) cm-1: 3500 (br COOH) 3420, (N-H), 3077(C=C-H Aromatic) 2858 (C-H aliphatic), 122

1701(C=O) 1666, 1614, 1586 (C=C benzene ring) 1480 (N-H bend.), 781 (o-disubstituted

1 Aromatic ring). H-NMR (300 MHz, DMSO-d6) δ (ppm): 12.07 (2H, br, s), 7.97 (2H, s), 7.36 (2H, d, J=7.8Hz), 6.96 (2H, d, J=7.8Hz), 6.74 (4H, br, s), 3.54 (2H, s). 13C-NMR (75 MHz,

DMSO-d6) δ (ppm): 170.40, 147.30, 134.51, 132.34, 130.49, 114.94, 112.43, 42.38. 5, 5'-Methylenebis (3-aminobenzoic acid) (1l) -1 Yellow solid (82%) m.p. 172-173°C, Rf = 0.73 FTIR (KBr) cm : 3600-3500 (br COOH) 3417, (N- H), 3023 (C=C-H Aromatic) 2830 (C-H aliphatic), 1700(C=O) 1670, 1631, 1587 (C=C benzene ring) 1488 (N-H bend.), 896, 835, 746 (C=C bending, Aromatic ring). 1H-NMR (300

MHz, DMSO-d6) δ (ppm): 11.95 (1H, br, s), 7.50 (2H, s), 7.23 (2H, s), 7.00 (2H, s), 5.12 (4H, br, 13 s ), 3.77 (2H, s). C-NMR (75 MHz, DMSO-d6) δ (ppm): 167.50, 146.97, 142.88, 131.44, 119.62, 119.50, 116.00, 42.74. 5, 5'-Methylenebis (4-aminobenzoic acid) (1m) -1 Dark yellow solid (81.5%) m.p. 245-247°C, Rf = 0.70 FTIR (KBr) cm : 3600-3500 (br COOH) 3417, (N-H), 3023 (C=C-H Aromatic) 2840 (C-H aliphatic), 1710 (C=O) 1665, 1620, 1590 (C=C benzene ring) 1488 (N-H bend.), 890, 828, 740 (C=C bending, Aromatic ring. 1H-NMR (300 MHz, DMSO-d6) δ (ppm): 12.12 (1H, br, s), 7.69 (2H, s), 7.26 (2H, d, J=7.86Hz), 6.90 (2H, d, 13 J=7.86Hz), 5.60 (4H, s), 3.41 (1H, s). C-NMR (75 MHz, DMSO-d6) δ (ppm): 167.79, 154.26, 133.34, 128.68, 124.84, 120.98, 115.51, 34.71. 2, 2'-Methylenebis (4-aminophenol) (1n) -1 Dark brown Solid (78%) m.p. 220-221°C, Rf0.67 FTIR (KBr) cm : 3600-3500 (br COOH) 3474, (N-H), 3023 (C=C-H Aromatic) 2825 (C-H aliphatic), 1665, 1603, 1514 (C=C benzene ring) 1471 1 (N-H bend.), 845, 833,(C=C bending, Aromatic ring). H-NMR (300 MHz, DMSOd6) δ: 9.53-9.50 (1H, br, s), 6.65 (2H, s), 6.46 (2H, d, J=7.75Hz), 6.42 (2H, d, J=7.75Hz), 4.84 13 (4H, s), 3.76 (1H, s). C-NMR (75 MHz, DMSO-d6) δ (ppm): 145.67, 141.53, 129.05, 118.97, 117.25, 115.77, 32.07. 4, 4'-Methylenebis (2-aminophenol) (1o) -1 Orange solid m.p.348-349, Rf=0.65, FTIR (KBr) cm : 3600-3500 (br COOH) 3385, (N-H), 3190 (C=C-H Aromatic) 2745 (C-H aliphatic), 1661, 1601, 1531 (C=C benzene ring) 1444 (N-H bend.), 1 836, 814, (o-disubstituted Aromatic ring). H-NMR (300 MHz, DMSO-d6) δ (ppm): 9.05 (2H, br, s), 7.67 (2H, s), 7.31 (2H, d, J=7.9Hz), 6.93 (2H, d, J=7.9Hz), 5.96 (4H, br, s), 3.54 (2H, s), 3.61 13 (2H, s). C-NMR (75 MHz, DMSO-d6) δ (ppm): 144.10, 135.16, 132.86, 132.86, 120.27, 116.75, 114.49, 42.38. 5, 5'-Methylenebis (2-methoxyaniline) (1p) o -1 Light yellow solid (85%) m.p.348-349 C, Rf=0.67, FTIR (KBr) cm : 3418, (N-H), 3050 (C=CH Aromatic) 2836 (C-H aliphatic), 1665, 1606, 1523 (C=C benzene ring) 1461 (N-H bend.), 833, 1 1 810, (o-disubstituted Aromatic ring). H-NMR (300 MHz, DMSO-d6) δ (ppm): H-NMR (300 MHz, DMSO-d6) δ: 7.57 (2H, s), 7.38 (2H, d, J=7.87Hz), 7.10 (2H, d, J=7.87Hz), 6.06 (4H, br, 13 s), 3.79 (6H, s), 3.68 (2H, s). C-NMR (75 MHz, DMSO-d6) δ (ppm): 145.52, 145.52, 136.72, 136.72, 134.11, 134.11, 120.04, 120.04, 116.73, 116.73, 111.94, 111.94, 56.79, 56.79, 42.38. 6, 6'-Methylenebis (3-chloroaniline) (1q) o -1 Light yellow solid (77%) m.p.172 C, Rf=0.76, FTIR (KBr) cm : 3418, (N-H), 3030 (C=C-H Aromatic) 2830 (C-H aliphatic), 1663, 1606, 1503 (C=C benzene ring) 1384 (N-H bend.), 830, 123

1 810,(C=C bending, Aromatic ring), 683 (C-Cl). H-NMR (300 MHz, DMSO-d6) δ (ppm): 7.06 (1H, s), 6.58 (2H, d, J=7.45Hz), 6.45 (2H, d, J=7.45Hz), 5.95 (4H, br, s), 3.98 (1H, s). 13C-NMR (75 MHz, DMSO-d6) δ (ppm): 145.21, 139.22, 127.31, 122.84, 122.23, 121.35, 41.57.

6, 6'-Methylenebis (2-amino-4-chlorophenol) (1r) o -1 Brown solid (74%) m.p.136-141 C, Rf=0.75 IFTR (KBr) cm : 3420, (N-H), 3070 (C=C-H Aromatic) 2858 (C-H aliphatic), 1662, 1614, 1586 (C=C benzene ring) 1455 (N-H bend.), 880, 1 808,(C=C bending, Aromatic ring), 761 (C-Cl) H-NMR (300 MHz, DMSO-d6) δ (ppm): 9.37 (2H, 13 br, s), 6.59 (2H, s), 6.43 (2H, s), 4.98 (4H, br, s), 3.79 (1H, s). C-NMR (75 MHz, DMSOd6) δ (ppm): 147.04, 133.37, 130.80, 127.08, 118.47, 117.35, 31.30. 6, 6'-Methylenebis (2-amino-4-nitrophenol) (1s) o -1 Orange solid (71%) m.p.189 C, Rf=0.80 FTIR (KBr) cm : 3417, (N-H), 3023 (C=C-H Aromatic) 2825 (C-H aliphatic), 1670, 1631, 1587 (C=C benzene ring) 1345 (N-H bend.), 1304 1 (NO2) 896, 838 (C=C bending, Aromatic ring), H-NMR (300 MHz, DMSO-d6) δ (ppm): 8.26 (2H, 13 br, s), 7.32 (2H, s), 7.18 (2H, s), 5.07 (4H, br, s), 3.80 (1H, s). C-NMR (75 MHz, DMSOd6) δ (ppm): 149.32, 140.96, 130.75, 129.54, 113.75, 110.21, 31.30. 5, 5'-Methylenebis (3-nitroaniline) (1t) -1 Yellow solid (83 %,) m.p.204-205, Rf=0.68 FTIR (KBr) cm : 3418(N-H), 2936 (C-H), 1666, 1523 (C=C) 1595 (N-H bend.), 1428 (N-H) 1128 (C-O), 860 (o-disubstituted Aromatic ring). 1H-

NMR (300 MHz, DMSO-d6) δ (ppm): 7.56 (2H, s), 7.40 (2H, s), 6.98 (2H, s), 5.19 (4H, br, s), 13 3.84 (1H, s). C-NMR (75 MHz, DMSO-d6) δ (ppm): 148.85, 144.13, 142.52, 122.09, 114.99, 108.49, 42.74. 6.5.2 General Procedure for the Synthesis of Reactive Azo Dyes (9a-j) Synthesis of reactive azo dyes involved three steps: i) Tetrazotization of 4,4’-Diamino diphenylmethane-2-sulfonic acid (F.C Acid) and Coupling with H-acid

FC-acid (2) (3.36 g, 0.01 mole) was suspended in H2O (80 mL). Hydrochloric acid (15 mL) was added drop wise to this well stirred suspension. The mixture was gradually heated up to 70 °C till clear solution obtained. The solution was cooled to 0-5 °C in an ice bath. A solution of NaNO2 (1g, 0.014mol) in H2O (10 mL) previously cooled to 0 °C, was then added over a period of 5 minutes with stirring. The stirring was continued for an hour maintaining the same temperature, with positive test for nitrous acid with required amount of a solution of a sulphamic acid. The clear tetrazo solution at 0-5 °C was used for subsequent coupling reaction. To a well-stirred solution of H-acid (3) (6.48 g, 0.02 mole), a freshly prepared solution of tetrazo F.C acid (3.36g, 0.01 mole) was added drop wise over a period of 10-15 minutes. The pH was maintained at 7.5 to 8.5 by simultaneous addition of sodium carbonate solution (10% w/v). During coupling the blue solution was formed. Stirring was continued for 3-4 hours, maintaining the temperature below 5 °C. The reaction mixture was heated up to 60oC and sodium chloride (15 g) added until the coloring material was precipitated. It was stirred for an hour, filtered and washed

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with a small amount of sodium chloride solution (5% w/v). The solid was dried at 8090oC and extracted with DMF. The dye was precipitated by diluting the DMF-extract with excess of chloroform. The blue colored dye (4) was then filtered, washed with chloroform and dried at 60oC. ii) Cynuration of Dye Cynuric chloride (3.7 g, 0.02 mol) was stirred in acetone (50 mL) at a temperature below 5°C for a period of an hour. A neutral solution of (4) (6.17 g, 0.01 mole) was then added in small lots in about an hour. Neutral pH was maintained below 5°C through this reaction. The reaction mass was then stirred at 0-5°C for further four hours when a clear solution was obtained. The cyanurated dye solution thus formed was salted out from 10% NaCl solution, filtered, washed and dried at 70oC (5) in 90% yield. Cyanurated dye was used for subsequent condensation with different bis-anilines. iii) Condensation of Cyanurated Dye with Bis-Anilines To the vigorously stirred aqueous solution of cyanurated dye (5) (1.5g, 0.0016 mol) at room temperature, was added a solution of 5,5'-methylenebis(3-nitroaniline), (0.228g 0.0008mol) in 15 ml water in acidic medium of HCl. The pH of reaction mixture was kept 4.0 and continued the stirring for 4h for completion of reaction which was checked by taking paper chromatography of reaction mixture. Dye was salted out from 15% solution of NaCl, filtered and dried in oven at 70oC keeping overnight. In this way prepared all 9a-j dyes by changing the bisanilines and keeping all other conditions identical.

[C53Cl2H34N15O21S5] (9a)

-1 Bluish crystal, (75.32%) λmax in nm (log ε): 599 (3.63), 396 (3.58), 355 (3.47). FTIR (KBr, cm ) νmax: 3526-2500 (O-H, COOH, N-H), 1750(C=O) 1619, 1591, 1529, (C=C aromatic), 1483 1 (CH2), 724 (Ar-H) 672 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.90 (s, 2H, CH2), 5.04(s, 2H, 2OH), 9.6(s, 2H, 2COOH), 9.8 (s, 2H, 2NH), 10.6 (s, 2H, 2NH), 7.6-8.3 (m, 19H, Ar-H) ppm. 13C- NMR (75 MHz, D2O) δ (ppm): 169.24, 151.43, 146.27, 145.21, 143.94, 142.89, 142.28, 137.05, 135.22, 134.47, 133.10, 132.84, 131.51, 130.55, 128.24, 125.54, 124.78, 123.97, 120.80, 120.27, 119.34, 117.64, 113.39, 42.38. [C53Cl2H34N15O21S5] (9b)

-1 Bluish crystal,(76.11%) λmax in nm (log ε): 614 (3.54), 396 (3.82), 355 (3.54). FTIR (KBr, cm ) νmax: 3471 br (OH, NH), 3072 (C=C-H) 1711 (C=O), 1660, 1590, 1502 (C=C aromatic), 723 (Ar- 125

1 H), 1483(CH2), 1070 S=O 772 (Ar-H) 672 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.89 (s, 2H, CH2), 5.01 (s, 2H, 2OH), 9.62(s, 2H, 2COOH), 9.79 (s, 2H, 2NH), 10.62(s, 2H, 2NH), 13 7.6-8.3(m, 19H, Ar-H) ppm. C-NMR (75 MHz, D2O) δ (ppm): 165.24, 150.31, 146.27, 145.21, 143.13, 142.89, 142.28, 141.04, 137.05, 132.84, 130.55, 129.67, 128.24, 127.60, 125.54, 123.97, 121.72, 119.34, 117.72, 116.29, 113.91, 42.34. [C53Cl2H34N15O21S5] (9c)

-1 Bluish crystal, (75.65%) λmax in nm (log ε): 608 (3.67), 396 (3.49), 355 (3.48). FTIR (KBr, cm ) νmax: 3443 br (OH, NH), 3071 (C=C-H) 2971 (CH2), 1747 (C=O), 1660, 1590, 1502 (C=C 1 aromatic), 1483(CH2), 1070, S=O 772 (Ar-H) 672 (C-Cl). H-NMR (300 MHz, D2O) δ: 1.95 (s, 2H, CH2), 5.02(s, 2H, 2OH), 9.59 (s, 2H, 2COOH), 9.78 (s, 2H, 2NH), 10.55(s, 2H, 2NH), 7.6- 1 8.3 (m, 19H, Ar-H) ppm. C-NMR (75 MHz, D2O) δ (ppm): 165.67, 152.43, 146.02, 145.27, 145.21, 142.28, 137.05, 134.28, 131.51, 130.55, 128.60, 128.24, 126.38, 126.31, 125.54, 124.78, 123.97, 120.80, 120.27, 119.34, 118.20, 117.64, 36.26. [C51Cl2H34N15O19S5] (9d)

-1 Bluish crystal (81.15%) λmax in nm (log ε): 643 (3.65), 396 (3.60), 355 (3.44). FTIR (KBr, cm ) νmax: 3441 (OH, NH), 3078 (C=C-H), 2835 (CH2), 1660, 1590, 1502 (C=C aromatic), 1070, 1 S=O, 723 (Ar-H), 672 (C-Cl). H-NMR (300 MHz, D2O) δ: 1.95 (s, 2H, CH2), 5.03 (s, 2H, 2OH), 5.3 (s, 2H, 2OH), 9.5 (s, 2H, 2NH), 10.56 (s, 2H, 2NH), 7.55-8.3(m, 19H, Ar-H) ppm. 13 C-NMR (75 MHz, D2O) δ (ppm): 165.32, 156.17, 151.43, 146.27, 146.21, 145.21, 142.89, 142.28, 137.05, 135.04, 133.90, 132.84, 131.51, 130.55, 128.24, 126.67, 124.78, 123.97, 120.80, 119.34, 117.64, 116.23, 114.42, 36.26. [C51Cl2H34N15O19S5] (9e)

Bluish crystal (80.72%). λmax in nm (log ε): 635 (3.72), 455 (3.61), 396 (3.60), 355 (3.52). FTIR -1 (KBr, cm ) νmax: 3448 (OH, NH), 1660, 1590, 1502 (C=C aromatic), 1070, S=O, 723 (Ar-H), 1 672 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.94 (s, 2H, CH2), 5.02 (s, 2H, 2OH), 5.33 (s, 2H, 2OH), 9.76 (s, 2H, 2NH), 10.57 (s, 2H, 2NH), 7.55-8.3 (m, 19H, Ar-H) ppm. 13C-NMR (75

MHz, D2O) δ (ppm): 164.91, 151.43, 147.03, 146.27, 145.21, 142.28, 137.05, 135.63, 133.04, 132.84, 131.51, 129.55, 128.24, 126.02, 125.54, 124.78, 123.97, 120.80, 120.50, 119.34, 115.57, 110.53, 42.38.

1 C-NMR (75 MHz, D2O) δ (ppm): 165.07, 164.12, 163.81, 153.70, 151.55, 150.52, 146.27, 143.64, 142.89, 142.28, 141.17, 137.18, 132.84, 131.65, 130.55, 129.30, 128.61, 125.54, 124.78, 123.97, 121.92, 120.80, 120.27, 119.34, 116.17, 114.44, 31.30.

[C51Cl4H32N15O19S5] (9g)

Bluish crystal (85.71%). λmax in nm (log ε): 615 (3.85), 420 (3.83), 396 (3.82), 355 (3.68). FTIR

-1 (KBr, cm ) νmax: 3440 (OH, NH), 2945 (CH2), 1660, 1585, 1507 (C=C aromatic), 1075, S=O, 720

1 (Ar-H), 672 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.87(s, 2H, CH2), 5.07(s, 2H, 2OH),

126

[C51Cl2H32N17O23S5] (9f)

Bluish crystal (83.22%). λmax in nm (log ε): 609 (3.49), 455 (3.72), 396 (3.67), 355 (3.47). FTIR -1 (KBr, cm ) νmax: 3422 (OH, NH), 2929 (CH2), 1660, 1590, 1502 (C=C aromatic), 1070, S=O, 1 723 (Ar-H), 672 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 3.41 (s, 2H, CH2), 5.05 (s, 2H, 2OH), 5.35 (s, 2H, 2OH), 9.71 (s, 2H, 2NH), 10.58(s, 2H, 2NH), 7.7-8.3(m, 17H, Ar-H) ppm. 5.31 (s, 2H, 2OH), 9.72 (s, 2H, 2NH), 10.53 (s, 2H, 2NH), 7.82-8.01(m, 17H, Ar-H). 13C-NMR

(75 MHz, D2O) δ (ppm): 164.91, 164.12, 151.43, 147.68, 146.27, 145.21, 142.89, 137.05, 133.95, 131.51, 130.55, 128.24, 127.88, 125.54, 125.30, 124.78, 123.97, 120.80, 119.34, 118.39, 117.64, 113.91, 31.30. [C51Cl2H32N17O21S5] (9h)

Bluish crystal (78.77%). λmax in nm (log ε): 601 (3.97), 420 (3.99), 396 (3.97), 355(3.81). FTIR -1 (KBr, cm ) νmax: 3465 (OH, NH), 2890 (CH2), 1648, 1570, 1510 (C=C aromatic), 1072, S=O, 1 735 (Ar-H), 670 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.88 (s, 2H, CH2), 5.1 (s, 2H, 2OH), 13 9.8 (s, 2H, 2NH), 10.75(s, 2H, 2NH), 7.85-8.30 (m, 19H, Ar-H) ppm. C-NMR (75 MHz, D2O) δ (ppm): 165.52, 163.25, 151.64, 149.08, 145.95, 142.81, 135.72, 131.62, 130.93, 128.45, 127.19, 125.54, 123.97, 121.41, 120.92, 119.54, 117.64, 113.63, 56.79, 42.38. [C51Cl2H36N15O19S5] (9i)

-1 Bluish crystal (84.57%). λmax in nm (log ε): 638 (3.95), 355 (3.94), 326 (3.95). FTIR (KBr, cm ) νmax: 3454 (OH, NH), 2929 (CH2), 1642, 1584, 1522 (C=C aromatic), 1125, S=O, 732 (Ar-H), 1 672 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.90 (s, 2H, CH2), 3.9 (s, CH3, OCH3) 5.0 (s, 2H, 13 2OH), 9.74 (s, 2H, 2NH), 10.53 (s, 2H, 2NH), 7.4-8.2 (m, 19H, Ar-H). C-NMR (75 MHz, D2O) δ(ppm): 165.24, 163.97, 151.43, 146.27, 145.73, 144.41, 142.28, 140.25, 132.84, 131.51, 130.55, 128.91, 128.24, 125.54, 124.78, 123.97, 122.57, 122.31, 120.80, 120.47, 120.27, 119.69, 117.73, 113.54, 41.12.

[C51Cl4H32N15O17S5] (9j)

-1 Bluish crystal, (80.43%). λmax in nm (log ε): 620 (3.82), 396 (3.86), 355 (3.66). FTIR (KBr, cm ) νmax: 3442 (OH, NH), 2890 (CH2), 1639, 1560, 1508 (C=C aromatic), 1127, S=O, 730 (Ar-H), 1 672 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.93 (s, 2H, CH2), 5.01 (s, 2H, 2OH), 9.73 (s, 2H, 13 2NH), 10.65 (s, 2H, 2NH), 7.6-8.2 (m, 19H, Ar-H) ppm. C-NMR (75 MHz, D2O) δ (ppm): 165.63, 163.97, 152.04, 149.72, 146.21, 145.21, 142.89, 140.22, 139.38, 131.51, 130.55, 128.24, 126.45, 125.54, 124.78, 123.97, 120.80, 119.34, 117.56, 114.11, 113.73, 42.74. 6.5.3 Dyeing method A laboratory model glycerin-bath high-temperature beaker dyeing machine was used. A paste of finely powdered dye (25 mg) was prepared with the dispersing agent EDTA (1ml, 10% W/V), in a ball mill for 10 min. To this paste, water (15 ml) was added under stirring and the pH was adjusted to 8.5-9.0, using Na2CO3 (1ml, 10% W/V). This dye suspension (100 ml) was added to a beaker provided with a lid and a screw cap. Before closing the lid and tightening the metal cap over the beaker, a wetted piece of cotton fiber was rolled into the beaker. The beaker was then placed 127

vertically on the rotatory carrier inside the tank. The rotatory carrier was then allowed to rotate in the glycerin-bath, the temperature of which was raised to 80 ºC (for cotton fiber) at the rate of 2 ºC/min. The dyeing was continued for 1h under pressure. After cooling for 1h, the beaker was removed from the bath and washed with water. The pattern was thoroughly washed with hot water at 50 ºC, and then with cold water, and finally dried at room temperature. 6.5.4 General Procedure for the Synthesis of Reactive Azo Dyes (10a-h) Same procedure was adopted for synthesis of reactive azo dyes 10a-h as mentioned at page 122-123 for synthesis of reactive dyes 9a-j. [C51Cl6H30N16O20S4] 10a

-1 Reddish violet crystal, (66%) λmax (nm) (log ε): 564, 305. FTIR (KBr, cm ) νmax: 3465 br (OH, NH), 3043 (C=C-H), 2955 (CH2), 1665, 1585, 1510 (C=C aromatic), 1465 (CH2), 1070 (S=O), 1 760 (Ar-H), 720 (C-Cl), 690 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.99 (2H, s), 7.17 (2H, s), 7.30 (2H, d, J=7.55Hz), 7.59 (2H, s), 7.56 (2H, s), 7.67(2H, s), 7.75 (2H, d, J=7.55 Hz), 7.78 (2H, s), 7.89 (2H, d, J=7.4 Hz), 8.10 (2H, d J=7.4 Hz), 8.36 (N-H, s), 8.42 (N-H, s), 8.79 (O-H, s), 13 9.49 (O-H, s ). C-NMR (75 MHz, D2O) δ (ppm): 170.0, 167.3, 158.2, 142.4, 140.4, 138.2, 137.5, 135.5, 134.5, 129.1, 128.3, 126.1, 124.4, 120.9, 120.5, 120.2, 118.6, 117.6, 115.6, 108.3, 41.1Anal. Calcd. For C51Cl6H30N16O20S4, C: 40.09, H: 1.98, N: 14.67, S: 8.97; Found: C: 40.05, H: 1.95, N: 14.60, S: 9.02. [C51H32Cl2N18O26S4] 10b

-1 Reddish violet crystal, (67%) λmax (nm): 567, 310. FTIR (KBr, cm ) νmax: 3447 br (OH, NH), 3065 (C=C-H), 2950 (CH2), 1662, 1586, 1507 (C=C aromatic), 1560 (NO2), 1427 (CH2), 1095 1 (S=O), 772 (Ar-H), 680 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.89 (2H, s), 7.15 (1H, s), 7.27 (2H, d, J=7.5Hz), 7.58 (2H, s), 7.56 (2H, s), 7.67 (2H, s), 7.75 (2H, d, J=7.5 Hz ), 7.78 (2H, d, J=7.45 Hz), 7.89 (2H, d, J=7.45 Hz), 8.30 (N-H, s), 8.38 (N-H, s), 8.75 (O-H, s), 9.43 (O-H, s 13 ). C-NMR (75 MHz, D2O) δ (ppm): 174.9, 158.8, 145.5, 141.8, 140.4, 138.3, 137.6, 136.5, 135.3, 131.2, 129.7, 128.7, 126.9, 120.9, 120.5, 120.2, 117.6, 117.1, 115.5, 108.6, 108.1, 44.9. Anal. Calcd. For C51H32Cl2N18O26S4, C, 40.51; H, 2.13; N, 16.67; S, 8.48; Found: C, 40.43; H, 2.10; N, 16.53; S, 8.52.

[C53H34Cl2N16O24S4] 10c

-1 Reddish violet crystal, (66%) λmax (nm): 560, 310. FTIR (KBr, cm ) νmax: 3526-3200 (O-H, COOH, N-H), 3078 (C=C-H), 2953 (CH2), 1735(C=O) 1627, 1587, 1523 (C=C aromatic), 1481 1 (CH2), 1078 (S=O), 732 (Ar-H), 672 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.95 (2H, s), 7.15 (2H, s), 7.27 (2H, d, J=7.55 Hz), 7.38 (2H, d, J=7.55 Hz), 7.54 (2H, s), 7.71 (2H, d, J=7.60 Hz), 7.78 (2H, s), 7.88 (2H, d, J=7.60 Hz), 7.97(1H, s), 8.33 (N-H, s), 8.41 (N-H, s), 8.70 (O-H, 13 s), 9.46 (O-H, s ), 12.74 (O-H, COOH, br, s). C-NMR (75 MHz, D2O) δ (ppm): 169.0, 168.5, 158.6, 140.3, 138.6, 138.1, 137.7, 135.3, 130.6, 129.9, 128.7, 127.1, 126.5, 121.1, 120.9, 120.2, 117.6, 116.8, 115.7, 108.5, 45.9. Anal. Calcd. For C53H34Cl2N16O24S4, C, 43.07; H, 2.32; N, 15.16; S, 8.68; Found: C, 43.02; H, 2.25; N, 15.09; S, 8.73.

128

[C51H32Cl2N18O24S4] 10d

-1 Reddish violet crystal, (71%) λmax (nm) : 557, 306. FTIR (KBr, cm ) νmax: 3480 (OH, NH), 3075 (C=C-H), 2830 (CH2), 1645, 1587, 1505 (C=C aromatic), 1550 (NO2), 1443 (CH2), 1107 1 (S=O), 733 (Ar-H), 672 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 2.01 (2H, s), 7.15 (2H, s), 7.27 (2H, d, J=7.53 Hz), 7.50 (2H, s), 7.66 (2H, s), 7.74 (2H, d, J=7.53 Hz), 7.78 (2H, s), 7.84 (2H, d, J=7.6 Hz), 8.07 (2H, d, J=7.6 Hz ), 8.33 (N-H, s), 8.41 (O-H, s), 8.65 (N-H, s), 9.38 (O- 13 H, s ). C-NMR (75 MHz, D2O) δ (ppm): 168.8, 158.7, 144.4, 140.3, 138.7, 137.6, 136.6, 135.3, 133.8, 129.4, 128.6, 126.2, 120.9, 120.5, 120.2, 119.1, 117.6, 116.3, 115.5, 112.1, 108.8, 55.1, 45.2 Anal. Calcd. For C51H32Cl2N18O24S4, C, 41.39; H, 2.18; N, 17.03; S, 8.66; Found: C, 41.33; H, 2.10; N, 17.00; S, 8.69. [C53H38Cl2N16O22S4] 10e

-1 Reddish violet crystal, (69%). λmax (nm): 570, 310. FTIR (KBr, cm ) νmax: 3465 (NH), 3063 (C=C- H), 2943 (CH2), 1663, 1595, 1512 (C=C aromatic), 1447 (CH2), 1075 (S=O), 1055 (C-O), 770 (Ar- 1 H), 673 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 4.07 (3H, s), 1.96 (2H, s), 6.75 (2H, d, J=7.15 Hz), 7.0 (2H, d, J=7.15 Hz), 7.15 (1H, s), 7.32 (1H, s), 7.50 (2H, s), 7.93(1H, s), 7.73 (2H, d, J=7.58 Hz), 7.86 (2H, d, J=7.58 Hz), 8.30 (N-H, s), 8.36 (O-H, s), 8.61 (N-H, s), 9.35

13 (O-H, s). C-NMR (75 MHz, D2O) δ (ppm): 45.2, 55.1, 108.8, 112.1, 115.5, 116.3, 117.6, 119.1, 120.2, 120.5, 120.9, 126.2, 128.6, 129.4, 133.8, 135.3, 136.6, 137.6, 138.7, 140.3, 144.4, 158.7, 168.8. Anal. Calcd. For C53H38Cl2N16O22S, C, 43.90; H, 2.64; N, 15.45; S, 8.84; Found: C, 43.81; H, 2.51; N, 15.40; S, 8.86.

[C53H34Cl2N16O24S4] 10f

-1 -1 Reddish violet crystal, (74%). λmax (nm): 568, 309. FTIR (KBr, cm ) νmax: FTIR (KBr, cm ) νmax: 3555-3265 (O-H, COOH, N-H), 3060 (C=C-H), 2962 (CH2), 1743 (C=O), 1629, 1587, 1540 (C=C aromatic), 1480 (CH2), 744 (Ar-H), 678 (C-Cl). 3422 (OH, NH), 2929 (CH2), 1660, 1590, 1502 1 (C=C aromatic), 1070, S=O, 723 (Ar-H), 672 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.79 (2H, s), 6.82 (2H, d, J=7.32 Hz), 7.29 (2H, d, J=7.32 Hz), 7.34 (2H, d), 7.45 (1H, s), 7.54 (2H, d, J=7.5 Hz), 7.69 (2H, d, J=7.5 Hz), 7.81 (2H, d, J=7.6 Hz), 8.08 (2H, d, J=7.6 Hz), 13 8.33 (N-H, s), 8.37(O-H, s), 8.38 (O-H, s), 9.38 (O-H, s ). C-NMR (75 MHz, D2O) δ (ppm): 180.32, 150.9, 149.2, 146.8, 139.4, 138.2, 136.4, 134.0, 131.7, 130.3, 129.2, 127.7, 126.3, 125.2, 123.4, 121.8, 119.0, 117.8, 116.1, 114.1, 118.1, 109.6, 38.15. Anal. Calcd. For

1 H-NMR (300 MHz, D2O) δ (ppm): 2.10 (2H, s), 6.58 (2H, d, J=7.24Hz), 6.76 (2H, d, J=7.24 Hz), 7.10 (2H, s), 7.32 (2H, s), 7.50 (2H, s), 7.69 (2H, d, J=7.6Hz), 7.81 (2H, d, J=7.60 Hz), 8.33

13 (N-H, s), 8.45 (N-H, s), 8.78 (O-H, s), 9.42 (O-H, s ). C-NMR (75 MHz, D2O) δ (ppm): 168.7, 158.4, 141.9, 140.3, 138.6, 137.6, 135.3, 134.9, 134.3, 129.4, 128.3, 126.5, 121.9,120.8, 120.2,

119.6, 117.5, 116.6, 115.4, 114.3, 108.4, 47.3. Anal. Calcd. For C51H34Cl2N16O22S4 , C, 43.08; H, 2.41; N, 15.76; S, 9.02; Found: C, 43.03; H, 2.35; N, 15.56; S, 9.10. 129

C53H34Cl2N16O24S4, C, 43.07; H, 2.32; N, 15.16; S, 8.68; Found: C, 43.01; H, 2.29; N, 15.10; S, 8.70.

[C51H32Cl4N16O20S4] 10g

-1 Reddish violet crystal, (74%). λmax (nm): 560, 310. FTIR (KBr, cm ) νmax: 3462 (OH, NH), 3055 (C=C-H), 2965 (CH2), 1633, 1586, 1522 (C=C aromatic), 1115 (S=O), 735 (Ar-H), 710 (C-Cl), 1 672 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.93 (2H, s), 7.02 (1H, s), 7.11 (2H, s), 7.30 (2H, s), 7.27 (2H, d, J=7.35 Hz), 7.50 (2H, s), 7.66 (2H, s), 7.69 (2H, d, J=7.35 Hz), 7.81 (2H, d, J=7.60 Hz), 8.08 (2H, d, J=7.6 Hz), 8.25 (N-H, s), 8.32 (O-H, s), 8.67(N-H, s), 9.36 (O-H, s ).

13 C-NMR (75 MHz, D2O) δ (ppm): 169.9, 158.6, 144.3, 143.7, 140.1, 138.7, 137.4, 135.9, 135.3, 129.5, 128.2., 126.3, 120.9, 120.5, 120.2, 118.0, 117.6, 115.3, 114.0, 113.5, 109.6, 45.3. Anal. Calcd. For C51H32Cl4N16O20S4 ,C, 41.99; H, 2.21; N, 15.36; S, 8.79; Found: C, 41.80; H, 2.11; N, 15.32; S, 8.83.

[C51H34Cl2N16O22S4] 10h

-1 Reddish violet crystal, (71%). λmax (nm): 568, 309. FTIR (KBr, cm ) νmax: 3466 (OH, NH), 3058

(C=C-H), 2875 (CH2), 1637, 1563, 1510 (C=C aromatic), 1120 (S=O), 737 (Ar-H), 672 (C-Cl).

6.5.5 General Procedure for the Synthesis of γ-acid Based Reactive Azo Dyes (15a-h) Same procedure was adopted for synthesis of reactive azo dyes 15a-h as mentioned at page 122123 for synthesis of reactive dyes 9a-j and 10a-h.

[C53H36Cl2N14O18S4] 15a

-1 Reddish yellow crystal, (65%) λmax (nm): 502, 386, 293. FTIR (KBr, cm ) νmax: 3560-3350 (OH, COOH, N-H), 3032 (C=C-H), 2915 (CH2), 1755 (C=O), 1625, 1587, 1525, (C=C aromatic), 1540 1 (N=N), 1465 (CH2), 1125 (S=O), 787 (Ar-H), 680 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 2.03 (2H, s), 6.83 (2H, s), 6.84 (4H, d, J=8.1Hz), 7.30 (2H, d, J=8.1Hz), 7.38 (2H, d, J=8.1Hz), 7.44 (1H, s), 7.56 (1H, s), 7.90 (4H, d, J=8.1Hz), 8.39 (N-H, s), 8.89 (N-H, s), 9.07(O- 13 H, s), 9.48 (O-H, s). C-NMR (75 MHz, D2O) δ (ppm): 168.5, 167.9, 166.0, 156.8, 156.5, 147.7, 144.3, 142.7, 135.3, 134.9, 131.3, 131.3, 128.8, 128.5, 126.7, 124.3, 123.8, 120.0, 118.4, 107.4, 45.5. Anal. Calcd. For C53H36Cl2N14O18S4, C, 46.94; H, 2.68; N, 14.46; S, 9.46; Found: C, 46.23; H, 2.54; N, 14.40; S, 9.50.

130

[C51H34Cl2N16O18S4] 15b

-1 Reddish yellow crystal, (68%) λmax (nm): 496, 389. FTIR (KBr, cm ) νmax: 3467 br (OH, NH), -1 3062 (C=C-H), 2920 (CH2), 1647, 1572, 1520 (C=C aromatic), 1565 cm 1462 (CH2), 1123 1 (S=O), 772 (Ar-H), 682 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.95 (2H, s), 6.84 (4H, d, J=8.1Hz), 7.15 (2H, s), 7.66 (2H, s), 7.78 (2H, s), 7.44 (2H, s), 7.68 (4H, d, J=8.1Hz), 7.90 (2H, d), 7.97 (2H, d), 8.11 (N-H, s), 8.20 (N-H, s), 8.34 (O-H, s), 9.47 (O-H, s). 13C-NMR (75 MHz,

D2O) δ (ppm): 169.0, 167.9, 156.5, 149.2, 147.7, 144.3, 143.8, 143.2, 142.7, 126.7, 131.9, 131.3, 128.8, 128.5, 124.3, 123.2, 121.6, 120.0, 117.4, 114.3, 107.4, 106.7, 42.3. Anal. Calcd. For C51H34Cl2N16O18S4, C, 45.11; H, 2.52; N, 16.50; S, 9.44; Found: C, 44.95; H, 2.30; N, 16.35; S, 9.51. [C51H34Cl4N14O16S4] 15c

-1 Reddish yellow crystal, (67%). λmax (nm) : 496, 394, 292. FTIR (KBr, cm ) νmax: 3435 br (OH, NH), 3043 (C=C-H), 2908 (CH2), 1627, 1577, 1523 (C=C aromatic), 1440 (CH2), 1070 (S=O), 1 767 (Ar-H), 680 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.78 (2H, s), 6.79 (2H, s), 6.84 (4H, d, J=8.1Hz), 7.34 (2H, s), 7.58 (2H, s), 7.68 (4H, d, J=8.1Hz), 7.729 (1H, s), 8.02 (N-H, s), 8.11 13 (N-H, s), 8.32 (O-H, s), 9.41 (O-H, s). C-NMR (75 MHz, D2O) δ (ppm): 170.4, 158.8, 154.9, 152.3, 143.3, 142.2, 133.9, 132.0, 130.4, 126.6, 123.8, 119.2, 116.2, 103.75, 101.4, 95.5, 39.01. Anal. Calcd. For C51H34Cl4N14O16S4, C, 44.75; H, 2.50; N, 14.32; S, 9.37; Found: C, 44.20; H, 2.32; N, 14.15; S, 9.40.

[C53H40Cl2N14O16S4] 15d

-1 Reddish yellow crystal, (70 %) λmax (nm): 492, 390, 283. FTIR (KBr, cm ) νmax: 3437 (OH, NH), 3072 (C=C-H), 2838 (CH2), 1653, 1574, 1525 (C=C aromatic), 1437 (CH2), 1070 (S=O), 1 755 (Ar-H), 683 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 4.05 (3H, s), 2.01 (2H, s), 6.75 (4H, d, J=8.1Hz), 7.00 (2H, d, J=8.15Hz), 7.15 (1H, s), 7.44 (1H, s), 7.77 (2H, d, J=8.15Hz), 7.90 (4H, d, J=8.1Hz), 7.27 (2H, s), 8.40 (N-H, s), 8.89 (N-H, s), 8.95 (O-H, s), 9.43 (O-H, s). 13 C-NMR (75 MHz, D2O) δ (ppm): 170.5, 167.8, 156.5, 147.7, 144.9, 144.3, 142.7, 134.6, 134.6, 133.6, 131.8, 131.3, 128.8, 128.5, 126.7, 124.3, 123.2, 121.7, 119.2, 118.4, 116.2, 112.6, 107.4, 55.8, 41.7. Anal. Calcd. For C53H40Cl2N14O16S4, C, 47.93; H, 3.04; N, 14.77; S, 9.66; Found: C, 47.11; H, 2.99; N, 14.50; S, 9.71.

[C51H34Cl4N14O14S4] 15e

-1 Reddish yellow crystal, (69%). λmax (nm): 498, 388, 292. FTIR (KBr, cm ) νmax: 3455 (OH,

NH), 3045 (C=C-H), 2890 (CH2), 1645, 1572, 1522 (C=C aromatic), 1433 (CH2), 1095 (S=O),

1 753 (Ar-H), 679 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 3.87 (3H, s), 2.05 (2H, s), 6.75 (4H, d, J=8.1Hz), 7.09 (2H, d, J=8.2Hz), 7.18 (1H, s), 7.27 (2H, s), 7.44 (1H, s), 7.77 (2H, d, J=8.20 Hz), 7.89 (4H, d), 7.98 (2H, d, J=8.1Hz), 8.32 (N-H, s), 8.92 (N-H, s), 9.10 (O-H, s), 9.33(O-H, 13 s). C-NMR (75 MHz, D2O) δ (ppm): 173.5, 168.6, 156.5, 147.7, 144.3, 143.7, 142.7, 135.6, 131.6, 131.2, 128.8, 128.5, 126.7, 124.3, 123.2, 120.5, 118.4, 114.2, 113.3, 107.4, 43.5. Anal. Calcd. For C51H34Cl4N14O14S4, C, 45.82; H, 2.56; N, 14.67; S, 9.59; Found: C, 45.35; H, 2.16; N, 14.45; S, 9.09.

131

[C51H32Cl6N14O14S4] 15f -1 Reddish yellow crystal, (74%). λmax (nm) (log ε): 492, 389, 289. FTIR (KBr, cm ) νmax: 3453

(OH, NH), 3053 (C=C-H), 2929 (CH2), 1637, 1586, 1512 (C=C aromatic), 1447 (CH2), 1122

1 (S=O), 750 (Ar-H), 680 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.94 (2H, s), 6.85 (4H, d, J=8.1Hz) 7.02 (2H, s), 7.15 (2H, s), 7.44 (1H, s), 7.77(2H, d, J=7.8Hz), 7.90 (4H, d, J=8.1Hz), 7.30 (2H, s), 8.02 (2H, d, J=7.8Hz), 8.34 (N-H, s), 8.83 (N-H, s), 8.97(O-H, s), 9.41 (O-H, s).

13 C-NMR (75 MHz, D2O) δ (ppm): 168.5, 167.9, 147.7, 156.5, 144.3, 142.7, 142.2, 139.3, 134.3, 131.9, 131.5, 130.1, 128.8, 128.5, 127.2, 126.7, 124.3, 123.2, 121.1, 120.0, 118.4, 107.4, 44.3. Anal. Calcd. For C51H32Cl6N14O14S4, C, 43.57; H, 2.29; N, 13.95; S, 9.12; Found: C, 43.10; H, 2.23; N, 13.34; S, 9.20.

[C51H32Cl6N14O14S4] 15g

-1 Reddish yellow crystal, (75%). λmax (nm) (log ε): 495, 392, 289. FTIR (KBr, cm ) νmax: 3435

(OH, NH), 3043 (C=C-H), 2923 (CH2), 1633, 1589, 1517 (C=C aromatic), 1453 (CH2), 1097

1 (S=O), 772 (Ar-H), 685 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 1.99 (2H, s), 6.86 (4H, d, J=8.1Hz), 7.17 (2H, s), 7.2 (2H, s), 7.44 (2H, s), 7.56 (2H, s), 7.77(2H, d, J=7.9Hz), 7.93 (4H, d, J=8.1Hz), 8.02 (2H, d, J=7.9Hz), 8.31 (N-H, s), 8.83 (N-H, s), 9.05 (O-H, s), 9.37 (O-H, s). 13C-

NMR (75 MHz, D2O) δ (ppm): 170.5, 167.9, 156.5, 147.7, 144.3, 142.7, 142.3, 137.1, 134.9, 131.6, 130.5, 128.8, 128.5, 126.7, 125.1, 124.3, 124.1, 123.2, 119.5, 118.4, 107.7, 42.1. Anal. Calcd. For C51H32Cl6N14O14S4, C, 43.57; H, 2.29; N, 13.95; S, 9.12; Found: C, 43.17; H, 2.20; N, 13.35; S, 9.25.

[C51H36Cl2N14O16S4] 15h

-1 Reddish yellow crystal, (70%). λmax (nm) (log ε): 499, 389, 289. FTIR (KBr, cm ) νmax: 3450

(OH, NH), 3064 (C=C-H), 2963 (CH2), 1639, 1568, 1526 (C=C aromatic), 1448 (CH2), 1152

1 (S=O), 755 (Ar-H), 682 (C-Cl). H-NMR (300 MHz, D2O) δ (ppm): 2.04 (2H, s), 7.15 (1H, s), 7.10 (1H, s), 6.58 (2H, d, J=8.1Hz), 6.78 (4H, d, J=8.15Hz), 7.44 (2H, s), 7.77 (2H, d, J=8.15 Hz), 7.87 (4H, d, J=8.1Hz), 8.41 (N-H, s), 8.83 (N-H, s), 9.06 (O-H, s), 9.45 (O-H, s). 13C-NMR

(75 MHz, D2O) δ (ppm): 171.2, 167.9, 156.5, 147.7, 144.3, 142.7, 141.9, 134.9, 131.3, 131.2, 128.8, 128.5, 126.7, 124.3, 123.2, 121.3, 119.6, 118.4, 116.6, 144.3, 107.8, 44.2. Anal. Calcd. For C51H36Cl2N14O16S4, C, 47.12; H, 2.79; N, 15.08; S, 9.86; Found: C, 47.05; H, 2.74; N, 14.95; S, 9.95.

132

6.5.6 General Procedure for the Synthesis of Heterocyclic Acid Dyes 4-Aminoacetanilide (0.01mol) was dissolved in water (15 mL) and 37% HCl (2 mL) was added at 20 oC with continuous stirring. The solution was cooled to 0-5oC in ice bath and an aqueous solution of sodium nitrite (0.01mol) in (5 mL) water was then added drop wise in 15 min. The stirring was continued for 1.5 h while maintaining the same temperature. The remaining nitrous acid was tested with the help of starch iodide paper while was killed by sulphamic acid solution until it showed negative test for nitrous acid. The solution of coupling component was prepared by dissolving it in sodium carbonate. To the well stirred coupler solution, above freshly prepared diazonium solution was gradually added drop wise keeping the same temperature over the period of half an hour. The reaction mixture was further stirred for 1.5 h at the same temperature, maintaining the pH at 8-8.5, by simultaneous addition of 10% sodium carbonate solution. Precipitates obtained, were filtered repeatedly washed with cold water and dried. The above prepared yellow color dyes (0.01 mol) component was dissolved in ethanol (20 mL) in a round bottom flask. The solution of hydrazine (0.02 mol) in 15 mL ethanol was added and was reflux for 3h. The resultant solution was cooled; filtered and yellow precipitate was obtained. Precipitate obtained was repeatedly washed, dried and collected to afford dyes (19a-i).

(E)-4-(4-((4-Acetamidophenyl) diazenyl)-5-methyl-3-(phenylamino)-1H-pyrazol-1 yl) benzenesulfonic acid (19a)

o -1 Yellow solid (60%). m.p>250 C. UV-Vis (ethanol) λmax/nm 388, FTIR (KBr) /cm 3330 (NH), 3077 (C=C-H Aromatic), 2858 (C-H aliphatic), 1693(C=O), 1666, 1590, 1523 (C=C benzene ring), 1 1440 (N=N str), 1250 (S=O). H-NMR (300 MHz, CDCl3) δ (ppm): 2.22 (s, 3H), 2.55 (s, 3H), 7.56 (d, 2H, J =9.0 Hz), 7.70 (d, 2H, J= 9.0 Hz), 7.02 (t, 1H), 7.33 (t, 2H), 7.75 (d, 2H), 7.80 (d, 2H, J=8.4), 7.96 (d, 2H, J=8.4), 8.55 (s, 1H, OH), 11.47 (s, 1H, NH ), 14.96 (s, 1H, 13 NH). C-NMR (75 MHz, CDCl3) δ (ppm):10.3, 24.2, 93.2, 117.0, 119.3, 122.4, 124.0, 126.9, 128.7, 129.6, 130.2, 138.5, 140.9, 143.0, 143.7, 158.3, 168.7. Anal.Calcd. For C24H22N6O4S1, C, 58.76; H, 4.52; N, 13.05; S, 6.54; Found: C, 58.40; H, 4.87; N, 12.93; S, 6.58. (E)-2-(4-((4- Acetamidophenyl) diazenyl)-5-methyl-3-oxo-2,3-dihydro-1H-pyrazol-1-yl) benzoic acid (19b)

o -1 Yellow solid (56%). m.p>250 C UV-Vis (ethanol) λmax/nm 389.5, FTIR (KBr) /cm 34503500 (COOH), 3298 (N-H), 3120 (C=C-H Aromatic), 2920 (C-H aliphatic), 1730 (C=O, str), 1695 (C=O) 1627, 1586, 1511 (C=C benzene ring), 1440 (N=N str), 1343 (C-H, bend), 1150 (C- 1 O). H-NMR (300 MHz, CDCl3) δ (ppm): 2.22 (s, 3H), 2.52 (s, 3H), 7.52 (d, 2H, J= 9.0 Hz), 7.74 (d, 2H, J= 9.0 Hz), 6.95 (t, 1H), 7.11 (t, 1H), 7.59 (t, 1H), 7.91 (t, 2H), 11.45 (s, 1H, NH), 13 13.11 (s, 1H, OH ), 14.75 (s, 1H, NH). C-NMR (75 MHz, CDCl3) δ (ppm): 13.1, 23.9, 97.5,107.1,113.1, 119.0, 130.2,130.8,134.4, 138.5, 143.5, 155.3, 165.2, 168.8, 169.4. Anal. Calcd. For C19H17N5O4, C, 60.15; H, 4.52; N, 18.46; Found: C, 59.93; H, 4.67; N, 18.33.

133

(E)-2-(4-((4-Acetamidophenyl) diazenyl)-3, 5-dimethyl-1H-pyrazol-1-yl) benzoic acid (19c)

o -1 Yellow solid (55%). m.p>250 C; UV-Vis (ethanol) λmax/nm 390, FTIR (KBr) /cm 3515 (COOH), 3390 (N-H), 3035(C=C-H Aromatic), 2928 (C-H aliphatic), 1735 (C=O, str), 1693 (C=O), 1640, 1581, 1517 (C=C benzene ring), 1437 (N=N str), 1173 (C-O). 1H-NMR (300 MHz,

CDCl3) δ (ppm): 2. 23 (s, 3H), 2.34 (s, 3H), 2.62 (s, 3H), 7.56 (d, 2H, J=8.7Hz), 7.62 (d, 2H, J 13 =8.7Hz), 7.72-7.96 (m, 4H), 14.81 (s,1H, NH), 12.75 (s, 1H, OH). C-NMR (75 MHz, CDCl3) δ (ppm): 10.6, 12.10, 24.0, 107.2, 119.0, 119.8, 122.9, 126.1, 126.8, 130.2, 133.1, 134.5, 137.4, 138.5, 150.6, 166.4, 168.9. Anal.Calcd. For C20 H19N5O3, C, 63.65; H, 5.07; N, 18.56; Found: C, 62.87; H, 5.18; N, 18.29. (E)-2-(4-((4-Acetamidophenyl) diazenyl)-5-methyl-3-(phenylamino)-1H-pyrazol-1-yl) benzoic acid (19d)

o -1 Yellow solid (62%), m.p>250 C; UV-Vis (ethanol) λmax/nm 389, FTIR (KBr) /cm 3510 (COOH), 3312 (N-H), 3123 (C=C-H Aromatic), 2950 (C-H aliphatic), 1738 (C=O, str), 1688(C=O), 1650, 1588, 1525 (C=C benzene ring), 1430 (N=N str), 1180 (C-O). 1H-NMR (300

MHz, CDCl3) δ (ppm): 2.24 (s, 3H), 2.62 (s, 3H), 7.45 (d, 2H, J =8.7Hz), 7.65 (d, 2H, J =8.7Hz), 7.02 (t, 1H), 7.33 (d, 2H, J= 9.0 Hz), 7.75 (d, 2H, J= 9.0 Hz), 7.78-7.90 (m, 4H), 11.41 (s,1H, 13 NH), 12.55 (s, 1H, OH), 14.80 (s, 1H, NH). C-NMR (75 MHz, CDCl3) δ (ppm): 15.3, 24.0, 93.7, 117.8, 119.0, 119.8, 122.4,122.9, 126.1, 126.9, 129.5, 130.2, 133.5, 134.9, 138.5, 140.9, 141.3, 158.3, 166.9, 168.4. Anal Calcd. For C25H22N6O3, C, 66.07; H, 4.88; N, 18.49; Found: C, 65.85; H, 4.97; N, 18.23. (E)-N-(4-((5-Methyl-1-phenyl-3-(phenylamino)-1H-pyrazol-4- yl)diazenyl)phenyl)acetamide (19e)

o -1 Yellow solid (59%), m.p>250 C. UV-Vis (ethanol) λmax/nm 387, FTIR (KBr) /cm 3255 (NH), 3132 (C=C-H Aromatic), 2943 (C-H aliphatic), 1688(C=O), 1653, 1596, 1511 (C=C benzene ring), 1 1420 (N=N str), 1362 (C-H bend), 1198 (C-O). H-NMR (300 MHz, CDCl3) δ (ppm): 2.21 (s, 3H), 2.59 (s, 3H), 7.57 (d, 2H, J=8.7Hz), 7.63 (d, 2H, J =8.7 Hz), 7.14-7.19 (m, 5H), 7.34-

13 7.40 (m, 5H), 11.48 (1H, NH), 14.81 (s, 1H, NH). C-NMR (75 MHz, CDCl3) δ (ppm): 24.59, 26.12, 116.45, 120.96, 121.11, 124.82, 125.87, 129.05, 135.39, 137.14, 138.10, 140.7, 163.16, 168.27. Anal. Calcd. For C24H22N6O, C, 70.23; H, 5.40; N, 20.47; Found: C, 70.05; H, 5.45; N, 20.20. (E)-N-(4-((5-Methyl-3-oxo-1-phenyl-2,3-dihydro-1H-pyrazol-4yl)diazenyl)phenyl) acetamide (19f)

o -1 Yellow solid (61%) m.p> 250 C; UV-Vis (ethanol) λmax/nm 387.5, FTIR (KBr) /cm 3303 (NH), 3153 (C=C-H Aromatic), 2881 (C-H aliphatic), 1696 (C=O, str), 1607, 1548, 1515 (C=C benzene 1 ring), 1427 (N=N str), 1320 (C-H bend), 1176 (C-O). H-NMR (300 MHz, CDCl3) δ (ppm): 2.20 (s, 3H), 2.58 (s, 3H), 7.57 (d, 2H, J =9.0 Hz), 7.62 (d, 2H, J =9.0Hz), 7.16 (t, 1H), 13 7.34-7.45 (m, 4H), 11.47 (s, 1H, NH), 14.80 (s, 1H, NH). C-NMR (75 MHz, CDCl3) δ (ppm): 24.56, 26.12, 116.42, 120.95, 121.15, 124.81, 125.86, 129.05, 135.42, 137.13, 138.08, 163.15, 168.36. Anal. Calcd. For C18H17N5O2, C, 64.47; H, 5.11; N, 20.88; Found: C, 64.07; H, 5.30; N, 20.50.

134

(E)-N-(4-((3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)diazenyl)phenyl)acetamide (19g)

o -1 Yellow solid (54%). m.p>250 C. UV-Vis (ethanol) λmax/nm 391.5, FTIR (KBr) /cm 3256 (NH), 3132 (C=C-H Aromatic), 2914 (C-H aliphatic), 1690(C=O), 1654, 1596, 1511 (C=C benzene ring), 1 1442 (N=N str), 1363 (C-H, bend), 1200(C-O). H-NMR (300 MHz, CDCl3) δ (ppm): 2.21 (s, 3H), 2.50 (s, 3H), 2.62 (s, 3H), 7.39 (d, 2H, J=9.0Hz), 7.58 (d, 2H, J=9.0Hz), 7.21-7.34 (m, 13 5H), 14.85 (s, 1H, NH). C-NMR (75 MHz, CDCl3) δ (ppm): 10.9, 12.5, 24.7, 107.5, 119.0, 124.9, 126.2, 126.9, 129.3, 130.3, 137.4, 138.3, 139.5, 143.5, 150.2, 165.2, 170.0. Anal.Calcd. For C19H19N5O, C, 68.45; H, 5.74; N, 21.01; Found: C, 68.10; H, 5.95; N, 20.80. (E)-4-(4-((4- Acetamidophenyl)diazenyl)-5-methyl-3-oxo-2,3-dihydro-1H-pyrazol-1-yl) benzenesulfonic acid (19h)

o -1 Yellow solid (57%), m.p>250 C. UV-Vis (ethanol) λmax/nm 395, FTIR (KBr) /cm 3415 (NH), 3065 (C=C-H Aromatic), 2950 (C-H aliphatic), 1698 (C=O) 1659, 1624, 1575 (C=C benzene ring) 1 1441 (N=N str), 1250 (S=O). H-NMR (300 MHz, CDCl3) δ (ppm): 2.02 (s, 3H), 2.26 (s, 3H), 7.54 (d, 2H, J=8.8Hz), 7.68 (d, 2H, J=8.8Hz), 7.25 (d, 2H, J= 8.35Hz ), 7.36 (d, 2H, J=8.4Hz), 7.75 (d, 2H, J =8.4Hz), 8.52 (s, 1H, OH), 11.43 (s, 1H, NH), 14.83 (1H, NH). 13C-

NMR (75 MHz, CDCl3) δ (ppm): 11.8, 24.7, 98.4, 119.0, 122.7, 127.0, 130.4, 138.5, 139.6, 140.0, 143.5, 155.1, 165.2, 168.5. Anal.Calcd. For C18H17N5O5S1, C, 52.04; H, 4.12; N, 16.86; S, 7.72; Found: C, 51.92; H, 4.35; N, 16.55; S, 7.90. (E)-4-(4-((4-Acetamidophenyl)diazenyl)-5-methyl-3-oxo-2,3-dihydro-1H-pyrazol-1-yl) benzoic acid (19i)

o -1 Orange Yellow solid (53%). m.p>250 C. UV-Vis (ethanol) λmax/nm 401, FTIR (KBr) /cm 3490 (COOH), 3380 (N-H), 3055 (C=C-H Aromatic), 2945 (C-H aliphatic), 1725 (C=O, str), 1687(C=O), 1653, 1622, 1590 (C=C benzene ring), 1455 (N=N str), 1145 (C-O).1H-NMR (300 MHz, CDCl3) δ (ppm): 2.24 (s, 3H), 2.61 (s, 3H), 7.50 ( d, 2H, J =9.0Hz), 7.65 (d, 2H, J =9.0 Hz ), 7.11 (d, 2H, J =8.5Hz), 7.86 (d, 2H, J =8.5Hz), 11.42 (s, 1H, NH), 12.71 (s, 1H, OH), 14.78 13 (1H, NH). C-NMR (75 MHz, CDCl3) δ (ppm): 12.7, 24.2, 98.8, 113.1, 119.0, 120.4, 130.2, 130.8, 138.5, 141.4, 143.5, 155.5, 163.2, 167.7. Anal.Calcd. For C19H17N5O4, C, 60.15; H, 4.52; N, 18.46; Found: C, 60.05; H, 4.70; N, 18.20. 6.5.7 Dyeing method: Dye solution (10 mL, 0.4 % w/v) was taken in a dye-bath. Glauber's salt solution (7mL, 20% w/v) was added to it. The pH of the dye-bath was adjusted to 6.5 by adding acetic acid solution (1.0 mL, 10% w/v) solution. The total volume of the dye-bath was adjusted to 100 mL by adding the required amount of water. The leather fabric was introduced into the dye-bath with stirring. The content of the dye-bath was stirred for 1h at 45-50oC. The temperature was then gradually raised to 80oC over a period of half hour and maintained for three hours. The dye-bath was kept rotating during the process of dyeing and was added formic acid 2.0 ml and pH adjusted to 2.0. After this, the dye liquor was taken in 250 mL volumetric flask. The fabric was washed with cold water and the combined solution of dye liquor and washings was then further diluted to 250 mL with water. From this diluted solution, 1mL was further diluted to 50 mL with water and the absorbance of this solution was measured to find out the exhaustion of the dye on fabric. The dyed 135

fabric was dried and mounted on shade card. A weighed amount of leather fabric was stirred in boiling acidified pyridine which dissolves the unfixed dye from fabric and from the absorbance of this solution percentage fixation was checked. 6.5.8 General Procedure for Synthesis of Calix [4] resorsoniarene Azo Dyes (23a-f) Into a three necked round bottom flash was taken 0.01mol o-hydroxy substituted aniline in 20mL water. It was added 5ml of 30% HCl into the reaction flask and cooled the flask to 05oC by keeping the flask in ice bath. Reaction mixture was added 5ml of aqueous solution of sodium nitrite 0.01mol with continuous stirring and maintaining temperature at 0-5oC. Reaction mixture was stirred for 1.5h until complete diazotization was evidence by TLC. In another flask calix [4] resorsoniarene (0.0025mol) was dissolved in alkaline water 20mL and started stirring while keeping the temperature 0-5oC. Started the addition of previously prepared diazonium salt to calix [4] solution at pH>8.5 and completed the addition in half hour. Continued the stirring of reaction mixture for 10h further unless coupling was completed evidenced by TLC of reaction mixture in 50:50 ethanol ethyl acetate. Dye solution was acidified to pH=2 with conc.HCl and precipitates were formed. Precipitates of dye were separated by filtration and recrystallized from methanol and chloroform mixture. In this way series of dyes 23a-f were synthesized.

Calix [4] resorcinoarenre Azo dye (23a)

o -1 Yellowish brown solid (63%), m.p>300 C. UV-Vis (DMSO) λmax/nm 425. FTIR (KBr) /cm 3475 (OH), 3043 (C=C-H Aromatic), 2977 (C-H aliphatic), 1641, 1622, 1590 (C=C benzene ring), 1 1448 (N=N str), 1160 (C-O). H-NMR (DMSO-d6, 300 MHz) δ (ppm): 10.16 (1H, br, s), 8.79 (1H, br, s), 8.27 (4H, s ), 7.27 (4H, s), 6.99 (4H, d, J= 8.1Hz ), 6.37 (4H, d, J= 8.35Hz), 13 3.83 (8H, s). C-NMR (DMSO-d6, 75 MHz) δ (ppm):163.05, 155.12, 143.16, 139.72, 138.24, 130.26, 126.44, 119.18, 118.90, 116.65, 31.30. Anal.Calcd. For C52H36N12O20, C, 54.36; H, 3.16; N, 14.63; Found: C, 54.30; H, 3.20; N, 14.52. Calix [4] resorcinoarenre Azo dye (23b)

o -1 Brown solid (68%), m.p>300 C. UV-Vis (DMSO) λmax/nm 445, FTIR (KBr) /cm 3450 (O-H), 3080 (C=C-H Aromatic), 2940 (C-H aliphatic), 1628, 1596, 1511 (C=C benzene ring), 1426 (N=N str), 1355 (C-H bend), 1188 (C-O).1H-NMR (600 MHz, DMSO) δ 8.40 (OH, s), 7.83 (4H, s), 7.33 (4H, s), 6.98 (4H, d, J= 8.15Hz), 6.78 (4H, d, J= 8.15Hz), 3.83 (2H, s). 13C-NMR 136

(DMSO-d6, 75 MHz) δ (ppm): 157.35, 155.12, 138.24, 136.70, 130.81, 130.26, 124.62, 120.99, 118.90, 117.85, 31.30. Anal.Calcd. For C52H36Cl4N8O12, C, 56.44; H, 3.28; N, 10.13; Found: C, 56.36; H, 3.32; N, 10.08. Calix [4] resorcinoarenre Azo dye (23c)

o -1 Brown solid (71%), m.p>300 C. UV-Vis (DMSO) λmax/nm 456, FTIR (KBr) /cm 3415 (O-H), 3054 (C=C-H Aromatic), 2962 (C-H aliphatic), 1634, 1623, 1570 (C=C benzene ring) 1446 1 (N=N str), 1250 (S=O). H-NMR (DMSO-d6, 300 MHz) δ (ppm): 10.25 (m, 1H), 9.49 (OH, s), 8.49-8.44 (OH, s), 7.89 (4H, s), 7.32 (4H, s), 7.03 (4H, d, J= 8.2Hz), 6.12 (4H, d, J= 8.2Hz),

13 3.79 (8H, s). C-NMR (DMSO-d6, 75 MHz) δ (ppm): 156.51, 155.12, 138.24, 137.07, 132.34, 130.37, 130.26, 123.81, 118.90, 117.40, 31.30. Anal.Calcd. For C52H40N8O24S4, C, 48.45; H, 3.13; N, 8.69; S, 9.95; Found: C, 48.36; H, 3.19; N, 8.58; S, 10.05. Calix [4] resorcinoarenre Azo dye (23d)

o -1 Yellowish brown solid (65%), m.p>300 C. UV-Vis (DMSO) λmax/nm 505, FTIR (KBr) /cm 3457(O-H), 3092 (C=C-H Aromatic), 2921(C-H aliphatic), 1637, 1591, 1545 (C=C benzene ring), 1 1438 (N=N str), 1347 (C-H, bend), 1200(C-O). H-NMR (DMSO-d6, 300 MHz) δ (ppm): 10.88 (OH, s), 7.83 (OH, s), 7.30 (4H, s) 7.04 (8H, d, J= 8.18Hz), 6.99 (8H, d, J= 8.18Hz), 3.85 13 (8H, s). C-NMR (DMSO-d6, 75 MHz) δ (ppm): 158.26, 155.12, 138.27, 138.24, 132.35, 130.26, 122.37, 121.15, 118.90, 117.11, 31.30. Anal.Calcd. For C52H40N8O12, C, 64.46; H, 4.16; N, 11.56; Found: C, 64.34; H, 4.04; N, 11.47. Calix [4] resorcinoarenre Azo dye (23e)

o -1 Yellow solid (60%), m.p>250 C. UV-Vis (ethanol) λmax/nm 460, FTIR (KBr) /cm 3330 (OH), 3070 (C=C-H Aromatic), 2898 (C-H aliphatic), 1638, 1583, 1529 (C=C benzene ring), 1443 (N=N 1 str), 1250 (S=O). H-NMR (DMSO-d6, 300 MHz) δ (ppm): 8.56 (OH, s), 8.27 (OH, s), 13 8.11-7.73 (16H, m), 8.07(4H, s), 7.63 (4H, s), 3.87 (8H, s). C-NMR (DMSO-d6, 75 MHz) δ (ppm): 155.72, 149.82, 139.07, 138.24, 136.84, 130.26, 129.63, 129.01, 128.30, 128.11, 127.97, 124.21, 118.90, 107.55, 31.30. Anal.Calcd. For C68H48N8O24S4, C, 54.84; H, 3.25; N, 7.52; S, 8.61, Found: C, 54.76; H, 3.33; N, 7.41; S, 8.69. Calix [4] resorcinoarenre Azo dye (23f)

o -1 Yellow solid (60%), m.p>250 C. UV-Vis (ethanol) λmax/nm 490, FTIR (KBr) /cm 3430 (OH), 3065 (C=C-H Aromatic), 2898 (C-H aliphatic), 1642, 1590, 1536 (C=C benzene ring), 1444 1 (N=N str), 1254 (S=O). H-NMR (DMSO-d6, 300 MHz) δ (ppm): 12.90 (OH, s), 9.12 (OH, s), 8.43 (2H, d. J= 8.20Hz), 7.96 (2H, d, J= 8.20Hz), 7.89 (1H, s), 7.68 (1H, s), 7.27 (4H, s), 3.76 13 (2H, s). C-NMR (DMSO-d6, 75 MHz) δ (ppm): 154.65, 151.39, 149.20, 138.24, 137.45, 136.05, 133.82, 130.26, 129.78, 128.81, 125.35, 118.90, 117.63, 110.22, 31.30. Anal.Calcd. For C68H44N12O32S4, C, 48.92; H, 2.66; N, 10.07; S, 7.68, Found: C, 48.83; H, 2.74; N, 9.98; S, 7.75.

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PART III

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Chapter 7 INTRODUCTION

7.1 Xanthene Dyes Fluorescent dyes containing at least a three-membered heterocyclic motif in their molecules are known as xanthene dyes. They are commonly found in large variety of natural products having diverse pharmacological activities. Presence and position of substituents have a large impact on the biological applications of synthetic and naturally occurring xanthones. The famous examples of xanthene dyes are fluorescein and rhodamine. Coumarin chromophores are also related to xanthene which can be seen as a structural part of rhodol fluorophore, the hybrid structure of fluorescein and rhodamine. The structural relationship between the xanthene dyes, coumarins and xanthones is illustrated in figure 7.1.

Figure 7.1 Structural relationships among xanthone, xanthene and coumarin chromophores Xanthenes are rare in natural plants and have been isolated from only two plant families, Compositae and Fabaceae [201-203]. Compounds I, II and III are examples of natural xanthenes (Figure 1.2). Blumeaxanthene I and blumeaxanthene II have been isolated from Blumea riparia (Compositae), a Chinese medicinal herb traditionally used to treat gynecological disorders [204] and the 3-Isopropyl-9a-methyl-1,2,4a,9-tetrahydroxanthene (III) has been isolated from Indigofera longeracemosa (Fabaceae) for use in traditional Indian medicine as an antidote for all snake venoms.

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Figure 7.2 Naturally occuring xanthenes in plants 7.2 Photophysical Properties Fluorescent dyes are widely employed in both qualitative and quantitative chemical and biological analyses and in other areas as well [206]. A great diversity of such dyes was used because the physicochemical properties of the dyes vary widely and different combinations of properties (e.g. absorption and emission maximum of chromophoric system, polarity and micro environmental dependence of the fluorescence) are suitable for different applications [207-209]. One property that is nearly always beneficial is high stability, both chemically and physically including photostability [210]. Here is a general structure of rhodamine derivatives where difference in photophysical properties - arises in the presence of substituents R1, R2, R3, R4, G and X (Figure 7.3).

Figure 7.3 General structure of xanthene derivatives with different photophysical properties. The activated processes in rhodamine derivatives seem to be associated with a non fluorescent twisted intramolecular charge-transfer (TICT) state characterized by an electron transfer from the amino groups to the xanthene ring followed by a rotation between them [211, 212]. The energy of the TICT state is higher than the energy of the first excited singlet state for the dyes without activated processes and lower for those with activated internal conversion. Then, the activated energy dissipation is explained by the population of the TICT state that is non-emissive and deactivates quickly to the ground state. The non-activated process involves energy dissipation by C–H and N– H stretching modes coupled with high frequency vibration modes of the solvent. The N–H vibration modes are found to be very effective in the dissipation of the electronic energy to polar solvents. Rhodamine 101 (Rho 101) and Rhodamine B (Rho B) are among the most used rhodamine and present an interesting behavior with pH and solvent polarity (Figure 7.4). In acidic solutions, the carboxyl group is protonated and the rhodamine dye is found in its cationic form. However, in basic solution, dissociation occurs and the rhodamine dye is converted into a zwitterion [213-214].

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Figure 7.4 Photophysical behavior expressed by Rhodamine 101 and Rhodamine B. 7.3 Derivitzation of Xanthene Dyes: A large variety of xanthene dyes and their derivatives have accomplished through harsh as well as green routes. A large share in xanthene derivatives belong to fluorescein and rhodamine derivatives because of their high photostability and application in diverse fields. 7.3.1 Fluorescein derivatives: Fluorescein is fluorescent molecule with conjugated framework in xanthene motif which was discovered in 19th century. There exist equilibrium between open quinoid form and closed lactone which leads to different absorption and emission over the pH range 5-9. At pH 2-4 closed lactone form dominates and at pH 7-9 open quinoid form prevails. Under mild acidic pH 5-7 a considerable population of fluorescein molecule exists in monoanionic and non fluorescent form. Under physiological pH 7.4 conditions dianionic and hydrophilic form of fluorescein dominates (Figure 7.5).

Figure 7.5 Two forms of fluorescein in the range of 6.31 to 6.80 phenolic pKa values

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To surmount the pH sensitivity and photo bleaching problems, the structure of fluorescein has been modified by substitution at phthalic anhydride and xanthene chromophore. For example 2',7'- dichlorofluorescein is less basic (pKa = 4.6) than fluorescein (pKa = 6.4), maintains fluorescein- like wavelengths and most important exhibits increased photostability relative to fluorescein (Figure 7.6) [215-216].

Figure 7.6 Structure of modified fluorescein 7.3.1.1 Fluorinated Benzo [c]xanthene Dyes Fluorinated benzo[c]xanthene dyes were synthesized from commercially available 6-hydroxy- 1naphthoic acid as exemplified by Scheme 7.1. Reaction of 6-Hydroxy-1-naphthoic acid with MeI/K2CO3 in THF followed by fluorination with Select Fluor [1-chloromethyl-4-fluoro- 1,4diazabicyclo [2.2.2]-octane bis(tetrafluoroborate)] provided product with some unreacted starting material. These compounds have been found to suitable for determination of intracellular pH, confocal laser scanning microscopy and flow cytometry.

Scheme 7.1. Synthesis of carboxy SNARF-4F dye. 7.3.1.2 Benzoxanthene Derivatives Benzoxanthene derivatives are important biologically active heterocycles, synthesized by mixing β-naphthol, an aromatic or aliphatic aldehyde, and a 1, 3-dicarbonyl substrate (Scheme 7.2) with various lewis acids. Similarly aryldibenzo dioxo-octahydroxanthene (Scheme 7.3), dioxohexahydroxanthene and dioxo-tetrahydrobenzoxanthene (Scheme 7.4) have been synthesized using lewis acids like H2SO4, Sulfamic acid or p-TSA, dodecylbenzenesulfonic acid [217], diammonium hydrogen phosphate [218], silica gel supported ferric chloride [219], Dowex- 50W [220], polyethylene glycol [221], indium (III) chloride or phosphorus pentoxide as catalyst

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[222]; (b) tetrabutyl ammonium fluoride in water [223]; (c) para-toluene sulfonic acid [224]; (d) solvent-free with iodine; (e) sodium hydrogen sulfate on silica gel in dichloromethane [225].

Scheme 7.2 Benzoxanthene derivatives active heterocycles synthesis.

Scheme 7.3 Dibenzoxanthenes derivatives synthesis

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Scheme 7.4 Dioxo-xanthenes derivatives synthesis

Scheme 7.5 Synthesis of 12-aryl-8,9,10,12-tetrahydrobenzo[a]xanthene-11-one derivatives in ionic liquid [NMP]H2PO4. 7.3.1.3 Imidazole Derivatives of fluorescein Several imidazole derivatives of fluorescein have been developed as diagnostic agents for positron emission tomography (PET) or optical imaging [226-230]. Recent development in synthesis of Nutlin-Glycine, has received eminence due to its usefulness regarding the imaging of tumor cells [231-233] (Scheme 7.6). Nutlin analogs have been developed that target to intracellular MDM2 and renders biological activities in tumor cells with wild type p53 but not cells with a mutant p53.

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Scheme 7.6 Synthetic scheme of Nutlin-Glycine-FAM conjugate. 7.3.1.4 Fluorescein-Based N-glycosylamines In recent years, fluorescein derivatives have played an important role in the field of drug discovery and gene delivery systems, cancer, [234] neurodegenerative diseases, [235] biosensors, [236-242] bioimaging, [243-245] and absorption studies of protein-based indicators [246-247]. These derivatives have been used as fluorescent tags for many biological molecules, such as proteins and DNA, as well as serving as a platform for many kinds of fluorescence probes [248]. Currently fluorescein-based N-glycosylamines are being explored for cell imaging studies (Scheme 7.7).

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Scheme 7.7 Synthesis of fluorescein-based N-glycosylamines. 7.3.2 Rhodamines Derivatives Rhodols and rhodamines have a widespread application as laser dyes, tracer agents, and biological probes. Different N-alkyl substitution patterns on the rhodamine correspond to different spectral

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characteristics. Attachment of alkyl moieties to the nitrogen core of rhodamine can tune absorption and fluorescent emission, which is here dependent on the number and type of alkyl groups. The simplest member of this class of fluorescein dyes, rhodamine 110 (Rh110),

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4 -1 exhibits fluorescein-like spectral properties with λmax = 496 nm, λem = 517 nm, ε = 7.4 × 10 M cm-1, and Φ = 0.92 in aqueous solution [249]. Substitution to tetramethylrhodamine (TMR) gives longer excitation and emission wavelengths (λmax/λem; 540/565 nm), but a lower quantum yield (Φ = 0.68). In general, quantum yields of rhodols and rhodamines decrease with increasing carbon number and the bulk of the substituents [250-251]. On the other hand, there is an exception where the julolidine ring incorporated into the rhodamine structure. Sulforhodamine (Rh101) shows improvement in quantum yields and exhibit longer excitation and emission wavelengths. The above mentioned rhodamines are illustrated in figure 1.7.

Figure 7.7 Structures of selected rhodamines Rhodols, rhodamines and their derivatives are usually prepared through acid-catalyzed condensation of an aminophenol with a phthalic anhydride (Scheme 1.8) [252].

Scheme 7.8 General synthesis of rhodols and rhodamines

The use of phthalic anhydrides bearing a substituent (R1) for bioconjugation yields products as intractable mixtures of 5- and 6-substituted regioisomers. Therefore functionalized commercially available rhodamines are often sold as regioisomeric mixtures. Synthesis of rhodamines by ZnCl2- catalyzed direct substitution of 3',6'-dichlorofluoresceins with amines was reported (Scheme 7.9) [253-255].

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Scheme 7.9 Synthesis of rhodamines via direct nucleophilic substitution from halogenated fluoresceins. Recently, a similar strategy for synthesis of rhodols was reported by Peng et al. in 2010. This new route consists of the mono-protection of the 3'-position of fluorescein by MOM, followed by the triflation of the 6'-position. This triflated intermediate was coupled with different amines under the catalysis of a palladium-phosphine complex, widely known as the Buchwald- Hartwig amination reaction (Scheme 7.10) [256].

Scheme 7.10 Synthesis of rhodol fluorophores via Buchwald-Hartwig amination reaction. In 2011 Grimm et al. used the above mentioned route for the preparation of rhodamines and N,Ndiacetylated rhodamines (Scheme 7.11) [257].

Scheme 7.11 Synthesis of rhodamines via Buchwald-Hartwig amination reaction. 7.4 Applications of Xanthene Dyes 7.4.1 pH Sensors To know about pH changes inside living cells is important for studying cellular internalization processes, such as phagocytosis [258] and endocytosis [259]. Similarly abnormal pH values inside the cell are noticed in some familiar disease types, such as cancer [260] and Alzheimers [261]. Some organelles, for example, endosomes [262] and plant vacuoles [263] show intracompartmental pH and the acidic environments in lysosomes (pH 4.5–5.5) [264] are known as a reason for the degradation of proteins in cellular metabolism. These studies have been made possible with help of fluorescent xanthene based probes. Figure 1.8 illustrates the mechanism of changes in detection response xanthene dye to pH changes.

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Figure 7.8 Mechanism of the xanthene probe response to pH changes 7.4.2 Metal Ion Detectors The design and development of highly sensitive and selective fluorescent probes for the detection of various metal ions in trace amounts in environmental and biological systems is of great interest to current researchers because of their powerful ability to improve the analytical sensitivity and capability for the sensing and visualization of analytes in living cells by utilizing molecular imaging techniques [265]. Xanthene derivatives for detection of toxic (Cd2+, Hg2+, Pb2+ ) as well as non for toxic metals (Cu2+, Fe3+, Zn2+) have developed which exhibit different colors with different metal ions (Figure 7.9).

Figure 7.9 Xanthene dyes detectors for Fe+3 and Hg+2 7.4.3 Fluorescence Imaging Fluorescence imaging is one of the most powerful techniques for visualizing temporal and spatial changes of biological phenomena in living cells, and many fluorescent probes have been developed. In particular, xanthene dyes such as fluorescein and rhodamines have favorable characteristics, such as high water solubility, high fluorescence quantum yield and high molar extinction coefficient, and they have been utilized as fluorescent cores for fluorescent probes working in the green to red wavelength region [266, 267]. Living cell imaging with chemosensor based on rhodamine derivatives has been achieved successfully (Scheme 1.12).

7.4.4 Xanthene Dyes as Dye Lasers Both cationic and anionic xanthene dyes are known to be efficient fluorescent dyes. Functional groups on the xanthene moiety control their fluorescent colors. Xanthene dyes are being applied

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as laser dye. A dye laser requires fluorescein and rhodamine dyes as an organic medium. They all oscillate in the visible area. Among these xanthene dyes, the rhodamine 6G is mainly used for a laser dye. (Scheme 7.13) [268]

Figure 7.10 Rhodamine 6G chloride solution in methanol emitting yellow light under the influence of a green laser

7.5 Summary of Applications and Derivatives of Xanthene Dyes Xanthene dyes are extremely important class of dyes because of their wide range of biological and pharmaceutical properties, such as agricultural bactericide activity, anti-inflammatory and anti viral, antioxidant, anti-cancer, cytotoxic and antiproliferative properties. These are being utilized as antagonists for paralyzing action of zoxazolamine and in photodynamic therapy. Beside this they are being used in dye lasers and in various photosensitized reactions. Due to the applicability of the xanthenes and benzoxanthenes, several synthetic protocols have been reported, including the reaction of alkylphenoxymagnesium halides with triethylorthoformate, the palladium-catalysed cyclization of polycyclic aryltriflate esters, the cyclocondensation reaction between 2-tetralone and 2-hydroxyarylaldehydes under acidic conditions, and the reaction of the condensation of cyclic 1,3-diketones with aryl aldehydes catalysed by molybdate sulphonic acid. Furthermore, 14-aryl- 14Hdibenzo [a, j] xanthene derivatives can be prepared by the condensation reaction of 2-naphthol with aryl aldehydes in the presence of different lewis acids and Bronsted acids [269-273]

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Objective and Plan of Research: The objective of this thesis was the development of a new synthetic strategy for the preparation of new xanthene derivatives. From the literature survey of xanthene dyes derivatives it was revealed that synthesis and studies of xanthene dyes based on double dianhydride was not done and in the well known methods the reactions were catalyzed by ZnCl2, HCl or H2SO4. So a strategy was adopted to synthesize xanthene dyes from double dianhydrides like biphenyl dianhydride, benzophenone dianhydride, oxidibenzyl dianhydride and naphthalene dianhydride in the presence of ammonium chloride which may act like latent catalyst. Selection of double dianhydride for xanthene dyes made keeping in view the following points To see the effect of double dianhydrides on the thermal and photochemical stability of xanthene dyes. To observe the changes in the absorption and emission properties of xanthene dyes utilizing double dianhydrides either they undergo bathochromic shift or hypsochromic shift. To study the relationship between structure and electrochemical properties by comparison with well known xanthene dyes like fluorescein and rhodamine.

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Chapter 8 RESULTS AND DISCUSSION 8.1 Synthesis of Biphenyl-3,3’,4,4’-tetracarboxylic dianhydride Based Xanthene Dyes (26ae) Xanthene dyes (26a-e) have been synthesized well in excellent yields via the short route following the schemes 1. The rational for the selection of these dyes for synthesis was to acquire various scaffolds of this nature by derivatization which will help in the future development of fluorescent materials for bioimaging. These xanthene derivatives have been synthesized from Biphenyl- 3,3’,4,4’-tetracarboxylic dianhydride condensation with 3,5-Dihydroxybenzoic acid, orcinol, o- Cresol, 3-N,N’-Dimethylamino phenol and Resorcinol. Reaction was catalyzed by ammonium o chloride NH4Cl. Intimate mixture of reactants was heated strongly at 180 C to achieve cyclization and removal of water. Ratio between dianhydrides and substituted phenol 25a-e was adjusted 1:4 for reaction on both sides of reactant 24 [274]. Indication of completeness of the reaction was the formation of solid mass at high temperature, which was soluble in alkali solution. Physical data of these dyes are shown in Table 8.1. Table 8.1 Physical characteristics of xanthene dyes (26a-e) Dye Colour in acidic medium Colour in Basic medium Melting point oC 26a Yellowish brown Yellowish brown >300 26b Yellowish Red Red 230-233 26c Colorless Violet 235-238 26d Pink Pink 240-245 26e Greenish Yellow Greenish Yellow >300

Scheme 8.1 Synthesis of Biphenyl-3,3',4,4'-tetracarboxylic dianhydrides based xanthene dyes (26a-e) 8.1.1 Spectral Characterization of Biphenyl-3,3’,4,4’-tetracarboxylic dianhydride Based

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Xanthene Dyes (26a-e) The structures of newly synthesized xanthene dyes (26a-e) were inveterated by UV, IR, NMR and LCMS studies. The UV-visible absorption spectra of the dyes 26a-e (1×10−7 M) were obtained at room temperature in water (Figure 2.1) and the selected spectral data are summarized in Table 8.2. Table 8.2 Wavelength of maximum absorption λmax of xanthene dyes (26a-e) in water Dye λmax (nm) 26a 447 26b 434 26c 541 26d 552 26e 493

UV-visible spectra of dyes were taken in alkaline water. The electronic transition in molecules provided two absorption maxima (λmax) in their UV.visible spectra (Figure 8.1) first at 262-288 nm and other at different wavelengths in visible region for different dyes. The bands at 260-300 nm for 26a-e were due to π-π* transition of the benzene rings common in all derivatives. This is in agreement with earlier report by Mielgo et al. as per benzenoid uv-visible absorption. The λmax for dye 26a was at 469 nm due to transitions of benzenoid structure formed in basic medium with increasing conjugation of rings along with opening of five membered lactone ring. Similarly the absorption band in 26d existed at 552 nm and imparted it pink color. This was due to more and more availability of electrons decreasing the energy difference between HOMO and LUMO energy levels and thereby increased λmax. The FTIR spectra of xanthene dyes (26a-e) showed absorption bands due to Ar-H, C=O of dianhydride, C=C and C-O, stretching and bending vibrations at 3160- 3448, 1820, 1760, 1589- 1637, 1230-1250 and 723-750 cm-1 respectively. Lactone ring formation was verified due to presence of peak at 1750-1780 cm-1 because of ester group. In particular the peak observed at 1150- 1100 cm-1 was as a result of C-O functionality. The absorption bands at 1618 cm-1 and 750 cm-1 depicted the present of C=C stretching and bending vibrations respectively. The 1H-NMR spectrum of compound 26e (Figure 8.2) showed downfield doublet patteren signal in the aromatic region at 7.53 and 8.249 ppm and singlet signal at 7.682 ppm due to biphenyl dianhydride nuclei. Similarly, two doublet and one singlet signals were observed at 7.93, 8.124 and 7.60 ppm respectively due to condensed substituted phenol 25e. In xanthene dye 26d biphenyl dianhydride peak splitting pattern was same like compound 26e but dye 26d showed singlet signal at 2.90 ppm due to presence of methyl groups on nitrogen atom of 25d nucleus used for synthesis of dye 26d. Difference among xanthene dyes series 26a-e was due to condensed substituted phenols with biphenyl dianhydride. In compound 26c at 2.24 ppm singlet signal was due to CH3 protons and multiplets at 9.3-8.20 (6H m), were seen due to 25c moiety condensed with dianhydride. Similarly for molecule 26b singlet peaks for CH3 and Ar-H were present at 2.4 and 7.83 ppm. 13C-NMR spectra of xanthene also provided the distinguishing signals for the

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synthesized compounds. In this way all xanthene dyes 26a-e synthesis was verified from 1H-NMR and13C-NMR spectra Synthesis of targeted xanthene dyes was also confirmed from LCMS analysis of dyes which showed strong M+1 adduct peaks for the molecular weights of compounds and other peaks at half molecular weight of compounds proving the symmetric nature of compounds (Figure 8.3).

Figure 8.1 Combined UV-Visible spectrum of Biphenyl-3,3',4,4'-tetracarboxylic dianhydride based xanthene dyes (26a-e)

Figure 8.2 1H-NMR spectrum of xanthene dye 26e

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Figure 8.3 LCMS spectrum of xanthene dye 26e 8.1.2 Electrochemical Studies of Biphenyl-3,3’,4,4’-tetracarboxylic Dianhydride Based Xanthene Dyes (26a-e) The electrochemical characterization of these dyes was made by cyclic voltammetry (Figure 8.4) using water having 0.1 M TBAPF6 as a supporting electrolyte. All redox potentials, HOMO (highest occupied molecular orbital), LUMO (lowest unoccupied molecular orbital) and band gap energies (Eg) were calculated from this technique. 8.1.2.1 Redox Potentials (E1/2) Synthesized xanthene dyes (26a-e) exhibited oxidation and reduction potentials on doing the cyclic voltammetric analysis. From the cyclic voltammograms oxidation and reduction potentials were displayed [275-276], which were used to determine redox potentials (E1/2) as shown in table 2.3. Lowest redox potential was observed for 26a which have 3, 5-dihydroxy benzoic acid condensed with biphenyl-3,3’, 4,4’-tetracarboxylic and highest redox potential was seen in 26d and 26e containing 3-N, N’-dimethylamino phenol and resorcinol. It could be visualized from the data in Table 2.3 that xanthene dyes containing electron withdrawing groups had low redox potentials while those containing electron donating groups had high redox potential values. 8.1.2.2 Lowest Unoccupied Molecular Orbital (LUMO) In order to calculate the absolute energies of LUMO level with respect to the vacuum level, the redox data were standardized to the ferrocene/ferricenium couple which had a calculated absolute energy of –4.8 eV [277]. The data related to LUMO level energies of dyes are presented in Table 8.3. Table 8.3 LUMO energy levels of xanthene dyes (26a-e) Dyes E1/2 (V) LUMO (eV) 26a -0.58 -4.22 26b -0.15 -4.65 26c -0.045 -4.755 26d +0.05 -4.85

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26e -0.07 -4.73

Figure 8.4 Cyclic voltammogram of xanthene dyes (26a-e) It was inferred from LUMO energy levels, which vary from -4.20 to -4.85 eV that the electron donating groups on the xanthene motif decreased the energy of LUMO levels while electron withdrawing groups increased the energy of LUMO energy levels. The energy of LUMO levels can be varied only by increasing the delocalization of electrons through alternating single and double bonds and it is noticed that energy difference decreases with increasing conjugation and vice versa. 8.1.2.3 Band Gap Energy (Eg) The optical band gap energies were calculated using the standard procedure. The band gap energy is the span of energies that lies between the valence and conduction bands for insulators and semiconductors. Every solid has its own characteristic energy-band structure. This variation in band structure is responsible for the wide range of electrical characteristics observed in various materials [278-279]. Band gap energy of dyes 26-e are given in Table 2.4. The band gap energy varied from 2.24 to 2.78 eV which was highest for dyes 26a and minimum for dyes 26d which was reliant upon substituent attached to xanthene chromophore. In case of dyes 26a carboxylic group was attached to xanthene motif while 26d had N, N’-dimethylamino group which increased the electron density of chromophore and energy levels got closed to each other and band gap energy was decreased. 8.1.2.4 Highest Occupied Molecular Orbital (HOMO) Table 8.4 depicts the highest occupied molecular orbital energy levels, which were calculated using the standard reported procedure. Considering the energy range from -6.86 to -7.32 eV for xanthene dyes 26a-e, it was observed that for dye 26b HOMO energy levels were at very low energy while 26a had high HOMO energy levels due to mesomorphic and inductive effect of carboxylic groups present in the xanthene chromophore. It was observed that there was a little difference in the effect of electron donating groups on the HOMO energy levels while electron withdrawing groups definitely decreased the energy of HOMO levels. Table 8.4 HOMO energy levels and band gap energy of xanthene dyes (26a-e)

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S. No. Eg(V) HOMO (eV) 26a 2.64 -6.86 26b 2.85 -7.50 26c 2.29 -7.04 26d 2.24 -7.09 26e 2.51 -7.24

8.1.3 Fluorescence Studies of Biphenyl-3,3’,4,4’-tetracarboxylic Dianhydride Based Xanthene Dyes (26a-e)

Fluorescence studies of all the dyes were made by preparing the aqueous solution of dyes (26a-e) but the four dyes 26d, and 26e were found to be highly fluorescent (Table 8.5) exhibiting yellowish green and reddish yellow fluorescence observable to naked eye. These dyes satisfied the requirement of fluorescence that the molecules should be highly conjugated devoid of rotational or vibrational motions as a whole molecule. Although other dyes had also conjugated system of bonds but molecules were flexible and undergoing rotational or vibrational motions which were contrary to fluorescence. The fluorescence spectrum showed only one emission for all dyes except 26a. Emission peak of largest λmax was seen for 26d in this series at 585 nm on excitation of aqueous solution at concentration of 1x10-7 M. Effect of using dianhydrides for the synthesis of xanthene dyes was manifested in their emission spectra which had undergone red shift as compared with rhodamine and fluorescein which had been synthesized from single anhydride (phthalic anhydride). On comparison with the emission spectrum of fluorescein and rhodamine it had been observed that they showed emission wavelength at 513 and 571 nm respectively, while these dyes exhibited emissions at 545-555 and 585-588 nm (Figure 8.5). So these dyes could be applied where usual xanthene fluorescent dyes are used with preference requiring lower energy source for excitation.

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Figure 8.5 Fluorescence spectrum of xanthene dyes 26a-e

Table 8.5 Absorption maximum λmax and emission maximum of xanthene dyes (26a-e) in water

Dye Emission wavelength λem (nm) Absorption wavelength λmax (nm) 26a 445 473

26b 434 486

26c 541 554

26d 552 588

26e 493 555

8.2 Synthesis of Benzophenone-3,3’,4,4’-tetracarboxylic Dianhydride Based Xanthene Dyes (28a-e) Xanthene dyes based upon benzophenone dianhydride have been synthesized following the Scheme 8.2. Reaction occurred through Friedel Crafts acylation mechanism which provided xanthene dyes 28a-e from reaction between benzophenone-3,3’,4,4’-tetracarboxylic dianhydride and substituted phenols (25a-e). Reaction mixture was fused at 180oC to achieve cyclic esters with elimination of water. Stoichiometric ratio between benzophenone dianhydride and substituted phenol 25a-e was adjusted at 1:4 to conduct reaction on both sides of reactants 27. Reaction was

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completed in 1.5h as determined by TLC of reaction mixture and solid mass was formed. Physical data of these dyes are shown in Table 8.6. Table 8.6 Physical characteristics of xanthene dyes (28a-e) Dye Colour in acidic medium Colour in Basic medium Melting point oC 28a Yellowish brown Yellowish brown >300 28b Yellowish Red Red 263-265 28c Colorless Violet 258-262 28d Pink Pink 265-267 28e Greenish yellow Greenish yellow >300

Scheme 8.2 Synthesis of Benzophenon-3,3',4,4'-tetracarboxylic dianhydrides based Xanthene dyes (28a-e) 8.2.1 Spectral Characterization of Benzophenone-3,3’,4,4’-tetracarboxylic dianhydride Based Xanthene Dyes (28a-e) The structures of newly synthesized xanthene dyes (28a-e) were elucidated by UV, FTIR, LCMS, 1H-NMR and 13C-NMR studies. The UV-visible absorption spectra of the dyes 28a-e (1×10−7M) were obtained at room temperature in water (Figure 8.6) and the selected spectral data is summarized in Table 8.7. Table 8.7 Wavelength of maximum absorption λmax of xanthene dyes (28a-e) in water Dye λmax (nm) 28a 465 28b 445 28c 531 28d 548 28e 490

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The derivatives of Benzophenon-3,3’,4,4’-tetracarboxylic dianhydride 28c and 28e provided only one absorption band, two and three for 28a and 28b respectively. The bands at 260-300 nm for 28a-e were due to π-π* transition of the benzene rings common in all derivatives. Similarly the absorption band in 28d was present at 548 nm. This was due to more and more delocalization of electrons causing the reduction in energy between HOMO and LUMO energy levels. There by it had lager λmax. In case of 28a λmax was 445 nm while for 28b, 28c and 28e λmax was at 478, 531 and 490 nm respectively. All this was ascribed to π-π* and n-π* transitions of lone pairs and πbonded electrons [280]. The FTIR spectra of xanthene dyes (28a-e) exhibited IR absorption peaks due to IR active functionalities within molecules which are Ar-H, C=O of dianhydride, C=C and C-O. These peaks are generated due to stretching and bending vibrations of functional groups. Lactone moiety was confirmed by the appearance of peaks in the range 1750-11770 cm-1 due to carbonyl group. In particular the peak observed at 1150-1100 cm-1 was as a result of carbon oxygen bond stretching vibrations. The absorption bands at 1618 and 750 cm-1 depicted the present of C=C stretching and bending vibrations respectively for aromatic nuclei. In case of 28a-e dyes benzophenone dianhydride was condensed with different substituted phenols 25a-e. The difference in this series was because of different phenols. In dye 28e two doublets and one singlet were present at 7.53, 8.249 and 7.60 ppm, respectively due to benzophenone dianhydride (27) nucleus. The splitting pattern of the central core remained same throughout this series, but position was varied little extent. A similar pattern was seen for 28d except for that of singlet peak at 2.92 ppm due to condensed 3-N,N’-Dimethylamino phenol. The dye 28b showed signals at 2.35 (s), 6.695 (s), and 6.45(s) ppm because of CH3 and Ar-H of phenolic component 25b and 28c represented the signal splitting pattern different from 28b owing to different phenolic isomer at 2.35 (6H s), 6.95 (2H d) 6.79 (2H d), 6.96 (2H t) 7.52 (2H s) ppm (Figure 8.7). In case of dye 28a aromatic signal splitting pattern was similar to that of 28b except to that of carboxylic peak present at 11.35 ppm. The 13C-NMR spectrum of compounds showed fifteen aromatic carbon atoms in the range 102.45-190.78 ppm (Figure 8.8). The synthesized dyes were also confirmed by their LCMS study which showed strong M+1 adduct peaks for the molecular weights of compounds (Figure 8.9).

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Figure 8.6 Combined UV spectrum of benzophenone-3,3',4,4'-tetracarboxylic dianhydride based xanthene dyes (28a-e)

Figure 8.7 1H-NMR of Benzophenone-3,3',4,4'-tetracarboxylic dianhydride based xanthene dye 28e

Figure 8.8 13C-NMR of Benzophenone-3,3',4,4'-tetracarboxylic dianhydride based xanthene dye 28e

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Figure 8.9 LCMS of Benzophenone-3,3',4,4'-tetracarboxylic dianhydride based xanthene dye 28e 8.2.2 Electrochemical Studies of Benzophenone-3,3’,4,4’-tetracarboxylic dianhydride Based Xanthene Dyes (28a-e) The electrochemical characterization of these dyes was made by cyclic voltammetry (Figure 8.10) using water having 0.1 M TBAPF6 as a supporting electrolyte. All redox potentials, HOMO (highest occupied molecular orbital), LUMO (lowest unoccupied molecular orbital) and band gap energies (Eg) were calculated from this technique.

Volt

Figure 8.10 Combined cyclic voltammogram of Benzophenone-3,3',4,4'-tetracarboxylic dianhydride based xanthene dye (28a-e)

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8.2.2.1 Redox Potentials (E1/2) Synthesized xanthene dyes (28a-e) exhibited redox potentials using the cyclic voltammetric analysis. From the cyclic voltammograms oxidation and reduction potentials were calculated to determine redox potentials (E1/2) as shown in Table 8.8. Lowest redox potential was observed for 28a dye which has 3, 5-Dihydroxy benzoic acid condensed with benzophenone- 3,3’,4,4’tetracarboxylic dianhydride and highest redox potential was seen in 28d and 28e containing 3-N, N’-dimethylamino phenol and resorcinol. It could be visualized from the data in table 2.8 that xanthene dyes containing electron withdrawing groups have low redox potentials while those containing electron donating groups have high redox potential values. 8.2.2.2 Lowest Unoccupied Molecular Orbital (LUMO) In order to calculate the absolute energies of LUMO level with respect to the vacuum level, the redox data were standardized to the ferrocene/ferricenium couple having energy –4.8 eV. The data related to LUMO level energies of dyes are presented in Table 8.8. Table 8.8 LUMO energy levels of xanthene dyes (28a-e) S. No. E1/2 (V) LUMO (eV) 28a -0.065 -4.735 28b -0.6 -4.20 28c -0.01 -4.79 28d +0.05 -4.85 28e -0.02 -4.78 It was apparant from LUMO energy levels, whose values are in range -4.20 to -4.85 eV, that the electron donating groups on the xanthene motif decreases the energy of LUMO levels while electron withdrawing groups increase the energy of LUMO energy levels. The energy of LUMO levels can be varied only by increasing the delocalization of electrons through alternating single and double bonds and it was noticed that energy difference decreases with increasing conjugation and vice versa. 8.2.2.3 Band Gap Energy (Eg) The optical band gap energies are calculated using the standard procedure. Every solid has its own characteristic energy-band structure. This variation in band structure is responsible for the wide range of electrical characteristics observed in various materials [281]. Band gap energy of dyes is given in Table 8.9. The band gap energy varied from 2.24 to 2.78 eV was highest for dye 28a and minimum for dye 28d which was due to substituents attached to xanthene chromophore. In case of dye 28a carboxylic group was attached to xanthene motif while 28d had N, N’dimethylamino group which increased the electron density of chromophore and energy levels got closed to each other and band gap energy decreased. 2.2.2.4 Highest Occupied Molecular Orbital (HOMO) Table 8.9 depicts the highest occupied molecular orbital energy levels, which were calculated using the Bredas equation. Considering the energy range from -6.86 to -7.32 eV for xanthene dyes 28a- e, it was observed that for dye 28b HOMO energy levels were at high energy while 28a had low

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energy HOMO levels due to mesomorphic and inductive effect of carboxylic groups present in the xanthene chromophore. It is observed that there is little difference in the effect of electron donating groups on the HOMO energy levels while electron withdrawing groups definitely increase the energy of HOMO levels. Table 8.9 HOMO energy levels and band gap energy of xanthene dyes (28a-e) Dyes Eg(eV) HOMO (eV) 28a 2.59 -7.32 28b 2.78 -6.98 28c 2.33 -7.12 28d 2.26 -7.11 28e 2.53 -7.31

8.2.3 Fluorescence Studies of Benzophenone-3,3’,4,4’-tetracarboxylic dianhydride Based Xanthene Dyes (28a-e) Fluorescence studies of all the dyes were made by preparing the aqueous solution of dyes (28a-e) but the two dyes 28d and 28e were found to be highly fluorescent (Table 8.10) exhibiting greenish yellow fluorescence under visible and UV.light observable to naked eye. The dyes 28ac were non fluorescent due to flexibility and exhibited rotational or vibrational motions which were contrary to fluorescence. Fluorescence spectrum of dyes 28a-e is shown below in figure 8.11 which was recorded by selecting different excitation wavelengths of the source [282]. The fluorescence spectrum showed only one emission for all dyes except 28a. Emission peak of lowest frequency was seen for 28d at 585nm on excitation of aqueous solution at concentration of 1x10-7 M. Table 8.10 Fluorescence data of xanthene dyes (28a-e) in water Dye Excitation wavelength (nm) Emission wavelength (nm) Stoke Shift 28a 455 473 18 28b 475 486 11 28c 543 554 11 28d 558 588 30 28e 500 555 55

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Figure 8.11 Fluorescence spectra of xanthene dyes (28a-e) in water Effect of using dianhydrides for the synthesis of xanthene dyes was manifested in their emission spectra which have undergone red shift as compared with rhodamine and fluorescein So these dyes can be applied where usual xanthene fluorescent dyes are used with preference requiring lower energy source for excitation. 8.3 Synthesis of 4, 4'-Oxydiphthalic Dianhydride Based Xanthene Dyes (30a-e) Symmetric xanthene dyes have been synthesized well in excellent yields and high purity in the solvent free conditions following the Schemes 8.3. The rational for selection of these dyes for synthesis, is to acquire various scaffolds of this nature by derivatization which will help in the development of dye lasers, solar cells and fluorescent bioimaging. Here xanthene dyes (30a-e) have been synthesized comprising 4,4'-Oxydiphthalic dianhydride condensed with 3,5Dihydroxybenzoic acid, Orcinol, o- Cresol, 3-N,N’-Dimethyl amino phenol and Resorcinol. Ammonium chloride was used as catalyst for condensation of substituted phenol with dianhydrides and reaction occured through friedel craft acylation pathway due to the presence of HCl provided o by the breakage of NH4Cl. Intimate mixture of reactants was heated strongly at 180 C to achieve cyclization and removal of water. Ratio between dianhydrides and substituted phenols 25a-e was adjusted 1:4 for reaction at both sides of reactant 29. Indication of completeness of reaction was the formation of alkali soluble solid mass at high temperature. Physical data of these dyes are shown in Table 8.11. Table 8.11 Physical characteristics of xanthene dyes (30a-e) Dyes Colour in Acidic medium Colour in Basic medium 30a Yellowish brown Yellowish brown 30b Yellowish Red Red 30c Colorless Violet 30d Pink Pink

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30e Greenish Yellow Greenish Yellow

Scheme 8.3 Synthesis of 4,4'-Oxydiphthalic anhydride based Xanthene Dyes (30a-e) 8.3.1 Spectral Characterization of 4, 4'-Oxydiphthalic Dianhydride Based Xanthene Dyes (30a-e) The structures of newly synthesized xanthene dyes (30a-e) were determined by UV, IR, NMR and LCMS studies. The UV-Visible absorption spectra of the dyes 30a-e (1×10−7 M) were obtained at room temperature in water (Figure 2.12) and the selected spectral data are summarized in Table 8.12. Table 8.12 Wavelength of maximum absorption (λmax/nm) of xanthene dyes (30a-e) in water Dye Solvent λmax (nm) 30a Water 453 30b Water 430 30c Water 558 30d Water 550 30e Water 501

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Figure 8.12 UV/visible spectrum of 4,4'-Oxydiphthalic anhydride based Xanthene Dyes (30a-e) UV-visible spectra of all dyes (30a-e) were taken in water. It was observed from their UV- visible spectra of dyes 30a-e, that dye 30a exhibited two absorption maxima one at 300 nm and other at 453nm, while all other dyes showed one absorption band in the visible region (Figure 8.12). The bands at 260-300 nm for 30a-e were due to π-π* transition of the benzene rings common in all derivatives [283]. The λmax for 30a is 453 nm was due to π-π* transitions of benzenoid structure formed in basic medium with increasing conjugation of rings with opening of five membered lactone ring. Similarly the absorption band in 30d lies at 550 nm giving them pink color. This is due more and more availability of electrons decreasing the energy difference between HOMO and LUMO energy levels. There by it has lager λmax. The FTIR spectra of xanthene dyes (30a-e) provided absorption bands due to different functional groups including Ar-H, C=O of lactone, C=O of carboxyl, C=C and C-O, stretching and bending vibrations at 3118-3140, 1782-1796, 1742-1754, 1620-1652, 1576-1594, 11211149, 836- 860 and 793-818 cm-1 respectively. In case of dye 30d, lactone formation was confirmed by the appearance of peak at 1789 cm-1 and opening of lactone peak at 1742 cm-1 due to carbonyl group of carboxyl, and are shifted to high frequency region because of five membered lactone ring formation and these peaks are common in all dyes. The absorption bands at 1630, 1592 and 859 cm-1 depicted the present of C=C stretching and bending vibrations respectively in aromatic moieties. The 1H-NMR spectrum of compound 30d showed 12H singlet peak at 2.83 ppm and 12H singlet at 2.99 ppm due to two CH3 substituents attached to nitrogen atom. Singlet peak at 6.52 ppm was due to aromatic proton adjacent to oxygen atom of xanthene chromophore. Doublets at 6.77 and 6.99 ppm were due to two mutually coupled aromatic protons attached to xanthenic part of molecule. Doublet signal at 7.34 and 7.73 ppm were because of dianhydride part of molecule, and a singlet signal at 7.05 ppm was because of aromatic isolated proton at phenyl ring of dianhydride. Difference among 30a-e series was due to condensed substituted phenols with biphenyl dianhydride. In xanthene 30c singlet signal at 2.24 ppm was due to CH3 protons and multiplet at

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93-8.20 (6H m), was seen due to 25c condensed with dianhydride. Similarly for dye 30b singlet signals for CH3 and Ar-H were present at 2.4 and 7.83 ppm. Molecular weight confirmation of the synthesized dyes was also achieved by the LCMS analysis of dyes which showed strong M+1 adduct peaks for compounds (Figure 8.13).

Figure 8.13 LCMS spectrum of 4,4'-Oxydiphthalic anhydride based Xanthene Dye 30d 8.3.2 Electrochemical Properties of 4, 4'-Oxydiphthalic dianhydride Based Xanthene Dyes (30a-e) The electrochemical characterization of these dyes was made by cyclic voltammetry (Figure 8.14) using water having 0.1 M TBAPF6 as a supporting electrolyte. All redox potentials, HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) were determined from cyclic voltammograms.

Figure 8.14 Combined cyclic voltammogram of xanthene dyes (30a-e) Synthesized xanthene dyes (30a-e) showed oxidation and reduction potentials on conducting cyclic voltammetric analysis. From the cyclic voltammograms oxidation and reduction potentials were calculated to determine redox potentials (E1/2) as shown in Table 8.13. Lowest

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redox potential was observed for 30a dyes which have 3, 5-Dihydroxy benzoic acid condensed with 4, 4'-Oxydiphthalic anhydride and highest redox potential was seen for 30c and 30d containing o-Cresol. It can be visualized from the data in table 2.13 that xanthene dyes containing electron withdrawing groups have low redox potentials while those containing electron donating groups have high redox potential values. The data related to LUMO level energies of dyes are presented in Table 8.13. Table 8.13 LUMO eV energy levels of 4,4'-Oxydiphthalic dianhydride based xanthene dyes (30a- e) S.No. E1/2 (V) LUMO (eV) 30a -0.350 -4.45 30b -0.125 -4.67 30c -0.04 -4.76 30d +0.07 -4.87 30e -0.02 -4.78

It was inferred from LUMO energy levels which varies from -4.16 to -4.87eV that there electron donating groups on the xanthene motif decreased the energy of LUMO levels while electron withdrawing groups increased the energy of LUMO energy levels. The energy of LUMO levels can be varied only by increasing the delocalization of electrons through alternating single and double bonds and it is noticed that energy difference decreases with increasing conjugation and vice versa. The optical band gap values were calculated using the standard procedure [284-285]. Band gap energy of dyes is given in Table 8.14. The band gap energy varies from 2.19 to 2.87 eV which is highest for dyes 30a, 30b and minimum for dyes 30c and 30d which depends upon substituents attached to xanthene chromophore as well as on the precursor (dianhydride) utilized for synthesis of dyes. In case of dye 30a carboxylic group was attached to xanthene motif while 30d has N, N’-Dimethylamino and hydroxyl groups which increases the electron density of chromophore and energy levels become close to each other and band gap energy was decreased. Table 8.14 depicts the highest occupied molecular orbital energy levels, which are calculated using the standard reported procedure [286]. Considering the energy range from -6.64 to -7.54 eV for xanthene dyes (30a-e) it was observed that for dye 30a, HOMO energy levels are at very low energy carboxylic groups present in the xanthene chromophore. It was observed that there is little difference in the effect of electron donating groups on the HOMO energy levels while electron withdrawing groups definitely increase energy of HOMO levels by increasing the energy gap between HOMO and LUMO. Table 8.14 HOMO eV energy levels and band gap energy of 4,4'-Oxydiphthalic dianhydride based xanthene dyes (30a-e) Dyes. Eg(eV) HOMO (eV) 30a 2.87 -7.54 30b 2.82 -7.27

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30c 2.19 -6.95 30d 2.25 -7.12 30e 2.53 -7.33

8.3.3 Fluorescence Studies of 4, 4'-Oxydiphthalic dianhydride Based Xanthene Dyes (30a-e) Fluorescence studies of all the dyes were made by preparing the aqueous solution of dyes (30a-e) and dyes 30d and 30e were found to be highly fluorescent (Table 8.15). These dyes has conjugated framework of single and double bonds and were devoid of rotational or vibrational motions as a whole molecule. Fluorescence spectrum of all dyes 30a-e is shown below in Figure 8.15 which was recorded by selecting different excitation wavelengths of the source. Fluorescence spectrum provided the intensity contribution to the observed emission at a given wavelength by different excitation wavelengths for the sample is exposed. The fluorescence spectrum showed only one emission for all dyes except 30a. Emission peak of largest wavelength was seen for 30d at 598 nm on excitation of aqueous solution at concentration of 1x10-7 M. Table 8.15 Fluorescence data of xanthene dyes (30a-e) in water Dye Excitation wavelength (nm) Emission wavelength (nm) Stoke Shift 30a 465 492 27 30b 488 505 17 30c 541 557 16 30d 560 598 38 30e 510 557 45

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Figure 8.15 Fluorescence spectrum of 4,4'-Oxydiphthalic anhydride based xanthene Dyes (30a- e) 8.4 Synthesis of 1, 4, 5, 8-Naphthalenetetracarboxylic dianhydride based xanthene Dyes (32a-e) Synthesis of naphthalene dianhydride based symmetric xanthene dyes has been achieved well in valuable yields under solvent free conditions following the route illustrated in scheme 8.4. The motive for selection of these dyes for synthesis is to achieve several molecules of this nature by derivatization which may help in the development of dye lasers, reprographic processes solar cells and fluorescent biolabelling. Symmetric xanthene dyes 32a-e were accomplished from condensation of 1, 4, 5, 8-Naphthalenetetracarboxylic dianhydride with different substituted phenols (25a-e) catalyzed by ammonium chloride. Ammonium chloride acted as latent catalyst which provided HCl after its breakage under fused reaction conditions to catalyze the reaction. Intimate mixture of reactants was fused at 180oC to obtain cyclization with removal of water. Mole ratio between dianhydride and substituted phenols 25a-e was kept at 1:4 to conduct the reaction at both sides of dianhydride. Reaction was completed in 1h as determined by TLC and resulted into a solid mass at 180oC. Products were purified by dissolving in alkaline solution and then acidified to regenerate the pure precipitate of dyes 32a-e. Physical data of these dyes are shown in Table 8.16. Table 8.16 Physical Characteristics of 1,4,5,8-Naphthalenetetracarboxylic dianhydride based Xanthene Dyes 32a-e Dyes Colour in Acidic Medium Colour in Basic Medium 32a Yellowish brown Yellowish brown 32b Yellowish Red Red 32c Colorless Violet 32d Pink Pink 32e Greenish yellow Greenish yellow

Scheme 8.4 Synthesis of 1,4,5,8-Naphthalenetetracarboxylic dianhydride based xanthene Dyes

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(32a-e) 8.4.1 Spectral Characterization of 1, 4, 5, 8-naphthalenetetracarboxylic dianhydride based xanthene Dyes (32a-e) The structures of newly synthesized compounds were confirmed by UV, FTIR, LCMS and NMR studies. The UV visible absorption spectra of the dyes 32a-e (1×10−7 M) were obtained at room temperature in water (Figure 8.16) and the selected spectral data are summarized in Table 8.17. Table 8.17 Wavelength of maximum absorption (λmax/nm) of xanthene dyes (32a-e) in water Dye Solvent λmax (nm) 32a Water 465 32b Water 481 32c Water 521 32d Water 545 32e Water 503

Figure 8.16 UV-Visible spectrum of xanthene dyes (32a-e) UV-visible spectra of all dyes (32a-e) were taken in water and one absorption band was provided by 32b and 32c and two bands were seen for 32d and 32e, and three for 32a. The bands at 260- 300 nm for 32a-e was ascribed to π-π* transition of the benzene rings common in all derivatives. Similarly the absorption band in 32e was present at 545 nm and this absorption imparted it yellowish color. This is due to more and more delocalization of electrons decreasing the energy difference between HOMO and LUMO energy levels and bathochromic shift in λmax was observed. In case of xanthene 32a λmax was at 465 nm while that for 32b, 32c and 32e absorption maxima

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were at 481, 521 and 503 nm respectively. All this was due to R-band and K-band transitions in these molecules. The FTIR spectra of xanthene dyes (32a-e) proved the presence of different functional groups in xanthene dyes. These were due to Ar-H, C=O of lactone, C=C and C- O, stretching and bending vibrations at 3118-3140, 1782-1796, 1742-1754, 1620-1652, 1576-1594, 1121-1149, 836-860 and 793-818 cm-1 respectively. FTIR spectrum of dye 32e, showed hydroxyl group peak at 3315 and 3118 cm-1 which were owing to hydroxyl group and C=C-H stretching vibrations (Figure 8.17). Five membered lactone ring formation and its opening was confirmed from the two peaks at 1790 and 1750 cm-1 in the spectrum. The other peaks in the spectrum were also in favour of the different functionalities in the molecule. In this way all other dyes functional groups have been confirmed from their respective FTIR spectra.

D:\Chem 320 Noor\CODE-33.0 CODE-33 Instrument type and / or accessory 06/04/2015

Page 1/1 Figure 8.17 FTIR spectrum of 1,4,5,8-Naphthalenetetracarboxylic dianhydride based dye 32e In these symmetric xanthene dyes based on naphthalene dianhydride condensed with different substituted phenols 25a-e difference lies in substituents at phenols. In 1H-NMR of dye 32e two doublets at 6.03 and 6.55 ppm and one singlet at 5.96 ppm respectively was due to xanthene chromophore of the molecule. Doublets at 7.22 and 7.44 ppm were due to two mutually coupled protons at naphthalene ring (Figure 8.18). Splitting pattern of the central core remained same throughout this series, but position was varied. Similar pattern was observed for 32d except to that of two singlet peak at 2.35 and 2.99 ppm due to condensed 3-N,N’-Dimethylamino phenol. The dye 32b showed signals at 2.35 (s), 6.695 (s), and 6.45(s) ppm because of CH3 and Ar-H of phenolic component 25b and 32c represented the signals splitting pattern different from 32b owing to different phenolic isomer at 2.35 (6H s), 6.95 (2H d) 6.79 (2H d), 6.96 (2H t) 7.52 (2H s) ppm. In dye 32a aromatic signals splitting pattern was similar to that of 32b except to that of carboxylic peak present at 11.35 ppm. The 13C-NMR spectrum of 32e showed ten aromatic carbons atoms in the range 102.42-169.40 ppm.

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Figure 8.18 1H-NMR spectrum of 1,4,5,8-Naphthalenetetracarboxylic dianhydride based dye 32e Confirmation of the synthesized dyes were also confirmed by the LCMS analysis of dyes which showed strong M+1 adduct peaks for the molecular weights of compounds (Figure 8.19).

Figure 8.19 LCMS spectrum of 1,4,5,8-Naphthalenetetracarboxylic dianhydride based xanthene dye 32e 8.4.2 Electrochemical Properties of 1, 4, 5, 8-Naphthalenetetracarboxylic dianhydride Based Xanthene Dyes (32a-e) Synthesized xanthene dyes (32a-e) showed oxidation and reduction potentials on conducting cyclic voltammetric analysis. From the cyclic voltammograms oxidation and reduction potentials were calculated to determine redox potentials (E1/2) as shown in Table 8.18. Behavior of dyes based on 1,4,5,8-Naphthalenetetracarboxylic dianhydride is different from dyes containing 4, 4'Oxydiphthalic anhydride precursor. Here lowest E1/2 was found for 32d containing dimethylamino group. Although this group is electron donating, but after donation of electron lactone ring is opened leaving two carboxylic groups directly linked to single aromatic nucleus which makes the ring highly deficient leading to lower redox values. Similar behavior was seen

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for dye 32e. Fused ring system in dyes 32a-e is responsible for their conversing behavior. The data related to LUMO level energies of dyes are presented in Table 8.18. Table 8.18 LUMO eV energy levels of 1,4,5,8-Naphthalenetetracarboxylic dianhydride based xanthene dyes (32a-e) S.No. E1/2 (V) LUMO (eV) 32a -0.095 -4.705 32b -0.64 -4.16 32c -0.05 -4.805 32d -0.820 -4.39 32e -0.225 -4.50

It is obvious from the energy of LUMO levels which range from -4.16 to -4.87eV that there electron donating groups on the xanthene motif decreased the energy of LUMO levels while electron withdrawing groups decreased the energy of HOMO energy levels. The optical band gap values are calculated using the standard procedure. The band gap energy is the span of energies that lies between the valence and conduction bands for insulators and semiconductors. Every solid has its own characteristic energy-band structure. This variation in band structure is responsible for the wide range of electrical characteristics observed in various materials. Band gap energy of dyes is given in Table 8.19. The band gap energy varied from 2.19 to 2.87 eV which is maximum for dye 32a and lowest for dyes 32c and 32e which depends upon substituents attached to xanthene chromophore as well as on the precursor (dianhydride) utilized for synthesis of dyes. In case of dye 32a carboxylic group is attached to xanthene motif while 32e have N, N’-Dimethylamino and hydroxyl groups which increases the electron density of chromophore and energy levels become close to each other and band gap energy is decreased. Table 8.19 depicts the highest occupied molecular orbital energy levels, which are calculated using the standard reported procedure Considering the energy range from -6.64 to -7.54 eV for xanthene dyes 32a-e, it is observed that for dye 32a HOMO energy levels are at very low energy carboxylic groups present in the xanthene chromophore while 32b and 32e have high HOMO energy levels due to resonance and inductive effect of hydroxyl groups present in dye molecule. It was observed that there was little difference in the effect of electron donating groups on the HOMO energy levels while electron withdrawing groups definitely increase energy of HOMO levels by increasing the energy gap between HOMO and LUMO. Table 8.19 HOMO/eV energy levels and band gap energy of xanthene dyes (32a-e) S.No. Eg(eV) HOMO (eV) 32a 2.61 -7.31 32b 2.59 -6.64 32c 2.25 -7.05 32d 2.46 -6.96

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32e 2.26 -6.65

8.4.3 Fluorescence Studies of 1, 4, 5, 8-Naphthalenetetracarboxylic dianhydride based xanthene Dyes (32a-e) Fluorescence studies of all the dyes were conducted by preparing the aqueous solution of dyes (32a-e) but there were only two dyes 32d and 32e which were found to be highly fluorescent (Table 8.20). Fluorescence spectrum of all dyes 32a-e is shown in Figure 8.20, which was recorded by selecting different excitation wavelengths of the source. Fluorescence spectrum provides the intensity contribution to the observed emission at a given wavelength by different excitation wavelengths for the sample is exposed. Emission peak of lowest frequency was present at 598 nm for 32d on excitation of aqueous solution at concentration of 1x10-7M.

Table 8.20 Fluorescence data of 1,4,5,8-Naphthalenetetracarboxylic dianhydride based xanthene dyes (32a-e) in water Dye Excitation wavelength (nm) Emission wavelength (nm) Stoke Shift 32a 465 492 27 32b 488 505 17 32c 541 557 16 32d 560 598 38 32e 510 557 45

Figure 8.20 Fluorescence spectra of xanthene dyes (32a-e) in water

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8.5 Synthesis of Xanthene Schiff Bases (35a-j) Synthesis of xanthene schiff bases 35a-j were synthesized from fluorescein whose synthesis was accomplished according to standard procedure utilizing phthalic anhydride and resorcinol in the presence of lewis acid catalyst ZnCl2 (Scheme 8.5). Fluorescein was treated with hydrazine in ethanol at reflux temperature to prepare xanthene imide intermediate. The intermediate 33 was reacted with different substituted aldehydes 34a-j to achieve targeted schiff bases 35a-j. The schiff bases were synthesized by following the usual procedure of schiff base formation according to scheme (Scheme 8.5). The reaction mixture was refluxed for 10-12h to obtain the products. Acidic medium increased the electrophilicity of carbonyl carbon of aldehydes and facilitated the attack of NH2 group of xanthene intermediate motif 33 and thereby made possible the synthesis of sterically hindered schiff bases [287].

Scheme 8.5 Synthesis of fluorescein Schiff bases 35a-j 8.5.1 Characterization of Xanthene Schiff Bases (35a-f) Xanthene schiff bases were characterized by spectroscopic techniques like UV.visible, FTIR and NMR. Strong absorptions of visible radiations in the range 470-485 nm ensured the presence of highly conjugated aromatic ring system in the schiff bases. All the schiff bases provided one band in the visible region which is due to π-π* and n-π* transitions. FTIR studies of schiff bases provided the absorptions peaks due to the presence of different functional groups stretching and bending vibrations. The common functional groups which are present in all schiff bases are, O-H, C-H, C=O, C=C, C=N, C-O and C-N whose peaks are present at respective positions in the FTIR spectra of compounds. Hydroxyl group IR absorption peaks are in the range 3630-3640/cm while the peaks for N-H group are absent due to schiff base formation. The carbonyl group of schiff bases showed peak in the range 1680-1690/cm due to imide formation and aromatic rings C=C peaks are present at 1624-1635, 1554-1560 and 15011510/cm. The peaks for CH=N group are seen in their FTIR spectra at 1485-1496/cm which are confirmation for

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synthesis of compounds and C-O, C-N peaks are present at their respective positions in the range 1050-1090 and 1010-1030/cm. In this way all schiff bases have been confirmed for their functional groups from their FTIR spectra. 1H-NMR and 13C-NMR provided the conclusive in favour of the synthesis of targeted compounds. The 1H-NMR of compound 35a showed the singlet signal at 8.12ppm due imine proton and a pair of doublets at δ 7.81 and 7.57 ppm due to mutually coupled protons of 4bromophenyl ring. Two sets of doublet of doublets at 7.38 and 7.23 ppm and doublet signals at 7.26 and 7.79 ppm are aromatic protons of phthalic anhydride ring of Schiff bases. Mutually coupled doublets at 6.87 and 6.35 ppm are resulted due to Ar-H of phenolic rings while a singlet peak at 6.29 ppm is to one isolated proton ortho to OH. C-NMR of compound 14a depicted the signals for C=O, C=N and methine carbon at 168.0, 155.9 and 60.5ppm respectively. Presence of imine signal and methine signals are confirmation for synthesized compounds, while the fourteen aromatic carbon signals at 152.3, 143.2, 139.3, 132.2, 131.7, 131.2, 128.3, 127.2, 126.3, 125.4, 117.3, 109.2, 105.9 ppm showed the aromatic skelton of molecule. In this way other compounds 35b-j have been confirmed for their structure from 1H-NMR and 13C-NMR studies. 8.6 Applications of Xanthene Dyes Synthesized dyes 26d, 26e, 28d and 28e were tested for cell staining. Dyes exhibited differential staining on onion cells. Dyes 26d and 28d were concentrated inside the cells nuclei while dyes 26e and 28e stained more the cell membrane. This differential staining was further judged from excitation of stained cells with UV-light, and yellowish light was emitted from cell membrane and cell wall. Dyes 26d and 28d have dimethylamino groups on xanthene chromophore which have more interaction with cells nuclei being proteinaceous in nature and dyes adsorbed and stained through lone pair interaction. Dyes 26e and 28e have hydroxyl groups on xanthene chromophore which have more interaction with cell wall and cell membrane which are made up of carbohydrates and lipids, dyes interact through hydrogen bonding and absorbed more toward cytoplasmic portion (Figure 8.21).

Figure 8.21 a) Dye 26e applied on onion cells, concentrated in cell membrane, b) Dye 26e emitting yellow fluorescence under UV-light.

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Chapter 9 EXPERIMENTAL 9.1 Materials Biphenyl-3,3’,4,4’-tetracarboxylic dianhydride, benzophenone- 3,3’,4,4’tetracarboxylic dianhydride, Phthalic anhydride, Hydrazine, 4, 4′-Oxydiphthalic anhydride and 1,4,5,8-Naphthalenetetracarboxylic dianhydride were obtained from sigma aldrich. Resorcinol, o-cresol, 3, 5-dihydroxy benzoic acid, orcinol, and 3-N, N’-dimethylamino phenol was obtained from BDH. Ammonium chloride and zinc chloride was purchased from Merck. Hydrochloric acid was purchased from Merck. Solvents such as ethanol, ethyl acetate, dichloromethane, DMSO, DMF, acetone, chloroform, and methanol were common laboratory grade chemicals and were purified before use. 9.2 Purification of Solvents Standard methods and procedures were followed for the purification and drying of solvents. The dried solvents were stored over type 4A° molecular sieves. Same purified solvents were used as discussed at page 36-37. 9.3 Instruments Used Melting points were determined using digital Gallenkamp (Sanyo) model MPD BM 3.5 with digital thermometer and are uncorrected. Infrared spectra were recorded using a Shimadzu IR 460 as KBr pellets and FTX 3000 MX spectrophotometer using ATR method. 1H-NMR and 13C- NMR spectra

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were obtained using a Bruker AM (300 MHz, 75 MHz) and (400 MHz, 100 MHz) spectrophotometers respectively in CDCl3, DMSO-d6, CD3OD-d4 solution using TMS as an internal reference. Chemical shift are given in δ-scale (ppm). Abbreviations s, d, dd, t, at, m have been used for singlet, doublet, double doublet, triplet, apparent triplet, multiplet respectively. Elemental analyses were performed on CHNS 932 LECO instrument. UV-Vis spectra were taken by CECIL-7400 UV/Visible Spectrophotometer and fluorescence spectra were recorded using the Hitachi FL solutions 7000 fluorescence spectrophotometer. Cyclic voltammetry was performed on CH-800 C potentiostate using 0.1M TBAPF6 as internal reference in DMSO on glassy carbon and platinum (0.2 mm diameter) as working electrodes versus Ag/AgCl reference electrode and platinum wire as counter electrode at room temperature. 9.4 Chromatographic Techniques 9.4.1 Thin Layer Chromatography (TLC) Same procedure and mobile phases were used to monitor the progress of reactions as discussed at pages 55-56. 9.5 Experimental Procedures 9.5.1 General Procedure for Synthesis of Biphenyl-3,3’,4,4’-tetracarboxylic dianhydride Based Xanthene dyes (26a-e) In a 250 ml round bottom flask a well mixed mixture of 0.294 gm (0.001mol) of biphenyl3,3’,4,4’- tetracarboxylic dianhydride (1) and substituted phenols (0.004mol) (25a-e Scheme 1) were fused in oil bath at 170oC. The fused melt was added 0.2 g of NH4Cl, and stirred the reaction mixture mechanically at 180-190oC until the solid mass obtained. The solid mass was dissolved in 10 ml of 5 % sodium hydroxide solution. The solution was filtered to remove any insoluble impurities and filtrate was treated with 3 ml of 30% hydrochloric acid and precipitated the dyes (26a-e).

6,6''-Oxybis (3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-1',8'-dicarb oxylic acid) (26a)

o Yellowish brown, m.p> 250 C; Rf: 0.34 (ethyl acetate: ethanol 1: 1); λmax (nm): 453; FTIR (Neat) ν: 3300-3500 (br, COOH, OH), 3130 (C=C-H, str), 1782 (lactone C=O), 1753 (carboxylic C=O), 1642 (C=C), 1588 (C=C), 1128 (C-O), 853 (Ar-H, bend), 810 (Ar-H, bend) cm-1. 1H-

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NMR (DMSO-d6, 300 MHz) δ (ppm): 12.24 (s, 4H, COOH), 8.40 (s, 2H, 4OH), 7.90 (d, 2H, J=8.3Hz), 7.30 (m, 2H), 7.13 (d, 2H, J=8.4Hz), 6.90 (s, 4H), 6.75 (s, 4H). 13C-NMR (DMSO 75 MHz) δ (ppm): 173.45, 167.81, 161.72, 156.64, 153.45, 132.62, 130.85, 123.46, 119.69, 116.12, 113.98, 110.13, 107.96. Anal. Calcd. For C44H22O19: C, 61.84 H, 2.59; Found: C, 61.96; H, 2.46. 6,6''-Oxybis(3',6'-dihydroxy-1',8'-dimethyl-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one) (26b)

o Brownish red, m.p> 250 C; Rf: 0.37 (ethyl acetate: ethanol 1: 1); λmax (nm): 430; FTIR (Neat) ν: 3323 (br, OH), 3123 (C=C-H, str), 1783 (lactone C=O), 1744 (carboxylic C=O), 1647 (C=C), 1570 -1 1 (C=C), 1145 (C-O), 853 (Ar-H, bend), 812 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 8.25 (m, 4H, 4OH), 7.91 (d, 2H, J=8.2Hz), 7.20 (m, 2H), 7.05 (d, 2H, J=8.2Hz), 13 6.40 (s, 4H), 6.34 (s, 4H), 2.27 (s, 12H). C-NMR (DMSO-d6 75 MHz) δ (ppm): 171.41, 161.54, 156.98, 155.78, 151.17, 138.19, 130.85, 123.46, 119.69, 115.06, 113.98, 112.02, 103.15, 19.89. Anal. Calcd. For C44H30O11: C, 71.93; H, 4.12; Found: C, 72.06; H, 4.05. 6, 6''-Oxybis (4', 5'-dimethyl-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one) (26c)

o Violet, m.p> 250 C; Rf: 0.43 (ethyl acetate: ethanol 1: 1); λmax (nm): 558; FTIR (Neat) ν: 3110 (C=C-H, str), 1788 (lactone C=O), 1742 (carboxylic C=O), 1638 (C=C), 1584 (C=C), 1133 (C- -1 1 O), 850 (Ar-H, bend), 818 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 7.92 (d, 2H, J=8.4Hz), 7.32 (d, 2H, J=8.4Hz), 7.25 (m. 1H), 7.18 (d, 4H, J=8.2Hz), 7.03 (d, 4H, 13 J=8.2Hz), 6.99 (m, 4H), 2.43 (s, 12H). C-NMR (DMSO-d6 75 MHz) δ (ppm): 172.98, 161.51, 151.89, 148.49, 131.61, 131.46, 130.51, 126.77, 126.20, 125.48, 121.57, 118.82, 112.64, 15.55. Anal. Calcd. For C44H30O7: C, 78.79; H, 4.51; Found: C, 78.91; H, 4.41. 6,6''-Oxybis(3',6'-bis(dimethylamino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one) (26d)

o Pink, m.p> 250 C; Rf: 0.36 (ethyl acetate: ethanol 1: 1); λmax (nm): 550; FTIR (Neat) ν: 3119 (C=C-H, str), 1789 (lactone C=O), 1742 (carboxylic C=O), 1630 (C=C), 1592 (C=C), 1149 (C- -1 1 O), 859 (Ar-H, bend), 793 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 7.82 (d, 2H, J=8.4Hz), 7.20 (m, 2H), 7.18 (d, 2H, J=8.4Hz), 6.98 (d, 2H, J=8.2Hz), 6.40 (d, 2H, 13 J=8.2Hz), 6.33 (s, 2H), 2.91 (s, 24H). C-NMR (DMSO-d6 75 MHz) δ (ppm): 170.88, 165.62, 153.89, 153.87, 148.92, 131.61, 125.44, 125.06, 118.82, 112.64, 109.36, 107.01, 41.91. Anal. Calcd. For C48H42N4O7: C, 73.27; H, 5.38; N, 7.12; Found: C, 74.70; H, 5.56; N, 7.32. 6,6''-Oxybis(3',6'-dihydroxy-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one) (26e)

o Yellow, m.p> 250 C; Rf: 0.40 (ethyl acetate: ethanol 1: 1); λmax (nm): 501; FTIR (Neat) ν: 3354 (br, OH), 3130 (C=C-H, str), 1788 (lactone C=O), 1754 (carboxylic C=O), 1633 (C=C), 1581 -1 1 (C=C), 1130 (C-O), 847 (Ar-H, bend), 801 (Ar-H6, bend) cm . H-NMR (DMSO-d6 300 MHz) δ (ppm): 8.29 (s, 2H), 7.95 (s, 2H), 7.20 (m, 2H), 6.90 (d, 2H, J=8.4Hz), 6.74 (d, 2H, J=8.4Hz), 13 6.68 (d, 2H, J=8.4Hz), 6.20 (d, 2H, J=8.4Hz), 6.17( m, 2H) 4.8 C-NMR (DMSO-d6 75 MHz) δ (ppm): 171.78, 161.45, 152.98, 153.33, 148.47, 131.14, 126.77, 125.06, 118.82, 117.10, 112.64, 109.36, 107.01. Anal. Calcd. For C40H22O11: C, 70.80; H, 3.27; Found: C, 70.91; H, 3.20.

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3.5.2 General Procedure for Synthesis of Benzophenone-3,3’,4,4’-tetracarboxylic dianhydride Based Xanthene Dyes (28a-e) Same procedure was adopted as discussed earlier at page 188

3,3'',6,6''-Tetrahydroxy-3',8'-dioxo-3',8'-dihydrodispiro[xanthene-9,1'- isochromeno[6,5,4def]isochromene-6',9''-xanthene]-1,1'',8,8''-tetracarboxylic acid (28a)

o Brown, m.p> 250 C; Rf: 0.37 (ethyl acetate: ethanol 1: 1); λmax (nm): 465; FTIR (Neat) ν: 33503500 (br, COOH, OH), 3133 (C=C-H, str), 1782 (lactone C=O), 1755 (carboxylic C=O), 1641 -1 1 (C=C), 1594 (C=C), 1128 (C-O), 836 (Ar-H, bend), 817 (Ar-H, bend) cm . H-NMR (DMSOd6, 300 MHz) δ (ppm): 14.12 (s, 2H, br ), 8.5 (s, 2OH, br). 8.35 (d, 2H, J=8.4Hz), 7.65 (d, 2H, J=8.4Hz), 6.97 – 6.99 (s, 4H), 6.80 (s, 4H). 13C-NMR (DMSO 75 MHz) δ (ppm): 168.92, 161.81, 156.72, 156.21, 135.45, 133.94, 133.48, 124.92, 124.47, 119.98, 115.90, 112.16, 108.46.

Anal. Calcd. For C42H20O18: C, 62.08; H, 2.48; Found: C, 62.15; H, 2.53. 3,3'',6,6''-Tetrahydroxy-1,1'',8,8''-tetramethyldispiro[xanthene-9,1'-isochromeno[6,5,4-def] isochromene-6',9''-xanthene]-3',8'-dione (28b)

o Yellowish red, m.p> 250 C; Rf: 0.48 (ethyl acetate: ethanol 1: 1); λmax (nm): 481; FTIR (Neat) ν: 3320 (br, OH), 3140(C=C-H, str), 1794 (lactone C=O), 1751 (carboxylic C=O), 1640 (C=C), -1 1 1580(C=C), 1145(C-O), 860(Ar-H, bend), 815 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 9.04 (s, 2OH), 8.24 (d, 2H, J=8.4Hz), 7.59 (d, 2H, J=8.4Hz), 6.44 (s, 4H), 6.29 (s, 4H), 2.24 (s, 12H). 13C-NMR (DMSO 75 MHz) δ (ppm): 163.41, 156.54, 156.07, 138.05, 135.16, 133.89, 124.92, 124.47, 119.98, 115.38, 114.84, 103.50, 19.89. Anal. Calcd. For C42H28O10: C, 72.83; H, 4.07; Found: C, 72.77; H, 4.03. 4,4'',5,5''-Tetramethyldispiro[xanthene-9,1'-isochromeno[6,5,4-def]isochromene-6',9''-xan thene]- 3',8'-dione (28c)

o Bluish violet, m.p> 250 C; Rf: 0.39 (ethyl acetate: ethanol 1: 1); λmax (nm): 521; FTIR (Neat) ν: 3118(C=C-H, str), 1788 (lactone C=O), 1742 (carboxylic C=O), 1620 (C=C), 1592 (C=C), 1121

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-1 1 (C-O), 858 (Ar-H, bend), 807 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 8.37 (d, 2H, J=8.2Hz), 7.71 (d, 2H, J=8.2Hz), 7.14 (d, 4H, J=8.1Hz), 7.03 (d, 4H, J=8.1Hz), 6.96 (m, 2H), 2.53 (s, 12H). 13C-NMR (DMSO 75 MHz) δ (ppm): 164.23, 152.65, 135.01, 134.94, 131.86, 131.41, 127.47, 125.88, 125.09, 124.89, 121.93, 119.34, 15.55. Anal. Calcd. For C42H28O6: C, 80.24; H, 4.49; O, 15.27; Found: C, 80.24; H, 4.49; O, 15.27. 3,3'',6,6''-Tetrakis(dimethylamino)dispiro[xanthene-9,1'-isochromeno[6,5,4-def]isochro mene- 6',9''-xanthene]-3',8'-dione (28d)

o Reddish pink, m.p> 250 C; Rf: 0.55 (ethyl acetate: ethanol 1: 1); λmax (nm): 545; FTIR (Neat) ν: 3130(C=C-H, str), 1786 (lactone C=O), 1745 (carboxylic C=O), 1652 (C=C), 1585(C=C), 1125 -1 1 (C-O), 847 (Ar-H, bend), 806 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 8.30 (d, 1H, J=8.2Hz), 7.67 (d, 2H, J=8.4Hz), 6.97 (d, 4H, J=8.2Hz), 6.48 (s, 4H), 6.32 (d, 4H, J=8.2Hz), 2.90 (s, 24H). 13C-NMR (DMSO 75 MHz) δ (ppm): 160.31, 157.33, 155.33, 132.82, 131.18, 129.93, 1135.45, 106.63, 103.54, 41.91. Anal. Calcd. For C46H40N4O6: C, 74.18; H, 5.41; N, 7.52; Found: C, 74.30; H, 5.33; N, 7.48. 3,3'',6,6''-Tetrahydroxydispiro[xanthene-9,1'-isochromeno[6,5,4-def]isochromene-6',9''xanthene]- 3',8'-dione (28e)

3.5.3 General Procedure for Synthesis of 4, 4'-Oxydiphthalic anhydride Based Xanthene Dyes (30a-e) Same procedure was adopted as discussed earlier at page 188.

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6,6''-Oxybis(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-1',8'-dicarb oxylic acid) (30a)

1 NMR. H-NMR (DMSO-d6, 300 MHz) δ (ppm): 12.24 (s, 4H, COOH), 8.95 (s, 4OH, br), 7.90 (d, 2H, J=8.4Hz), 7.30 (m, 2H), 7.13 (d, 2H, J=8.4 Hz), 6.90 (s, 4H), 6.75 (s, 4H), 13C-NMR (DMSO 75 MHz) δ (ppm): 170.41, 168.92, 159.14, 154.83, 150.08, 133.65, 126.85, 123.46, 117.69, 116.12, 113.98, 110.13, 107.96. Anal. Calcd. For C44H22O19: C, 61.84 H, 2.59; Found: C, 61.96; H, 2.46. 6,6''-Oxybis(3',6'-dihydroxy-1',8'-dimethyl-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one) (30b)

o Brownish red, m.p> 250 C; Rf: 0.37 (ethyl acetate: ethanol 1: 1); λmax (nm): 430; FTIR (Neat) ν: 3323 (br, OH), 3123 (C=C-H, str), 1783 (lactone C=O), 1744 (carboxylic C=O), 1647 (C=C), 1570 -1 1 (C=C), 1145 (C-O), 853 (Ar-H, bend), 812 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 9.35 (m, 4OH, br), 7.91 (d, 2H, J=8.15 Hz), 7.20 (m, 2H), 7.05 (d, 2H, J=8.15Hz), 6.40 (s, 4H), 6.34 (s, 4H), 2.27 (s, 12H). 13C-NMR (DMSO 75 MHz) δ (ppm): 172.98, 163.19, 156.05, 155.78, 151.17, 138.19, 130.85, 123.46, 119.53, 112.02, 103.15, 19.89. Anal. Calcd. For C44H30O11: C, 71.93; H, 4.12; Found: C, 72.06; H, 4.05. 6,6''-Oxybis(4',5'- dimethyl-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one) (30c)

o Violet, m.p> 250 C; Rf: 0.43 (ethyl acetate: ethanol 1: 1); λmax (nm): 558; FTIR (Neat) ν: 3110 (C=C-H, str), 1788 (lactone C=O), 1742 (carboxylic C=O), 1638 (C=C), 1584 (C=C), 1133 (C- -1 1 O), 850 (Ar-H, bend), 818 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 7.92 (d, 2H, J=8.4Hz), 7.32 (d, 2H, J=8.4Hz), 7.25 (m. 1H), 7.18 (d, 4H, J=8.1Hz), 7.03 (d, 4H, J=8.1Hz), 6.99 (m, 4H), 2.43 (s, 12H). 13C-NMR (DMSO 75 MHz) δ (ppm): 169.53, 160.42, 152.63, 147.47, 131.61, 129.51, 123.54, 121.20, 119.48, 117.57, 116.32, 111.14, 15.55. Anal. Calcd. For C44H30O7: C, 78.79; H, 4.51; Found: C, 78.91; H, 4.41. 6,6''-Oxybis(3',6'-bis(dimethylamino)-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one) (30d)

o Pink, m.p > 250 C; Rf: 0.36 (ethyl acetate: ethanol 1: 1); λmax (nm): 550; FTIR (Neat) ν: 3119 (C=C-H, str), 1789 (lactone C=O), 1742 (carboxylic C=O), 1630 (C=C), 1592 (C=C), 1149 (C- -1 1 O), 859 (Ar-H, bend), 793 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 7.82 (d, 2H, J=8.4Hz), 7.20 (m, 2H), 7.18 (d, 2H, J=8.3Hz), 6.98 (d, 2H, J=8.4Hz), 6.40 (d, 2H, J=8.4Hz), 6.33 (s, 2H), 2.91 (s, 24H). 13C-NMR (DMSO 75 MHz) δ (ppm): 169.86, 158.51, 153.87, 147.49, 133.61, 125.77, 125.06, 118.82, 112.64, 109.36, 107.01, 41.91. .Anal. Calcd. For C48H42N4O7: C, 73.27; H, 5.38; N, 7.12; Found: C, 74.70; H, 5.56; N, 7.32. 6,6''-Oxybis(3',6'-dihydroxy-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one) (30e)

o Yellow, m.p> 250 C; Rf: 0.40 (ethyl acetate: ethanol 1: 1); λmax (nm): 501; FTIR (Neat) ν: 3354 (br, OH), 3130 (C=C-H, str), 1788 (lactone C=O), 1754 (carboxylic C=O), 1633 (C=C), 1581

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-1 1 (C=C), 1130 (C-O), 847 (Ar-H, bend), 801 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 7.91 (s, 2H), 7.20 (s, 2H), 7.11 (d, 2H J=8.4Hz), 7.03(d, 2H J=8.3Hz), 6.55 (d, 2H, J=7.5Hz), 6.48 (s, 2H, J=7.5Hz). 13C-NMR (DMSO 75 MHz) δ (ppm): 172.98, 161.51, 153.89, 153.87, 148.49, 131.61, 126.61, 125.76, 118.82, 117.10, 112.98, 109.83, 107.01. Anal. Calcd. For C40H22O11: C, 70.80; H, 3.27; Found: C, 70.91; H, 3.20. 3.5.4 General Procedure for synthesis of 1,4,5,8-Naphthalenetetracarboxylic dianhydride Based Xanthene Dyes (32a-e) Same procedure was adopted as discussed earlier at page 188.

3,3'',6,6''-Tetrahydroxy-3',8'-dioxo-3',8'-dihydrodispiro[xanthene-9,1'- isochromeno[6,5,4def]isochromene-6',9''-xanthene]-1,1'',8,8''-tetracarboxylic acid (32a)

o Brown, m.p> 250 C; Rf: 0.37 (ethyl acetate: ethanol 1: 1); λmax (nm): 465; FTIR (Neat) ν: 33503500 (br, COOH, OH), 3133 (C=C-H, str), 1782 (lactone C=O), 1755 (carboxylic C=O), 1641 -1 1 (C=C), 1594 (C=C), 1128 (C-O), 836 (Ar-H, bend), 817 (Ar-H, bend) cm . H-NMR (DMSOd6, 300 MHz) δ (ppm): 14.12 (s, 2H, br), 8.55 (s, 2OH, br). 8.35 (d, 2H, J=8.4 Hz), 7.65 (d, 2H, J=8.4 Hz), 6.99 (s, 4H), 6.80 (s, 4H), 13C-NMR (DMSO 75 MHz) δ (ppm): 168.92, 161.42, 156.11, 135.40, 133.89, 124.92, 121.47, 118.78, 115.90, 112.16, 108.46. Anal. Calcd. For C42H20O18: C, 62.08; H, 2.48; Found: C, 62.15; H, 2.53. 3,3'',6,6''-Tetrahydroxy-1,1'',8,8''-tetramethyldispiro[xanthene-9,1'-isochromeno[6,5,4-def] isochromene-6',9''-xanthene]-3',8'-dione (32b)

o Yellowish red, m.p> 250 C; Rf: 0.48 (ethyl acetate: ethanol 1: 1); λmax (nm): 481; FTIR (Neat) ν: 3320 (br, OH), 3140(C=C-H, str), 1794 (lactone C=O), 1751 (carboxylic C=O), 1640 (C=C), -1 1 1580(C=C), 1145(C-O), 860(Ar-H, bend), 815 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 9.45 (s, 2OH, br), 8.37 (d, 2H, J=8.35 Hz), 7.59 (d, 2H, J=8.35Hz), 6.44 (s, 4H), 6.29 (s, 4H), 2.24 (s, 12H). 13C-NMR (DMSO 75 MHz) δ (ppm): 169.23, 157.54, 156.07, 138.05, 135.16, 133.89, 124.92, 119.98, 115.38, 114.84, 103.50, 19.89. Anal. Calcd. For C42H28O10: C, 72.83; H, 4.07; Found: C, 72.77; H, 4.03. 4,4'',5,5''-Tetramethyldispiro[xanthene-9,1'-isochromeno[6,5,4-def]isochromene-6',9''xanthene]- 3',8'-dione (32c)

o Bluish violet, m.p> 250 C; Rf: 0.39 (ethyl acetate: ethanol 1: 1); λmax (nm): 521; FTIR (Neat) ν:

186

3118(C=C-H, str), 1788 (lactone C=O), 1742 (carboxylic C=O), 1620 (C=C), 1592 (C=C), 1121

-1 1 (C-O), 858 (Ar-H, bend), 807 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 8.37 (d, 2H, J=8.5 Hz), 7.71 (d, 2H, J=8.5 Hz), 7.14 (d, 4H, J=8.2 Hz), 7.03 (d, 4H, J=8.2 Hz), 6.96 (m, 2H), 2.53 (s, 12H). 13C-NMR (DMSO 75 MHz) δ (ppm): 170.42, 158.65, 135.73, 134.03, 131.86, 127.47, 125.88, 124.20, 122.89, 121.93, 119.34, 15.55. Anal. Calcd. For C42H28O6: C, 80.24; H, 4.49; O, 15.27; Found: C, 80.24; H, 4.49; O, 15.27. 3,3'',6,6''-Tetrakis(dimethylamino)dispiro[xanthene-9,1'-isochromeno[6,5,4-def]isochrom ene- 6',9''-xanthene]-3',8'-dione (32d)

o Reddish pink, m.p> 250 C; Rf: 0.55 (ethyl acetate: ethanol 1: 1); λmax (nm): 545; FTIR (Neat) ν: 3130(C=C-H, str), 1786 (lactone C=O), 1745 (carboxylic C=O), 1652 (C=C), 1585(C=C), 1125 -1 1 (C-O), 847 (Ar-H, bend), 806 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 8.30 (d, 2H, J=8.4 Hz), 7.67 (d, 2H, J=8.4 Hz), 6.97 (d, 4H, J=8.1 Hz), 6.48 (s, 4H), 6.32 (d, 4H, J=8.1 Hz), 2.90 (s, 24H). 13C-NMR (DMSO 75 MHz) δ (ppm): 172.23, 160.41, 154.73, 135.01, 134.94, 126.16, 125.09, 124.89, 119.72, 110.30, 106.89, 41.91. Anal. Calcd. For C46H40N4O6: C, 74.18; H, 5.41; N, 7.52; Found: C, 74.30; H, 5.33; N, 7.48. 3,3'',6,6''-Tetrahydroxydispiro[xanthene-9,1'-isochromeno[6,5,4-def]isochromene-6',9''xan thene]-3',8'-dione (32e)

o Reddish yellow, m.p> 250 C; Rf: 0.53 (ethyl acetate: ethanol 1: 1); λmax (nm): 503; FTIR (Neat) ν: 3315 (br, OH), 3118(C=C-H, str), 1790 (lactone C=O), 1750 (carboxylic C=O), 1650 (C=C), -1 1 1576 (C=C), 1140 (C-O), 858 (Ar-H, bend), 822 (Ar-H, bend) cm . H-NMR (DMSO-d6, 300 MHz) δ (ppm): 7.75 (d, 2H, J=9.0Hz), 7.64 (d, 2H, J= 9.0Hz), 6.97 (d, 2H, J=9.0Hz), 6.49 (s, 1H), 6.31 (d, 4H, J=9.0Hz). 13C-NMR (DMSO 75 MHz) δ (ppm): 177.23, 160.27, 156.10, 139.02, 134.94, 128.24, 126.02, 124.89, 119.34, 116.02, 113.98, 104.89. Anal. Calcd. For C38H20O10: C, 71.70; H, 3.17 Found: C, 71.83; H, 3.10. 3.5.5 General Procedure for Synthesis of Xanthene Schiff Bases (35a-j) In a 100 ml two neck round bottom flask, 0.5 g resorcinol (2.1mmol) and 0.25 g phthalic anhydride o (1.0 mmol) were fused at 180 C in the presence of 0.07 g (0.28 mmol) ZnCl2. The flask is fitted with a reflux condenser. On complete fusion of the components, the resulting fused mass was dissolved in 10 mL (20%) NaOH solution and was then filtered, the filtrate was treated with 2 mL (36%) HCl and was filtered again. The resulting solid mass (Fluorescein) was dried and recrystallized with propanol. In the second step, 0.33 g fluorescein (1.6 mmol) and hydrazine 0.2 mL were refluxed for 12 hrs in the presence of ethanol to synthesize fluorescein imide intermediate. In third step, substituted aldehyde (0.245g, 1.2 mmol) was dissolved in hot ethanol (20 ml) and heated to reflux in an oil bath. Then, a solution of fluorescein hydrazine (0.36 g, 1mmol) in ethanol (15ml) was added drop wise to the flask in one hour at reflux. Finally, the precipitate produced was filtered and dried and purified by recrystallization from ethyl alcohol and ethyl acetate and in this way a series of xanthene Schiff bases 35a-j was synthesized.

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3-Bromophenylimine Derivative of Fluorescein Hydrazine (35a) o -1 Orange white solid ; yield : 66 % ; m.p : 260 C ; Rf : 0.36, IR (neat) : ѵ/cm ; 3631 (O-H), , 1691 1 (C=O), 1502, 1547 (Ar-C=C), 1016 (C-O), 803 (C-Cl); H-NMR (CD3)2CO, 300 MHz) δ (ppm): 8.08 (s, 1H, -CH=N-), 7.81 (d, 1H, J= 7.5 Hz, Ar-H),7.60 (d,1H, J= 7.5 Hz, Ar-H), 7.52 (m,1H, Ar-H),7.38 (dd,1H, Ar-H),7.35 (dd,1H, Ar-H),7.28 (dd,1H, Ar-H),7.25 (dd,1H, Ar-H), 7.20 (dd,1H, Ar-H), 6.82 (d, 2H, J=7.1 Hz, Ar-H), 6.32 (d, 2H, J=7.1 Hz Ar-H), 6.23 (s, 2H, Ar- 13 H); 5.03 (s, 2H, phenol); C-NMR (CD3)2CO, 75 MHz) δ (ppm): 168.4, 156.6, 143.6, 139.4, 135.2, 134.4, 132.1, 131.4, 131.2, 130.3, 129.7, 129.3, 128.6, 128.6, 128.4, 127.3, 126.3, 117.3, 109.4, 105.4, 60.2. 4-Bromophenylimine Derivative of Fluorescein Hydrazide (35b) o -1 Orange solid ; yield : 52% ; m.p : 198 C ; Rf :0.09, IR (neat) : ѵ/cm ; 3632 (O-H), 1683 (C=O), 1 1504, 1551 (Ar-C=C), 1009 (C-O), 556 (C-Br) ; H-NMR (CD3)2CO, 300 MHz) δ (ppm): 8.12 (s, 1H, -CH=N-), 7.81 (d, 1H, J= 6.9 Hz, Ar-H), 7.57 (d, 2H, J=6.9Hz, Ar-H),7.38 (d,1H, J=7.45 Hz Ar-H), 7.26 (d, 1H, J=7.45 Hz Ar-H), 7.23 (dd, 1H, Ar-H), 6.87 (d, 2H, J= 7.1 Hz, Ar-H), 6.35 (d, 2H, J=7.1 Hz Ar-H), 6.29 (s, 2H, Ar-H), 5.54 (s, 2H, phenol); 13C-NMR (CD3)2CO, 75 MHz) δ (ppm): 168.0, 155.9, 152.3, 143.2, 139.3, 132.2, 131.7, 131.2, 128.3, 128.2, 126.3, 125.4, 117.3, 109.2, 105.9, 60.5. 2-Chlorophenylimine Derivative of Fluorescein Hydrazine (35c) o -1 Yellow solid ; yield : 61 % ; m.p: 217 C ; Rf: 0.34, IR (neat) : ѵ/cm ; 3633 (O-H), 1684(C=O),

1 1504, 1553 (Ar-C=C), 1010 (C-O), 801 (C-Cl); H-NMR (CD3)2CO, 300 MHz) δ (ppm): 8.04 (s, 1H, -CH=N-), 7.80 (d, 1H, J= 6.95 Hz, Ar-H), 7.61 (d,1H, J= 6.95 Hz, Ar-H), 7.36 (dd, 1H, ArH), 7.34 (d, 1H, J= 7.3Hz, Ar-H),7.25 (dd, 1H, Ar-H),7.23 (dd, 1H, Ar-H), 6.86 (d, 1H, J= 7.3 Hz, Ar- H), 6.84 (d, 2H, J=7.1 Hz, Ar-H), 6.37 (d, 2H, J=7.1 Hz, Ar-H), 6.25 (d, 2H, Ar-H),5.04 13 (s,2H, phenol); C-NMR (CD3)2CO, 75 MHz) δ (ppm): 168.2, 156.3, 152.2, 143.1, 134.1, 133.4, 132.7, 132.5, 130.6, 129.4, 129.0, 128.1, 128.0, 127.0, 126.1, 117.2, 109.2, 105.6, 60.0.

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3-Clorophenylimine Derivative of Fluorescein Hydrazine (35d) o -1 Orange white solid ; yield : 66 % ; m.p : 260 C ; Rf : 0.36, IR (neat) : ѵ/cm ; 3631 (O-H), , 1691 1 (C=O), 1502, 1547 (Ar-C=C), 1016 (C-O), 803 (C-Cl); H-NMR (CD3)2CO, 300 MHz) δ (ppm): 8.08 (s, 1H, -CH=N-), 7.81 (d, 1H, J= 6.9 Hz, Ar-H),7.60 (d,1H, J= 6.9, Ar-H), 7.52 (d,1H, Ar-H),7.38 (dd,1H, Ar-H),7.35 (dd,1H, Ar-H),7.28 (dd,1H, Ar-H),7.25 (dd,1H, ArH),7.20 (dd,1H , Ar-H),6.82 (d,2H, J=7.4 Hz, Ar-H), 6.32 (d, 2H, J=7.4 Hz, Ar-H), 6.23 (s, 2H, 13 Ar-H) ; 5.03 (s, 2H, phenol); C-NMR (CD3)2CO, 75 MHz) δ (ppm): 168.4, 156.6, 143.6, 139.4, 135.2, 134.4, 132.1, 131.4, 131.2, 130.3, 129.7, 129.3, 128.6, 128.6, 128.4, 127.3, 126.3, 117.3, 109.4, 105.4, 60.2. 4-Clorophenylimine Derivative of Fluorescein Hydrazine (35e) o Brown solid; yield 69%, m.p; 168 C, Rf: 0.31, FTIR (neat) v/cm: 3635 (OH), 1685 (C=O),

1 1548, 1503 (Ar-C=C), 1018 (C-O), 807 (C-Cl), H-NMR (CD3)2CO, 300 MHz) δ (ppm): 8.05 (1H, s, CH=N), 7.82 (1H, d, J=6.9Hz, Ar-H), 7.60 (1H, d, J=6.9Hz, Ar-H),7.38(1H, dd, Ar-H), 7.26(1H, dd, Ar-H), 7.24(1H, dd, Ar-H),6.90 (1H, d, J=7.1Hz, Ar-H), 6.32 (1H, d, J=7.1Hz, Ar-

13 H),6.26 (1H, s, Ar-H), 5.06 (1H, s, phenolic OH). C-NMR (CD3)2CO, 75 MHz) δ (ppm): 168.1, 156.2, 152.2, 143.2, 139.5, 136.6, 132.6, 131.9, 131.6, 130.6, 129.7, 129.0, 128.6, 128.3, 126.5, 117.5, 109.1, 105.6, 60.1. 2,4-Dichlorophenylimine Derivative of Fluorescein Hydrazine (35f) o -1 Golden yellow solid ; yield : 43% ; m.p: 210 C ; Rf : 0.24, IR (neat) : ѵ/cm ; 3637 (O-H),

1 1691 (C=O), 1502, 1553 (Ar-C=C), 1024 (C-O), 812 (C-Cl); H-NMR (CD3)2CO, 300 MHz) δ (ppm):8.14 (s, 1H, -CH=N-), 7.84 (m, 1H, J= 7.15Hz, Ar-H), 7.52 (d,1H, J= 7.15Hz, Ar-H), 7.37 (dd,1H, Ar-H), 7.30 (d,1H, J=7.1Hz, Ar-H), 7.26 (dd,1H, Ar-H), 7.23 (d,1H, J=7.4Hz ArH),7.21 (d, 1H, J=7.1Hz, Ar-H), 6.83 (d,2H, J=7.1Hz, Ar-H), 6.34 (d, 2H , Ar-H), 6.26(s,2H, 13 Ar-H), 5.01 (s, 2H, phenol); C-NMR (CD3)2CO, 75 MHz) δ (ppm): 168.3, 155.7, 143.0, 139.4, 138.0, 135.4, 132.6, 132.0, 131.5, 131.3, 130.5, 129.4, 129.3, 128.4, 127.1, 126.2, 117.1, 109.5, 105.7, 61.0. 3-Methoxyphenylimine Derivative of Fluorescein Hydrazine (35g) o -1 3 Yellow solid ; yield : 59% ; m.p: 233 C ; Rf : 0.19, IR (neat) : ѵ/cm ; 3632 (O-H), 2857 (Csp H),

3 1 1683 (C=O), 1504, 1557 (Ar-C=C),1372 (Csp -H,-CH3), 1016 (C-O); H-NMR (CD3)2CO,

300 MHz) δ (ppm): 8.04 (s, 1H, -CH=N-), 7.82 (d, 1H, J= 6.9 Hz, Ar-H), 7.80 (d, 1H J= 6.9 Hz, Ar-H), 7.37 (dd,1H, Ar-H), 7.26 (dd, 1H, Ar-H), 7.24 (dd, 1H, Ar-H), 7.21 (dd,1H, Ar-H),7.12 (s, 1H, Ar-H), 6.90 (d, 2H, J=7.2Hz, Ar-H), 6.8 (d,1H , J=7.2Hz, Ar-H), 6.32 (m,2H, Ar-H) , 13 6.26 (s,2H, Ar-H), 5.01 (s,2H, phenol), 3.73 (s, 3H, -CH3) ; C-NMR (CD3)2CO, 75 MHz) δ (ppm): 168.0, 160.8, 155.2, 152.5, 143.2, 139.4, 134.8, 132.4, 131.5, 131.0, 129.9, 129.8, 128.2, 126.5, 117.5, 116.3, 113.4, 109.1, 105.3, 61.2, 55.7. 2-Nitrophenylimine Derivative of Fluorescein Hydrazine (35h) o -1 Brown solid ; yield : 53% ; m.p: 227 C; Rf : 0.21, IR (neat) : ѵ/cm ; 3635 (O-H), 1681 (C=O),

1 1505, 1548 (Ar-C=C), 1350,1553(-NO2),1017 (C-O) ; H-NMR (CD3)2CO, 300 MHz) δ (ppm):

189

8.21 (d, 1H, Ar-H ), 8.04 (s, 1H, -CH=N-), 7.90 (d, 1H, J= 6.9 Hz, Ar-H), 7.82 (d, 1H, J= 6.9 Hz, Ar-H), 7.72 (dd, 1H, Ar-H), 7.63 (dd, 1H, Ar-H), 7.35 (dd, 1H, Ar-H), 7.24 (dd,1H, Ar-H), 6.86 (d, 1H, J=7.1Hz, Ar), 6.83 (d, 2H, J=7.1Hz, Ar-H), 6.28 (s, 2H, Ar), 5.06 (s,2H, phenol);

13 C-NMR (CD3)2CO, 75 MHz) δ (ppm): 168.4, 155.7, 152.3, 148.9, 143.0, 139.6, 135.0, 132.0, 131.4, 130.1, 129.4, 129.3, 128.0, 126.4, 126.3, 121.2, 116.2, 105.1, 61.3. 3-Nitrophenylimine Derivative of Fluorescein Hydrazine (35i) o -1 Brown solid ; yield : 67% ; m.p: 203 C; Rf : 0.27, IR (neat) : ѵ/cm ; 3639 (O-H), 1686 (C=O), 1 1502, 1549 (Ar-C=C), 1346,1557(-NO2),1019 (C-O) ; H-NMR (CD3)2CO, 300 MHz) δ (ppm): 8.61 (s, 1H, Ar-H ),8.23 (d,1H, J=6.9Hz Ar-H),8.07 (d,1H, J=6.9Hz Ar-H), 8.02 (s, 1H, -CH=N), 7.85 (d, 1H, J= 7.3 Hz, Ar-H), 7.62 (dd,1H, Ar-H), 7.41 (dd,1H, Ar-H), 7.25 (dd,1H, Ar-H), 6.86 (d,1H, J=7.3Hz, Ar-H),6.83 (d, 2H, J=6.9Hz Ar-H), 6.32 (d,2H, J=6.9Hz Ar-H), 6.26 (s,

13 2H, Ar-H), 5.15 (s,2H, phenol); C-NMR (CD3)2CO, 75 MHz) δ (ppm): 168.1, 155.4, 148.5, 143.0, 138.5, 135.3, 134.7, 133.6, 131.4, 129.8, 129.7, 128.4, 128.2, 126.1, 124.1, 123.4, 117.3, 109.3, 105.7, 61.2. 3-Hydroxyphenylimine derivative of fluorescein hydrazine (35j) o -1 Yellow brown solid ; yield : 69% ; m.p: 231 C ; Rf : 0.41, IR (neat) : ѵ/cm ; 3631 (O-H), 1684

1 (C=O), 1501, 1554 (Ar-C=C), 1015 (C-O); H-NMR (CD3)2CO, 300 MHz) δ (ppm): 8.03 (s, 1H, -CH=N- ), 7.82 (d, 1H, J= 7.1 Hz, Ar-H), 7.36 (dd,1H, Ar-H), 7.32 (dd, 1H, Ar-H), 7.21 (d, 1H, Ar-H),7.10 (dd, 1H, Ar-H), 7.08 (s, 1H, Ar-H), 6.85 (d, 1H, J=7.4Hz, Ar-H), 6.80 (d, 1H , J=7.4Hz, Ar-H), 6.72 (d, 1H, J=7.1Hz, Ar-H), 6.65 (d, 1H, J=7.1Hz, Ar-H), 6.32 (d, 2H, 13 J=6.9Hz, Ar-H), 6.23 (d, 2H, J=6.9Hz, Ar-H), 5.02(s, 3H, phenol). C-NMR (CD3)2CO, 75 MHz) δ (ppm): 168.3, 158.6, 155.7, 153.2, 143.2, 138.5, 135.2, 132.7, 131.1, 130.3, 129.4, 128.0, 126.4, 121.8, 118.2, 117.4, 115.0, 109.5, 106.3, 60.2.

190

CONCLUSIONS Four series of a new rylene dyes were successfully synthesized and characterized by FTIR, NMR and elemental analysis and their optical, thermal and electrochemical properties have been investigated. Synthesis was achieved by taking the advantage of reactivity of bay position and peri position of tetrachloro perylene dianhydride and perylene dianhydride. Solubility of rylene dyes was achieved by introducing azo dyes and aliphatic alcohols at bay positions. These bay substituted azo dyes exhibited absorption in UV in range 425-450 nm and their emissions were in the range 500-515 nm. Substitution of rylene at the dianhydride position with different Schiff bases shifted their absorption maximum to 526 nm and emission to 550 nm but created solubility issue in water. Electrochemical analysis showed that bay azo and alkyl substituted dye showed only the oxidation potential while the redox potential was observed for dye where extension of delocalization was made along the molecular axis. The low LUMO level estimated from UV and cyclic voltammetry account for their possible use as electron transport and emissive material for OLEDs. Different types of azo dyes which include reactive azo dyes, heterocyclic azo dyes and calix azo dyes were successfully synthesized. Newly synthesized dyes were characterized by FTIR, NMR, elemental analysis studies and their exhaustion, fixation and fastness properties have been investigated. In reactive azo dyes the reactive functionality has been introduced in the form of cynuric chloride. Bridging bis anilines have used as linkers to produce dimers of reactive dyes which have high solubility and substantivity with cellulosic fibers. These bismonochlorotriazine dyes have exhaustion and fixation values upto 92%. They showed high light fastness, rub fastness and wash fastness, 6-7, 4-5 and 4-5 respectively after application on cotton fibers having different color shades in different schemes. Heterocyclic (pyrazolone) azo dyes have been synthesized by changing the reaction pathway. In these dyes first azo dyes are synthesized from different active methylene compounds and then their heterocycles have been synthesized by treating them with different hydrazines. Application of heterocyclic dyes on leather showed high light fastness, wash fastness and rub fastness values 4-5, 4-5 and 3-4 respectively which are moderate to good.

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Exhaustion and fixation values of these dyes are also very high on account of their high attraction and substantivity with cotton fibers except to that of few dyes having no suitable functional groups for interaction. Furthermore, the dyes containing sulfonic and carboxyl substituents exhibited an incredible degree of levelness after washing demonstrating the good diffusion and excellent affinity to the fabric. In calix azo dyes calix [4] resorsoniarene was used as coupler with different azo components having hydroxyl group at adjacent position to azo linkage. These dyes were synthesized to behave as multifunctional metal ion sequestrants and so their metal ion interactions were determined for Cu (II), Fe II), Co (II), Ni (II), Hg (II) and Cr (III) in their aqueous solutions but these probes proved to very sensitive for Cu+2 exhibiting bathochromic shift in absorption maxima. Five series of a new xanthene dyes and their derivatives were successfully synthesized by an efficient and cost effective method devoid of any metal catalyst. Synthesis of these dyes was catalysed by NH4Cl which acted as latent catalyst and provided the pure product as compared to usual Lewis acid catalyst used for xanthene dyes. The dyes were characterized by UV-visible, FTIR, NMR, LCMS and elemental analysis and their photophysical, thermal and electrochemical properties have been investigated. The newly synthesized xanthene dyes are thermally and photochemically stable. The xanthene dyes based upon double dianhydrides condensed with 3N,N’-dimethylaminophenol and resorcinol are highly fluorescent. Their absorption and emission maxima have undergone a bathochromic shift as compared to their counterpart xanthene dyes, rhodamine and fluorescein and are potentially applicable where usual xanthene fluorescent dyes are used with preference requiring lower energy source for excitation. Fluorescence quantum yields have been reduced to little extent in these compounds due to more flexibility in the molecules between two aromatic rings of dyes. These dyes exhibited redox potential in their electrochemical study and they have small band gap energies between HOMO and LUMO levels. Newly synthesized dyes have applied for staining study of onion cells and differential scanning was observed for nucleus and cytoplasm by these dyes and they retained their fluorescent properties after staining the cells.

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