DESIGN AND SYNTHESIS OF FLUORESCENT PROBES A dissertation submitted to Kent State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy by Prabin Rai August, 2013 Dissertation written by Prabin Rai M.Sc. Tribhuvan University, 1994 Ph.D., Kent State University, USA, 2013 Approved by , Chair, Doctoral Dissertation Committee Dr. Robert J. Twieg , Members, Doctoral Dissertation Committee Dr. Paul Sampson Dr. Scott Bunge Dr. Brett Ellman ______________________________ Dr. Hiroshi Yokoyama Accepted by , Chair, Department of Chemistry Dr. Michael Tubergen , Associate Dean, College of Arts and Sciences Dr. Raymond Craig ii TABLE OF CONTENTS LIST OF FIGURES ......................................................................................................... ix LIST OF SCHEMES .................................................................................................... xvii LIST OF TABLES ........................................................................................................ xxv LIST OF ABBREVIATIONS .................................................................................... xxvii ACKNOWLEDGMENTS .......................................................................................... xxxii CHAPTER 1 Introduction ............................................................................................... 2 1.1.A brief introduction of Super Resolution Imaging ....................................................... 2 1.2 Single molecule detection and its application in super resolution imaging .................. 4 1.3 Single molecule imaging............................................................................................... 5 1.4 Super-resolution imaging by single molecule localization ........................................... 8 1.5 Fluorescent probes for super resolution imaging ........................................................ 10 1.6 Photoswitchable fluorescent probes utilized in super resolution imaging .................. 12 1.7 Problems faced by the existing photoswitchable probes. ........................................... 18 1.8 Scope of this thesis...................................................................................................... 19 CHAPTER 2 Design and Synthesis of rhodamine spirolactams ................................ 22 2.1 A brief overview of Rhodamine Spirolactams ............................................................ 22 2.2 Rhodamine spirolactam derivatives: ........................................................................... 26 2.3 Synthesis of rhodamine derivatives: ........................................................................... 28 2.4 Synthesis of tetra-N-ethyl-spiro[benzo[4,5]imidazo[2,1-a]isoindole-11,9'-xanthene]- 3',6'-diamine (P-71) ........................................................................................................... 28 2.5 Literature approaches to 1H-isoindolo<1,2-a>benzimidazole-1-one (2.6) ................ 29 iii 2.6 Attempts on synthesis of rhodamine spirolactams (2.17) based on 7H- Benzimidazo[2,1 a]benz[de ] isoquinolin-7-one .............................................................. 39 2.7 Synthesis of 7H-Benzimidazo[2,1 a]benz[de ] isoquinolin-7-one (2.15) ................... 40 2.8 Attempts on synthesis of rhodamine spirolactams (2.20) based on 12H-phthaloperin- 12-one ............................................................................................................................... 41 2.9 Synthesis of 12H-phthaloperin-12-one (2.18) following the literature ...................... 41 2.10 Synthesis of Rhodamine salts following literature procedure .................................. 43 2.11 Synthesis of Rhodamine spirolactams from N-(9-(2-carboxyphenyl)-6- (dialkylamino)-3H-xanthen-3-ylidene)-N-methylmethanaminium perchlorate ............... 45 2.12 Rhodamine Spirolactam incorporating other dyes .................................................... 59 2.13 Synthesis of Rhodamine derivatives with different amine donor groups: ................ 63 2.13.1 Synthesis of (1-(9-(2-carboxyphenyl)-6-(pyrrolidin-1-yl)-3H-xanthen-3-ylidene) pyrrolidinium chloride) ..................................................................................................... 63 2.13.2 Synthesis of Rhodamine 101 spirolactams ............................................................ 64 2.13.3 Attempt in synthesizing Rhodamine spirolactam incorporated with 7- azanorbornane as donor group .......................................................................................... 65 2.13.4 Synthesis of 7-azanorbornane hydrogen chloride by Mitsunobu reaction following literature procedure: .......................................................................................................... 66 CHAPTER 3 Photophysical properties of rhodamine spirolactams .......................... 70 3.1 Photophysical properties of rhodamine spirolactams ................................................. 70 3.2 UV-Vis absorption of the rhodamine lactam in their closed state .............................. 70 3.3 Effect of substitution on the absorbance of the closed form ....................................... 74 3.4 Preliminary study of photoswitching efficiency of rhodamine spirolactams ............. 78 3.5 Rhodamine spirolactams under the influence of pH ................................................... 81 3.6 Single molecule detection in neutral and acid conditions ........................................... 93 3.7 Summary ..................................................................................................................... 94 iv CHAPTER 4 Modifications of DCDHF azides and strained alkenes for efficient 1,3 dipolar cycloadditions ..................................................................................................... 97 4.1 Introduction ................................................................................................................. 97 4.2 A brief introduction to azido functionalized chemistry .............................................. 98 4.3 A different approach of azide chemistry with strained alkenes ................................ 102 4.4 Some Preliminary Studies on functionalized strained alkenes. ................................ 107 4.5 A 1,3-dipolar reaction between azido functionality and norbornene and to understand the kinetics of the addition. ............................................................................................. 119 4.6 Synthesis and Result ................................................................................................. 127 4.6.1 Synthesis of norbornene with ester functional group ............................................ 127 4.6.2 Synthesis of norbornene with benzo-group: .......................................................... 128 4.6.3 Synthesis of norbornene with alkyl group on a bridge head position .................... 128 4.6.4 Synthesis of norbornene with cyclopropane bearing alcohol group ...................... 131 4.7 Synthesis of DCDHF dyes with an azido group ....................................................... 132 4.8 Synthesis of DCDHF dyes incorporating CF3 group with azido group on it. .......... 134 4.9 Synthesis of DCDHF incorporating two F atoms in π-system ................................. 138 4.10 Synthesis of DCDHF incorporating CF3 and Ph ring. ............................................ 138 4.11 Attempts on the synthesis with two CF3 groups on DCDHF head ......................... 147 4.12 Synthesis of the DCDHF unit incorporating a thiazole ring: .................................. 148 4.13 Conclusion: ............................................................................................................. 154 CHAPTER 5 Some Cell Imaging technique By Chemical activation ...................... 157 5.1 Introduction ............................................................................................................... 157 5.2 A brief overview of Point Accumulation for Imaging in Nanoscale Topography (PAINT)63: ...................................................................................................................... 158 5.3 Factors Essential to PAINT fluors ............................................................................ 159 v 5.4 Rationale for screening PAINT fluors ...................................................................... 162 5.5 Photophysical properties of some PAINT fluors ...................................................... 163 5.6 Spectra in Phosphate Buffer Saline (PBS) (~µM of dye concentration) .................. 165 5.7 Fluorescence of some individual PAINT fluor candidates: ...................................... 166 5.7.1 Nile red sulfate (P193), NL03008, and Nile Red ................................................... 166 5.7.2 Fluorescence QY increases with viscosity ............................................................. 170 - 5.7.3 A comparison of fluorescence properties of Nile red-SO3 (P-193), NL03008 and Nile red with respect to viscosity .................................................................................... 171 5.8 Preliminary Comparison Results .............................................................................. 173 5.9 Results and discussion .............................................................................................. 174 5.10 Synthesis of hydroxy-Nile red phenol: ..................................................................