W&M ScholarWorks Undergraduate Honors Theses Theses, Dissertations, & Master Projects 5-2017 Tuning the pKa of Fluorescent Rhodamine pH Probes via Substituent Effects Sarah G. Stratton College of William and Mary Follow this and additional works at: https://scholarworks.wm.edu/honorstheses Part of the Physical Chemistry Commons Recommended Citation Stratton, Sarah G., "Tuning the pKa of Fluorescent Rhodamine pH Probes via Substituent Effects" (2017). Undergraduate Honors Theses. Paper 1105. https://scholarworks.wm.edu/honorstheses/1105 This Honors Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Undergraduate Honors Theses by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. Tuning the pKa of Fluorescent Rhodamine pH Probes via Substituent Effects A thesis submitted in partial fulfillment of the requirement for the degree of Bachelors of Science in Chemistry from The College of William and Mary By Sarah Graham Stratton Accepted for ______________________________________ ____________________________________________ Dr. Elizabeth J. Harbron, Director ____________________________________________ Dr. Robert J. Hinkle ____________________________________________ Dr. John C. Poutsma ____________________________________________ Dr. John Conlee ii Table of Contents List of Tables and Figures ............................................................................................................................ i Abstract ...................................................................................................................................................... 1 Introduction ................................................................................................................................................ 2 Results and Discussion .............................................................................................................................. 11 Section I. Synthesis of di-ortho RB and R6G derivatives ........................................................................ 11 Section II. pKa studies of ortho-substituted RB and R6G derivatives ..................................................... 24 Section III. Crystal structure and Gaussian modeling ............................................................................ 61 Section IV. Nano incorporation of RB-DIA dyes .................................................................................... 82 Conclusions ............................................................................................................................................... 94 Experimental ............................................................................................................................................. 95 Acknowledgements ................................................................................................................................ 114 References .............................................................................................................................................. 116 Appendix A .............................................................................................................................................. 119 Appendix B .............................................................................................................................................. 188 Appendix C .............................................................................................................................................. 248 Appendix D ............................................................................................................................................. 275 List of Tables and Figures Figure 1. Jablonski diagram depicting absorption in blue and fluorescence in red 4 Figure 2. Absorption (left) and fluorescence (right) spectra. 4 Figure 3. Rhodamine dye structures (RB and R6G) 5 Scheme 1. Rhodamine spirolactam ring opening 5 Figure 4. Rhodamine para substituents 8 Figure 5. Rhodamine B-adamantanamine 8 Figure 6. RB and R6G di-ortho substituents and RB mono-ortho substituents 9 Scheme 2. Rhodamine 6G synthesis and derivatives 13 Scheme 3. Rhodamine 6G synthesis and derivatives 19 Table 1. Comparison of syntheses for RB and R6G di-ortho substituted derivatives 23 Figure 7. A fluorescence spectra of R6G-DMA 25 Figure 8. Graph of fluorescence peak intensity versus pH for R6G-DMA 26 Table 2. Average pKa values for the RB and R6G di-ortho series. 26 Table 3. Average pKa values for the additional titrations 28 Table 4. Average pKa values for the RB mono-ortho series 31 Table 5. Steric parameters for modeling di-ortho series 34 Table 6. Electronic parameters for modeling di-ortho series 35 Figure 9. RB and R6G pKa data for di-ortho substituents versus their A values 36 Figure 10. RB and R6G pKa data for electron withdrawing di-ortho substituents versus their A values 36 Figure 11. RB and R6G pKa data for electron donating di-ortho substituents versus their A values 37 Figure 12. R6G A value predicted pKa versus experimental pKa 38 Table 7. R6G regression analysis 39 Figure 13. R6G Es predicted pKa versus experimental pKa 45 Figure 14. R6G Es–σI-F regression predicted pKa versus pKa of di-ortho derivatives 47 Figure 15. RB Es-σp-F regression predicted pKa versus pKa of di-ortho derivatives 47 Table 8. Analysis of VIFs of R6G three-parameter regression models 47 Figure 16. R6G Es–σp-R regression predicted pKa versus pKa of di-ortho derivatives 48 Figure 17. R6G Es–σp-F-R regression predicted pKa versus pKa of di-ortho derivatives 49 Table 9. Analysis of VIFs of RB three-parameter regression models 50 Table 10 RB regression analysis 51 Figure 18. RB Es-σp-R regression predicted pKa versus pKa of di-ortho derivatives 54 Table 12. Electronic parameters for modeling mono-ortho series 56 Figure 19. RB pKa data for mono-ortho substituents versus their σp values 57 Table 11. Mono-ortho regression analysis 58 Figure 20. RB pKa data for mono-ortho substituents versus their σo values 59 Figure 21. RB σo regression predicted pKa versus pKa of mono-ortho derivatives 60 ii Figure 22. RB-DIA aliphatic region 61 Table 12. Crystal growing experiment results based on solution 62 Figure 23. Crystal structure of RB-DIA 63 Figure 24. Optimized structure of R6G-A (left) front view and (right) bird’s eye view 64 Figure 25. Optimized structure of R6G-DFA (left) front view and (right) bird’s eye view 64 Figure 26. Optimized structure of R6G-DCA (left) front view and (right) bird’s eye view 65 Figure 27. Optimized structure of R6G-DEA (left) front view and (right) bird’s eye view 65 Figure 28. Optimized structure of R6G-DMA 65 Figure 29. Optimized structure of R6G-DIA 66 Figure 30. Crystal structure of RB-A 66 Figure 32. Optimized structure of RB-DCA 67 Figure 33. Optimized structure of RB-DEA 67 Figure 34. Optimized structure of RB-DMA 67 Figure 35. Optimized structure of RB-DIA 68 Table 13. RB and R6G derivatives by pKa, xanthene ring twist, A value, σp value, and Es 68 Figure 36. The angles measured to calculate the xanthene twist 69 Figure 37. Xanthene ring torsion for RB and R6G derivatives versus the Es parameter 69 Figure 38. Xanthene ring torsion for RB and R6G derivatives versus the A value parameter 70 Figure 39. Xanthene ring torsion for RB and R6G derivatives versus the σp parameter 71 Figure 40. RB and R6G derivatives pKa values versus their respective xanthene ring torsion 71 Table 14. Regression analysis on R6G and RB using xanthene twist as a parameter 72 Figure 41. The angle between C20-N3-C21-C22 73 Figure 42. pKa versus RB and R6G derivatives measure of coplanarity with the carbonyl 73 Table 15. RB and R6G derivatives by pKa and coplanarity with the carbonyl 73 Figure 43. RB and R6G derivatives measure of coplanarity with the carbonyl versus the Es parameter 74 Figure 44. RB and R6G derivatives measure of coplanarity with the carbonyl versus the σp parameter 75 Figure 45. Optimized structure of RB-2CA 76 Figure 46. Optimized structure of RB-2MA 76 Figure 47. Optimized structure of RB-2IA 76 Table 16. RB mono-ortho derivatives by pKa, xanthene ring twist, A value, σp value, and Es 77 Figure 48. Xanthene ring torsion for RB mono-ortho derivatives versus A values 77 Figure 49. Xanthene ring torsion for RB mono-ortho derivatives versus the Es parameter 78 Figure 50. Xanthene ring torsion for RB mono-ortho derivatives versus the σp parameter 78 Table 17. RB mono-ortho derivatives by pKa and coplanarity with the carbonyl 79 Figure 51. RB mono-ortho derivatives measure of coplanarity with the carbonyl versus Es parameter 80 Figure 52. RB mono-ortho derivatives measure of coplanarity with the carbonyl versus σp parameter 80 iii Figure 53. Structure of PFBT 82 Figure 55. The addition of acid switches the fluorescence of the CPNs from the green region to the red as FRET occurs between PFBT and RB-DIA 83 Figure 56. PFBT UV-Vis spectrum 85 Figure 57. Post-argon bubbling addition of dye fluorescence, excitation at 450 nm 86 Figure 58. Post-argon bubbling addition of dye fluorescence, excitation at 535 nm 86 Figure 59. Pre-argon bubbling addition of dye fluorescence, excitation at 450 nm 88 Figure 60. Pre-argon bubbling addition of dye fluorescence, excitation at 535 nm 88 Figure 61. Post-argon bubbling addition of dye UV-Vis spectra 89 Figure 62. Pre-argon bubbling addition of dye UV-Vis spectra 89 Figure 63. Two-day old nanos