Synthesis and Fluorescence Studies of Ph-Responsive Rhodamine B Derivatives
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W&M ScholarWorks Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects 2013 Synthesis and Fluorescence Studies of pH-Responsive Rhodamine B Derivatives William Lawrence Czaplyski College of William & Mary - Arts & Sciences Follow this and additional works at: https://scholarworks.wm.edu/etd Part of the Inorganic Chemistry Commons Recommended Citation Czaplyski, William Lawrence, "Synthesis and Fluorescence Studies of pH-Responsive Rhodamine B Derivatives" (2013). Dissertations, Theses, and Masters Projects. Paper 1539626948. https://dx.doi.org/doi:10.21220/s2-8aff-cq35 This 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 Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. Synthesis and Fluorescence Studies of pH-Responsive Rhodamine B Derivatives William Lawrence Czaplyski Herndon, Virginia Bachelor of Science, The College of William and Mary, 2012 A thesis presented to the Graduate Faculty of the College of William and Mary in Candidacy for the Degree of Master of Science Department of Chemistry The College of William and Mary August, 2013 APPROVAL PAGE This thesis is submitted in partial fulfillment of the requirements for the degree of Master of Science William Lawrence Czaplyski Approved by the Committee, July 2013 Committee Chair Professor Elizabeth J. Harbron, Chemistry The College of William and Mary Professor Jonathan Scheerer, Chemistry The C ollege of William and Mary Professor KfTstin WuMbg^, Chemistry The C ollege of William and Mary ABSTRACT We report the synthesis of a series of pH-sensitive fluorescent dyes from rhodamine B andpara -substituted anilines, which were selected to span a wide range of electronic properties, as given by their Hammett constants. The rate of ring-opening of the rhodamine spirolactams upon exposure to acid was studied by fluorescence spectroscopy. Correlations between the substituent Hammett constant and the properties of the dye were observed and suggest general applicability to the design of rhodamine-based pH probes. TABLE OF CONTENTS Acknowledgements iii Dedication iv List of Tables v List of Figures vix Chapter 1. Background 1 Absorbance and Fluorescence 3 Rhodamine Dyes as pH Sensors 5 Hammett Substituent Theory 10 Kinetics Models 12 Works Cited 15 Chapter 2. Introduction 17 Design and Synthesis 17 Studies of Rhodamine B Benzaldehyde Derivatives in 1:1 MeOH:H20 19 pH Titrations of Rhodamine B Benzaldehyde Derivatives in 1:9 Et0H:H20 22 Studies in 1:1 EtOH:H20 25 NMR Studies 29 Conclusions 31 Works Cited 33 Experimental Section 34 i Chapter 3. Design and Synthesis 42 Response of Rhodamine B Aniline Derivatives to Acid 43 Kinetic Studies 45 pH Titration Studies 49 Activation Energy Studies 57 13C NMR Studies 59 Rational Design and Synthesis of a pH Sensitive Rhodamine B Derivative 62 Dichloronitro Derivative Studies 63 Conclusions 64 Works Cited 66 Experimental Section 67 Appendix Supporting Information for Chapter 2 and 3 76 ii ACKNOWLEDGEMENTS I would first and foremost like to thank Professor Elizabeth Harbron for taking me into her lab as a freshman and allowing me to continue with the master’s program here. She has been nothing short of an amazing mentor and has helped me grow into an independent researcher while also always being there for when I needed help. Her willingness to provide support and a listening ear extended beyond the experiments in her lab, and I am very fortunate to have worked with her. I would also like to express my gratitude to Professor Jonathan Scheerer and Professor Kristin Wustholz for being on my committee. Their classes here were extremely challenging as well as rewarding, and I am glad to have learned so much from them. I would also like to thank Professor Carey Bagdassarian for challenging and supporting me since his class in the first semester of my freshman year. I am grateful for his constant reminders to step back and enjoy everything, not just the chemistry and lab work. I want to acknowledge the other members of the Harbron lab, both past and present, for providing a healthy environment in which to learn, teach, and take intellectual risks, especially the group of Summer 2010. Becca Allred laid the foundation for the rhodamine pH project, Courtney Roberts made tremendous progress on the initial synthesis and testing of the benzaldehyde derivatives, and Grace Purnell helped carry the benzaldehyde project and do the initial data acquisition for the aniline project. All three of them helped our pH projects evolve to where they are today, and I am indebted to their work. I would also, especially, like to thank Beth Childress and Emily Eklund for their constant friendship and support. Their presence helped to brighten up days where research did not go as envisioned, and I enjoyed being able to spend time with them outside of lab as well. I am also grateful for all of my friends, who have helped me get to where I am today, and my parents for supporting me, especially in my undergraduate career here. I would finally like to thank Kaila Margrey and Val Tripp for their unwavering friendship and support. They have been the best friends I could ever ask for, and I could not have gotten through all the lab work and writing without them. I want to thank Kaila especially for all her assistance in overcoming my NMR limitations and for tolerating my odd work habits. She also provided many invaluable suggestions regarding my work, especially for the use of a f-butyl aniline. For Kaila, my best friend. iv LIST OF TABLES 2.1 Summary of kinetics data for derivatives 2-5 in 1:1 MeOH:H20 21 2.2 Calculated pKa values in 1:9 EtOH:H20 24 2.3 Summary of rhodamine benzaldehyde kinetics data in 1:1 EtOH:H20 28 3.1 Summary of kinetics data for the rhodamine B aniline derivatives 47 3.2 Calculated pKa values for derivatives 1-8 in 1:1 EtOH:H20 51 3.313CNMR shifts for the spirolactam carbonyl for derivatives 1-8 59 v LIST OF FIGURES 1.1 Structures of several xanthene dyes 2 1.2 Equilibrium of closed and open forms of rhodamine B 2 1.3Structures of rhodamine hydrazides 3 1.4 Jablonski diagram showing absorption and fluorescence 4 1.5 Proposed equilibrium for rhodamine 6G oxaldehyde derivative 6 1.6 Structure of hydrazone 5 6 1.7 Proposed mechanism for ring-opening of rhodamine hydrazides 8 1.8 Summary of spirolactam substituent effect on pKa of ring- opening 9 2.1 Synthesis of rhodamine B benzaldehyde derivatives 2-6 18 2.2 Synthesis of rhodamine 6G benzaldehyde derivatives 8^11 19 2.3 Fluorescence kinetic studies of 2-5 in 1:1 MeOH:H20 with rapid equilibrium fit 20 2.4 Hammett plot of rate constant in 1:1 MeOH:H20 21 2.5 Absorbance pH titrations for derivatives 2-5 1:9 Et0H:H20 23 2.6 Fluorescence pH titrations for derivatives 2-5 1:9 EtOH:H20 24 2.7 Absorbance pH titrations for derivatives 1, 4-6 in 1:1 EtOH:H20 26 2.8 Fluorescence pH titrations for derivatives 1, 4-6 in 1:1 EtOH:H20 26 2.9 Fluorescence kinetic studies of derivatives 4-6 in 1:1 EtOH:H20 27 2.10 Fluorescence kinetic studies of derivatives 8-10 in 1:1 EtOH:H20 28 vi 2.11 Proposed mechanism of fluorescence for rhodamine B benzaldehyde derivatives 30 3.1 Synthesis of rhodamine B aniline derivatives 42 3.2 Change of absorbance and fluorescence of 8 with decreasing pH 44 3.3 Proposed mechanism of ring-opening for rhodamine aniline derivatives 45 3.4 Kinetics scans of derivatives 8-10 with first order kinetics fits 46 3.5 Hammett plot of rate constant vs op 48 3.6Hammett plot of rate constanto 'vs 49 3.7 Normalized absorbance curves for pH titrations 50 3.8 Absorbance pH titration curves for 1-8 corrected for dilution 51 3.9 Hammett plot of final absorbance intensities vs a- 53 3.10 Fluorescence pH titration curves for 1-8 corrected for dilution 54 3.11 Hammett plot of final fluorescence intensities vs. a- 56 3.12 Arrhenius plot for derivatives 4 and 8 58 3.13 Hammett plot of carbonyl chemical shift vs. a- 60 3.14 Rate constant vs. carbonyl chemical shift 61 3.15 Synthetic scheme for dichloronitro derivative 62 3.16 Kinetics scans of 8 and 9 in 1:1 EtOH:H20 63 vii Chapter 1: Introduction to Rhodamine Dyes Background Fluorescent dyes have been synthesized and studied for over a century and have been employed in a variety of disciplines. The fluorescent compounds known as rhodamine dyes were first synthesized in 1905 by Noelting and Dziewonsky .1 Although many different rhodamine derivatives have been synthesized, there have been comparatively few efforts to examine exactly how their stereoelectronic properties impact their function as fluorescent chemosensors. Both projects described in the subsequent chapters are united under the overarching theme of determining the contribution of electronics to how rhodamines respond to changes in pH. This is demonstrated through kinetics and titration studies utilizing both absorbance and fluorescence. Elucidation of the impact of electronics possesses implications for the rational design of rhodamine derivatives that respond both rapidly and strongly to acidic stimuli. Rhodamines are a subset of the larger class of xanthene dyes, which are # ^ characterized by the presence of the tricyclic xanthene core within the molecule. Several of these xanthene dyes are commercially available and differ primarily with respect to the substituents on the core; rhodamines possess amino functionalities, while fluoresceins possess hydroxy or carbonyl substituents.