University of Tennessee, Knoxville Trace: Tennessee Research and Creative Exchange Masters Theses Graduate School 12-2009 Design and Synthesis of FRET-Based Boronic Acid Receptors to Detect Carbohydrate Clustering and Development of Diacylglycerol-Based Lipid Probesto Investigate Lipid-Protein Binding Interactions Manpreet Kaur Cheema University of Tennessee - Knoxville Recommended Citation Cheema, Manpreet Kaur, "Design and Synthesis of FRET-Based Boronic Acid Receptors to Detect Carbohydrate Clustering and Development of Diacylglycerol-Based Lipid Probesto Investigate Lipid-Protein Binding Interactions. " Master's Thesis, University of Tennessee, 2009. https://trace.tennessee.edu/utk_gradthes/517 This Thesis is brought to you for free and open access by the Graduate School at Trace: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of Trace: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a thesis written by Manpreet Kaur Cheema entitled "Design and Synthesis of FRET-Based Boronic Acid Receptors to Detect Carbohydrate Clustering and Development of Diacylglycerol-Based Lipid Probesto Investigate Lipid-Protein Binding Interactions." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Chemistry. Michael Best, Major Professor We have read this thesis and recommend its acceptance: David Baker, Frank Vogt Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) To the Graduate Council: I am submitting herewith a thesis written by Manpreet Kaur Cheema entitled “Design and Synthesis of FRET-Based Boronic Acid Receptors to Detect Carbohydrate Clustering and Development of Diacylglycerol-Based Lipid Probes to Investigate Lipid ‒Protein Binding Interactions.” I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Chemistry. Michael Best Major Professor We have read this thesis and recommend its acceptance: David Baker Frank Vogt ` Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduat e School (Original signatures are on the file with official student record) Design and Synthesis of FRET-Based Boronic Acid Receptors to Detect Carbohydrate Clustering and Development of Diacylglycerol-Based Lipid Probes to Investigate Lipid-Protein Binding Interactions A Thesis Presented for The Master of Science Degree University of Tennessee, Knoxville Manpreet Kaur Cheema December 2009 Acknowledgements First of all, I would like to thank my parents, without whose guidance and perseverance, I would not have been able to reach this stage in my life. Thank you, Dr. Best, for being a constant source of motivation and inspiration throughout the course of this degree. The things that I have learned as a graduate student in your research lab have helped me develop into a more confident chemist. I also want to take pleasure in thanking my group members for constantly being there for me and helping me get through tough times. I will really cherish the good times that we shared in this lab. I would like to take an opportunity to thank Irene Abia (Baker’s group) for synthesizing an important molecule for our research design. I also want to thank my family members for providing me all the moral support whenever I needed it the most. Thank you Mandy for making me have faith in me. Last but not the least; I would like to thank my friends, Simi, Shibani and Ashutosh for being my pillars of support during my stay here. You guys are like my family away from home. ii Abstract Carbohydrate–protein interactions play vital roles in various biochemical processes such as signal transduction and cell surface recognition events. The clustering of carbohydrates into dense domains such as lipid rafts regulates recognition by multivalent receptors (i.e. lectins). These regions are known to play important roles in biological processes such as cellular transduction and trafficking. In order to characterize the clustering of glycans on cell surfaces, detection of domains with high carbohydrate density is of great interest. In this thesis, we present the work based on a modular strategy to design and synthesize boronic acid-based carbohydrate receptors, which are termed as boronolectins because of their similarly in functions with lectins, in order to understand the molecular basis of carbohydrate–protein interactions. These receptors will then be employed for binding studies with carbohydrate based guests and catechol derivatized diol target molecules in order to study the binding interactions between the boronic acid receptors and diol moieties present in guest molecules. A second project that is described in this thesis is based on developing diacylglycerol-based lipid probes, which could be employed for studying protein ‒lipid binding interactions. Due to involvement of protein ‒lipid binding interactions in the onset of various pathophysiological conditions, it is of paramount importance to investigate these interactions at the molecular level. DAG (diacylglycerol) represents an important class of signaling lipids and members of the Protein kinase C (PKC) family are described as the main iii responsive receptors of DAG. PKCs are known to be involved in tumorigenesis. In order to elucidate the exact correlation between PKC activity and carcinogenesis, it will be beneficial to design and synthesize DAG-based lipid analogs. In this thesis, hence, a modular strategy to design and synthesize a class of DAG-based lipid analogs by appending reporter groups such as polyaromatic fluorophores in the sn -1 acyl chains via the traceless Staudinger ligation is described. In both the projects, the synthetic strategy adopted is based on a modular design in order to generate a common scaffold which undergoes modification at the last step to generate a class of fluorophore tagged analogs to be employed for studies. iv Table of Contents Chapter Page Chapter 1: Design, Synthesis and Binding studies of Boronic Acid Based Carbohydrate Sensors Background and significance ................................................................................1 Significance of carbohydrates in biological processes..........................................1 Boronic acids as receptor/lectin mimics................................................................4 Research design of boronic acid-based receptors..............................................12 FRET-based detection........................................................................................12 Design and synthesis of fluorophore tagged boronic acid sensors .....................15 Design and synthesis of divalent carbohydrate guest.........................................25 Binding studies....................................................................................................30 Experimental.......................................................................................................35 Chapter 2: Design and Synthesis of Azide-Labeled Diacylglycerol Analogs Background and significance .............................................................................53 Modular design of lipid probes ............................................................................57 Modular approach to synthesize fluorophore tagged lipid analogs .....................65 Experimental.......................................................................................................74 Appendix References .........................................................................................................90 NMR spectra.......................................................................................................96 Vita....................................................................................................................109 v List of Figures Figure Page Figure 1.1 Carbohydrate recognition by lectins, toxins, viruses etc. 3 Figure 1.2 Illustration of interaction between boronic acid and saccharide 5 Figure 1.3 Formation of reversible covalent linkages to diols by boronic acids 6 Figure 1.4 The binding process between phenylboronic acid and a diol 7 Figure 1.5 Examples of monovalent and bivalent boronic acid receptors 8 Figure 1.6 PET-derivatized boronic acid-based receptors 9 Figure 1.7 Various pyrene-appended boronic acid sensors 10 Figure 1.8 Examples of various types of boronic acid sensors 11 Figure 1.9 Diagrammmatic representation of boronic acid FRET pairs employed for carbohydrate recognition 13 Figure 1.10 FRET- conceptual description 15 Figure 1.11 Synthesis of pyrene-tagged boronic acid receptor 17 Figure 1.12 Alternate synthesis of pyrene-tagged protected boronic acid 18 Figure 1.13 Synthetic schemes for synthesis of FRET-dervatized boronic acid-based receptors 19 Figure 1.14 Modular approach using click reaction 21 Figure 1.15 Alternate synthesis of fluorophore-tagged receptors using click reaction 22 Figure 1.16 Attempted approach to synthesize fluorophore-tagged receptors 23 Figure 1.17 Alternate coupling reaction approach 24 Figure 1.18 Diagrammatic representation of design of divalent guests 26