STRUCTURE ELUCIDATION AND ISOTOPIC LABELING EXPERIMENTS PROVIDE INSIGHTS INTO THE BIOSYNTHESIS OF THREE PHYTOCHEMICALS: ALKAMIDES, AVENACIN, AND HYDROXYLATED FATTY ACIDS by Alicen M. Teitgen A Thesis Submitted to the Faculty of Purdue University In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy Department of Chemistry and Chemical Biology Indianapolis, Indiana December 2017 ii THE PURDUE UNIVERSITY GRADUATE SCHOOL STATEMENT OF COMMITTEE APPROVAL Dr. Robert E. Minto, Chair Department of Chemistry and Chemical Biology Dr. Eric Long Department of Chemistry and Chemical Biology Dr. Sébastien Laulhé Department of Chemistry and Chemical Biology Dr. John Goodpaster Department of Chemistry and Chemical Biology Approved by: Dr. Eric Long Head of the Graduate Program iii To Jake – Thank you for always believing in me. iv ACKNOWLEDGMENTS I am forever grateful to so many people who helped me along my journey. First, I want to thank Dr. Robert E. Minto for mentoring me over the past 5 years. Dr. Minto’s knowledge still intimidates me, but his wisdom and support has challenged me to become a better chemist, writer, and learner. I also want to thank Dr. Brenda Blacklock for her guidance and encouragement along the way. I’ve enjoyed sharing my love for running with both Dr. Minto and Dr. Blacklock, and they have been extremely compassionate towards my family and me. Thank you to Dr. Michael R. Shepard for helping me to get started in lab and for your friendship. I want to thank all the undergraduates I have had the privilege to work with over the years: Paola, Ngun, Jermell, and Bailey. A special thanks to Bailey, whose fun-loving spirit and contagious attitude helped keep my spirits up during the last year of graduate school. I also owe a special thanks to all our collaborators who made our projects possible. It truly takes a village to raise a family, especially when you start a family during graduate school. For that, I owe enormous thanks to all the family and friends who were there while I pursued my dreams. To my mom and step-dad, Nancy, and Tim, and to my parents-in-law, Barb and Tom, thank you for all the support especially in helping to take care of Jules and Eve. Thank you to my dad, Mike, for always being my #1 fan and for Sunday pizza nights. I treasure that Jules and Eve have such a special relationship with their grandparents. Thank you to the best bro ever, Kyle, for your witty personality and for always making me smile. Thank you to our community of friends, especially those from Our Lady of Grace, for unrelenting support and encouragement. Each of you has played an important role in our lives. Finally, thank you to Jules and Eve for your endless love. There is nothing better then coming home to your hugs, kisses, and the excitement you have when I walk through the door. When school was the most difficult, you were my biggest inspiration to keep working hard. Though you might not remember this time in your life, I hope you’ll always remember that no dream is too big. To Jake, thank you for never giving up on me and for always believing in me. It wasn’t easy and, without you, I’m sure I wouldn’t have made it. Thank you for being my best friend and for helping me to achieve mydreams. v TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix LIST OF ABBREVIATIONS ........................................................................................ xviii ABSTRACT .......................................................................................................................xx CHAPTER 1. INTRODUCTION ...................................................................................21 1.1 Evolution of Plant Natural Product Biosynthesis .....................................................21 1.2 Research Objectives .................................................................................................26 CHAPTER 2. IDENTIFICATION OF HYDROXYLATED FATTY ACIDS FOUND IN ORYCHOPHRAGMUS VIOLACEUS ............................................................27 2.1 Introduction ..............................................................................................................27 2.1.1 Lipids from specialized metabolism ............................................................27 2.1.2 Specialized fatty acids in the seed oils of the Brassicaeae ...........................35 2.2 Materials and Methods .............................................................................................36 2.2.1 GC/MS protocols for the structure elucidation of 7,18-(OH)2-24:2Δ15, 21 37 2.2.1.1 FAME and pyrrolidide GC/MS methods ..................................................37 2.2.1.2 Metathesis and racemic 3-cyclohexen-1-ol GC/MS methods ...................38 2.2.1.3 GC/MS method for chiral separations .......................................................40 15,21 2.2.2 Isolation of 7,18-(OH)2-24:2Δ from seed oil ..........................................41 15,21 2.2.3 Derivatives of 7,18-(OH)2-24:2Δ ...........................................................41 15,21 2.2.4 Configuration of C-18 in 7,18-(OH)2-24:2Δ ..........................................42 15,21 2.2.5 Route 1 for the attempted synthesis of 7,18-(OH)2-24:2Δ .....................44 15,21 2.2.6 Route 2 for the synthesis of 7,18-(OH)2-24:2Δ ......................................47 15,21 2.2.7 Configuration of C-7 in 7,18-(OH)2-24:2Δ ............................................53 2.3 Results and Discussion .............................................................................................55 2.3.1 Serendipitous discovery of novel oils in O. violaceus seeds ........................55 2.3.2 Structural identification of novel fatty acids from O. violaceus seeds .........56 15,21 2.3.3 Configuration at the C18 stereocenter of 7,18-(OH)2-24:2Δ .................68 15,21 2.3.4 Configuration at the C7 stereocenter of 7,18-(OH)2-24:2Δ ...................70 vi 2.3.5 Oil composition ............................................................................................78 2.4 Conclusion ................................................................................................................82 CHAPTER 3. THE CONVERGENCE OF TWO PATHWAYS IN THE BIOSYNTHESIS OF ALKAMIDES FROM ECHINACEA .............................................83 3.1 Introduction ..............................................................................................................83 3.1.1 Convergence of two metabolic pathways .....................................................83 3.1.2 Alkamide biosynthesis in Echinacea ...........................................................87 3.2 Materials and Methods .............................................................................................96 3.2.1 Germination and growth of E. purpurea seedlings ......................................98 3.2.2 Alkamide analysis ........................................................................................98 3.2.3 Isotopic labeling experiments .......................................................................99 3.2.4 Equations used to calculate incorporation levels .......................................100 3.2.5 Fatty acid methyl ester preparation for MS analysis ..................................100 3.2.5.1 Protocols for alkamide and FAME analysis by GC/MS and LC/MS .....101 3.2.6 Synthesis of aldehydes and [d3]-aldehydes ................................................104 3.2.7 Synthesis of alkamides and [d3]-alkamides ................................................117 3.2.8 Synthesis of fatty acids and [d3]-fatty acids ...............................................121 3.2.9 Synthesis of (E,E)-2,4-dodecadienoic acid ................................................127 3.2.10 Synthesis of (2E,4E,8Z,10E)-dodecadienoic acid ......................................130 3.2.11 Synthesizing acyl-CoAs .............................................................................133 3.2.12 Calculating concentrations of acyl-CoAs ...................................................133 3.3 Results and Discussion ...........................................................................................135 3.3.1 Isotope labeling experiments of amino acids, Val and Ile .........................136 3.3.2 Synthesis of deuterated aldehydes ..............................................................141 3.3.3 Synthesis of α,β-unsaturated alkamides .....................................................143 3.3.4 Synthesis of α,β-unsaturated fatty acids .....................................................145 3.3.5 Determination of isotope distribution patterns in alkamides isolated from plants provided deuterated α,β-unsaturated fatty acids and α,β-unsaturated alkamides ..................................................................................................................146 3.3.6 Synthesis of 12:1, 12:2, and 12:4 used for acyl-CoAs ...............................147 3.3.7 Concentrations of acyl-CoAs .....................................................................151 vii 3.3.8 Activity assays of acyl-CoAs with EpLigase .............................................152 3.4 Conclusion ..............................................................................................................154 CHAPTER 4. STRUCTURE ELUCIDATION