Exploring the Monoterpene Cyclization Mechanism by Studying (+)-Limonene Synthase Using Novel Fluorinated Substrate Analogues Master’s Thesis Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Department of Biochemistry Daniel Oprian, Advisor In Partial Fulfillment of the Requirements for the Degree Master of Science in Biochemistry by Qi Yu May 2017 Copyright by Qi Yu © 2017 ACKNOWLEDGEMENT I would like to give my most sincere gratitude to every single person who has been guiding me and helping me along this journey. I would first like to thank my thesis advisor and research mentor, Dr. Daniel Oprian, for this great opportunity being able to work in his lab, which brings me the most amazing experience in science. I thank him for always being so patient and inspiring as a mentor. The door to Dr. Oprian’s office was always open whenever I ran into a trouble spot or had a question about my research or writing. He consistently allowed this thesis to be my own work, but steered me in the right the direction whenever he thought I needed it. I would like to give a big thank to my Ph.D. student mentor, Ben, who has been helping and advising me during the entire project, as well for always being available for help and being so encouraging. I would also like to acknowledge Dr. Isaac Krauss and chemistry department Ph.D. student Leiming, for providing me professional guidance on organic chemistry synthesis. I would also like to thank all the members from Oprian lab, both past and present, my fellow classmates in the biochemistry department. Nobody has been more important to me in the pursuit of this project than the members of my family. I must express my profound gratitude to my parents, whose love and support are with me in whatever I pursue. I also would like to give a big thank to my friends who have been always encouraging me and caring about me. Finally, I would like to thank my little cat, Yangyang, for just being so cute. iii ABSTRACT Exploring the Monoterpene Cyclization Mechanism by Studying (+)-Limonene Synthase using Novel Fluorinated Substrate Analogues A thesis presented to the Department of Biochemistry Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By Qi Yu A proposed universal monoterpene synthases (cyclases) cyclization mechanism topologically requires an isomerization step of the substrate geranyl diphosphate (GPP) to the formation of the proposed universal intermediate linalyl diphosphate (LPP), which has never been directly observed. The study described here was designed to investigate the cyclization mechanism by studying a model enzyme, (4R)-(+)-limonene synthase ((+)-LS), a monoterpene cyclase that produces (+)-limonene by using substrate GPP. The study synthesized two novel difluorinated substrate analogues, 8,9-difluorogeranyl diphosphate (DFGPP) and 8,9-difluorolinalyl diphosphate (DFLPP), which were designed to retard the cyclization step in order to observe the intermediate at the enzyme active site. Both analogues are proved to be substrates for (+)- -1 LS. Compared to GPP, which has a kcat value of 0.0905 ± 0.0070 s , DFGPP has a reduced kcat -1 value at 0.00144 ± 0.00002 s , showing a 69-fold slower rate in the catalytic reaction. The KM value of DFGPP is 20.50 ± 1.05 µM, and does not change much compared to that of the nonfluorinated substrate with 29.79 ± 7.83 µM. Both DFGPP and DFLPP are catalyzed to make the same acyclic product and the structure of the product has been investigated through NMR. Our results indicate that LPP might be an intermediate in the ring closure mechanism for (+)- LS. Additionally, LPP is also proved to be a substrate for (+)-LS and makes (+)-limonene. iv TABLE OF CONTENTS ABSTRACT iv TABLE OF CONTENTS v LIST OF SCHEMES AND FIGURES vi INTRODUCTION Terpenes 1 Monoterpene Synthases 2 Limonene Synthase 4 Fluorinated Substrate Analogues 6 MATERIALS AND METHODS Difluorinated Substrate Analogues Synthesis 9 Enzymatic Activity Study 24 Fluorinated Terpene Product Structural Determination 26 Crystallization 26 RESULTS AND DISCUSSION Difluorinated Substrate Analogues Synthesis 27 Enzymatic Activity Study 33 Future Directions 41 APPENDIX: SUPPLEMENTARY FIGURES 43 REFERENCES 46 v LIST OF SCHEMES AND FIGURES List of Schemes 1. DFGPP and DFLPP Synthesis 27 List of Figures 1. Generation of Acyclic Precursors 2 2. Proposed Monoterpene Synthase Cyclization Mechanism & Monoterpene diversity 4 3. Limonene Enantiomers 5 4. DFGPP and DFLPP 7 5. 1H-NMR of DFGPP 31 6. 1H-NMR of DFLPP 31 7. 19F-NMR of DFGPP 32 8. 31P-NMR of DFGPP (decoupled) 32 9. Single Vial Method 33 10. DFGPP Product GC, DFLPP Product GC and Standard Limonene GC 34 11. LPP Product GC and Standard Limonene GC 34 1 12. (a) H-NMR of the DFGPP and DFLPP Product in C6D6 35 19 (b) F-NMR of the DFGPP and DFLPP Product in C6D6 35 13. 8,9-difluoro-β-ocimene 36 14. (a) Possible Product Fluorines Structure on C7 36 (b) Deprotonated Terminal Di-methyl Group 36 15. Michaelis-Menten Plot for Reaction of DFGPP and (+)-LS 38 16. Michaelis-Menten Plot for Reaction of GPP and (+)-LS 38 17. Figure 17. Michaelis-Menten Plot for Reaction of DFLPP and (+)-LS 39 18. Figure 18. Michaelis-Menten Plot for Reaction of LPP and (+)-LS 39 19. 8,9-difluoro-β-ocimene Formation 40 20. A1: DFGPP 19F-NMR 43 21. A2: DFGPP 31P-NMR (coupled) 43 22. A3: DFGPP 13C-NMR 43 23. A4: DFLPP 19F-NMR 44 vi 24. A5: DFLPP 31P{1H}-NMR 44 25. A6: DFLPP 13C{1H}-NMR 44 26. A7: LPP 1H-proton NMR 45 27. A8: DFGPP/DFLPP Product GC Overlapping with Commercially Available Ocimene GC 45 vii Introduction Terpenes Terpenes represent one of the most widely distributed and structurally diverse classes of secondary metabolites in nature, with more than 55,000 members identified. Many terpenes are essential for plants in their secondary metabolism like plant hormones, and for ecological interactions, such as chemical defenses, and pollinator attractants.1 Besides, terpenes have economic and industrial applications ranging from flavorings and fragrances to solvents, medicines, and natural rubber, etc.2 Recently, scientists have considered terpenes as potential biofuels as alternatives to existing fuel due to their high-energy content and physiochemical similarities to petroleum-based fuels.3,4,5 In the pharmaceutical field, some terpenes have also found use as anti-inflammatory, antitumor, and anti-metastatic agents.6, 7 Despite their structural diversity, terpenes are constructed with a repetitive and “head- to-tail” joining of C5 isoprenoid units. All terpenes are derived from a common precursor, isopentenyl diphosphate (IPP), which is isomerized to dimethylallyl diphosphate (DMAPP) using IPP isomerase. Prenyltransferases catalyze the condensation reaction of one DMAPP with one IPP unit to form the monoterpene (C10) precursor, geranyl diphosphate (GPP). An addition of one IPP unit to GPP will give farnesyl diphosphate (FPP), the precursor for sesquiterpenes (C15), and one addition of IPP to FPP will then give geranylgeranyl diphosphate (GGPP), which 1 is the precursor for diterpenes (C20) (Figure 1). Most terpenes are found to be only derived from those three acyclic precursors. - 1 - Figure 1. Generation of Acyclic Precursors: DMAPP being sequentially elongated by prenyltransferases to generate geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP). Adapted from Davis & Croteau (2000).1 Monoterpene Synthases Monoterpenes are a class of terpenes containing two isoprene units with the molecular formula C10H16, and they can be either cyclic or acyclic. This study focuses on the study of limonene synthase, which is a monoterpene cyclase that produces the cyclic monoterpene - 2 - limonene using substrate GPP. Over many years, Croteau and co-workers have established a common monoterpene cyclization mechanism, which involves several high-energy carbocationic intermediates.8-10 The stereochemistry of monoterpene products is determined at the very first stereoselective binding of distinct GPP conformation at the enzyme active site.10 The cyclization reaction is then initiated with divalent metal ion-dependent (Mn2+ or Mg2+) ionization of the allylic diphosphate on GPP, forming a resonance-stabilized allylic carbenium ion. A syn-migration of the diphosphate to C3 gives the tightly enzyme-bound and undetectable intermediate, linalyl diphosphate (LPP), which geometrically allows the newly formed C2-C3 single bond to rotate under a lower energy barrier compared to the geranyl cation. Therefore, C1 is now in a preferred position for C6-C1 cyclization from an anti-endo conformation. A second ionization of the allylic diphosphate on LPP is then followed by C6-C1 cyclization to give the proposed universal monoterpene intermediate, the α-terpinyl cation. Finally, reactions are terminated with deprotonation, nucleophile capture, hydride shifts, etc. Monoterpene basic skeletons then go through a series of oxidation reactions to achieve great cyclic monoterpene diversity (Figure 2).13 Divalent metal ions (Mn2+ or Mg2+) are found to be required for monoterpene synthases catalysis, and an absolutely conserved aspartate-rich DDxxD motif in monoterpene synthases active site is believed to be involved in those divalent ions coordination for substrate binding.11,12 - 3 - Figure 2. Proposed Monoterpene Synthase Cyclization Mechanism & Monoterpene diversity. Starting from GPP, monoterpene cyclization requires ionization and syn-migration of the allylic diphosphate to give the proposed intermediate, LPP, which