Limonene Synthase Using Novel Fluorinated Substrate Analogues Ma

Home , Monoterpene, Sabinene

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

Qi Yu

May 2017

Qi Yu

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 is capable of cyclization, forming the universal α-terpinyl cation (mechanism highlighted in grey), which goes through various reaction 13 pathways and forms diverse monoterpenes. Adapted from Degenhardt (2009) .

Limonene Synthase

Limonene synthase (LS) catalyzes the simplest cyclization among all terpenoid cyclizations, terminating the reaction to produce limonene through deprotonation on a terminal

- 4 - methyl group from the α-terpinyl cation intermediate. Therefore, limonene synthase is often used as a model for terpenoid cyclization studies.1

In nature, limonene appears as two different enantiomers, with (4R)-(+)-LS ((+)-LS) giving (4R)-(+)-limonene, and (4S)-(-)-LS ((-)-LS) giving (4S)-(-)-limonene (Figure 3). The stereochemistry of the limonene product is determined by initial folding of GPP at the active site.14,18

Figure 3. Limonene Enantiomers: limonene appears as two enantiomeric forms: (4R)-(+)-limonene, commonly found in citrus fruits, and (4S)-(-)-limonene, found in plants, such as pine, sage, etc.

Previous studies on (-)-LS suggest the same cyclization mechanism for the (+)-LS, in which the GPP goes through a series of carbocation-pyrophosphate anion paired intermediates to give the universal α-terpinyl cation.1,14 Croteau and co-workers have done a large numbers of studies on (-)-LS and its reaction mechanism,14-16 but to date (+)-LS has never been explored in detail. Additionally, researchers have never directly observed LPP in the reaction pathway, even though studies have shown linalyl disphosphate as an alternative substrate to GPP for (-)-

LS, indicating LPP as a possible intermediate in the cyclization catalyzed by monoterpene synthases. Here, we are interested in the cyclization mechanism of (+)-LS from Citrus sinensis

(Navel orange), which will provide insights into the reaction mechanism for both LS enantiomers and other terpene synthases.

- 5 - Several studies have determined crystal structures of (+)- and (–)-LS in both apo- and substrate analogue-bound forms14,17. Morehouse et al. determined the structure of (+)-LS in the apoprotein form (without cofactors or ligands bound) in an “open” conformation at 2.3 Å resolution (5UV0) and results show that there are very few differences in active site amino acid residues between the two enantiomer producing enzymes17. In addition,Morehouse and co- workers have characterized the enzymatic activity for the (+)-LS with GPP and both KM and kcat values fall within the typical range observed for other terpene synthases, and they have also shown that (+)-LS, similar to (-)-LS, prefers divalent metal ion Mn2+ to Mg2+.17

Fluorinated Substrate Analogues

Scientists have used fluorinated substrate analogues to study different terpene synthase reaction mechanisms.19-23 In the study of prenyl-transfer reactions, Poulter and co-workers used

FGPP as an analogue to GPP, substituting the C2 hydrogen of GPP with fluorine, which posed an electron-withdrawing effect on the developing allylic carbocation, and caused a slow down on the ionization of the initial allylic diphosphate. The experiment indicated the requirement of the allylic carbenium ion for the prenyl-transfer reaction which is highly similar in principle to the initial steps of the proposed monoterpene synthase reaction.19,20

Croteau and co-workers have used FGPP and 2-fluorolinayl diphosphate (FLPP) to study

(-)-LS and (+)-bornyl diphosphate synthase. In the study of (-)-LS co-crystallized with FLPP, a right-handed screw conformation was observed for the fluorinated analogue that agrees with prediction for stereoselective binding to (-)-LS.21,22 In the most recent study of (+)-LS structural characterization, Ramasamy and co-workers have determined the crystal structures of apo-(+)-

- 6 - LS soaked with the monofluorinated substrate analogues 2-fluorogeranyl diphosphate (FGPP) and 2-fluoroneryl diphosphate (FNPP) at 2.4 and 2.2 Å resolution, respectively.23 In the crystal bound with FGPP, a single left-handed screw conformation was observed, which agrees with the predictions for substrate binding stereoselectivity.23 Those studies also showed their fluorinated substrate analogues as competitive inhibitors to their corresponding terpene synthases, with Ki similar to their KM, indicating the binding affinity was likely not affected by the fluorine substitution.19-23

This thesis will focus on the study of (+)-LS from naval orange, which catalyzes the committed step in producing the monoterpene (+)-limonene. The goal is to study the LS cyclization mechanism and prove LPP as an intermediate in the course of cyclization catalysis using two novel difluorinated substrate analogues: 8,9-difluorogeranyl diphosphate (DFGPP) and 8,9-difluorolinalyl diphosphate (DFLPP) (Figure 4). In order to trap LPP before the cyclization step, we designed a competitive inhibitor, DFGPP, which allows the allylic diphosphate to go through the ionization/syn-migration step to form DFLPP but then stops because the electron-withdrawing fluorine atoms block the subsequent ring closure step.

Characterization of the reaction requires that we have authentic DFLPP for comparison.

F O O 8 F 8 5 1 O O O P P O F 7 3 5 O P P O F 7 O O 9 6 4 2 3 O O 9 6 4 1 2 8,9-difluorogeranyl diphosphate (DFGPP) 8,9-difluorolinalyl diphosphate (DFLPP)

Figure 4. DFGPP and DFLPP: two difluorinated substrate analogues that we designed for the limonene synthase cyclization mechanism study to prove LPP’s existence. We anticipated that DFGPP will slow down the cyclization reaction and we will, therefore, be able to see DFLPP at the enzyme active site.

Both DFGPP and DFLPP were successfully synthesized and their structures have been confirmed with nuclear magnetic resonance spectroscopy (NMR). Both analogues functioned

- 7 - as substrates for (+)-LS and made the same final product, but interestingly the product information from gas chromatography/ mass spectroscopy (GC/MS) and the structure determination from NMR (details shown in results and discussion) showed that their product was not cyclic, but rather an acyclic monoterpene. Enzymatic studies with DFGPP were conducted, with its KM of 20.50 ± 1.05 µM, which is similar to GPP KM of 29.79 ± 7.83 µM.

-1 The kcat of DFGPP was 0.00144 ± 0.00002 s , 69-fold smaller than GPP kcat of 0.0905 ± 0.0070 s-1. DFLPP was only synthesized in small quantity and therefore a full characterization of this analog has not been possible. However, the preliminary measured turnover rate is roughly 13- fold less than that observed for the non-fluorinated counterpart, LPP, which was also investigated in this thesis. Apo-(+)-LS crystal trays have been set and a future direction for this project is to attempt to co-crystallize both analogs with the apoprotein, in order to understand the structural elements involved in this enzyme’s cyclization mechanism. The cyclization mechanism is universal to all monoterpene cyclases, with the isomerization to make proposed

LPP and and then gets cyclized to α-terpinyl cation. As a result, proof of LPP being an intermediate for (+)-LS can thus be applied to all monoterpene cyclases.

- 8 - Materials and Methods

Difluorinated Substrate Analogues Synthesis

Experimental details:

All chemicals were purchased from Sigma-Aldrich and Fischer Scientific and used as received unless otherwise stated. Tetrahydrofuran (THF), acetonitrile, diethyl ether and dichloromethane (DCM) were anhydrous unless otherwise stated. Reactions carried out at room temperature were open to air unless otherwise stated. Solvent was removed by rotary evaporation under reduced pressure using BÜCHI Rotavapor R-114 model. The purity and identity of each chemical were assessed by proton, fluorine, phosphorus, and carbon nuclear magnetic resonance (NMR) spectroscopy. NMR spectra were recorded on a Varian 400-MR spectrometer (9.4 T, 400 MHz) in D2O adjusted to pH ~8.0 with ND4OD for phosphorylation

1 13 products, or in CDCl3 for the rest of the chemicals. H and C chemical shifts are reported in parts per million (ppm) downfield from TMSP (trimethylsilyl propionic acid). 31P chemical shifts are reported in ppm relative to 85% ο-phosphoric acid. 19F chemical shifts are referenced to NaF dissolved in D2O (-125.3ppm). J coupling constants are reported in units of frequency

(Hertz) with multiplicities listed as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), m (multiplet), br (broad), and app (apparent).

- 9 - Synthesis of 8,9-Difluorogeranyl Diphosphate (DFGPP) and 8,9-Difluorolinalyl

Diphosphate (DFLPP)

5-Bromopentan-2-one

Br O

(1)

In a 250 mL oven-dried round-bottom flask, a solution of α-acetylbutyrolactone (6.5 mL, 60.0 mmol) in toluene (40mL) was stirred at 80 °C under N2 atmosphere as hydrobromic acid (48% in H2O, 10.2 mL, 90.0 mmol) was added drop-wise via syringe. Under N2, the reaction mixture was stirred and heated at 80 °C in a mineral oil bath for 14 hours. To the reaction mixture, water

(30 mL) and diethyl ether (30 mL) were added and the organic layer was separated from the aqueous layer through a 250 mL separation funnel. The aqueous layer was extracted with ether

(2 × 20 mL), and the organic layers were combined and washed with water (2 × 20 mL) and then brine (20 mL). The washed organic layer was dried over anhydrous MgSO4, filtered and then concentrated under reduced pressure. After concentration, the dark brown crude material was purified over flash chromatography on silica column with hexane and ethyl acetate (8:1) mobile phase. Fractions were analyzed by thin-layer chromatography (TLC) developed in 8:1 hexane/ethyl acetate and visualized with iodine staining. Fractions containing Br-Ketone were pooled and concentrated under reduced pressure, giving (1) as a pale yellow oil (8.87g, 92.8%);

1 Rf 0.43 (Hexane:ethyl acetate = 8:1); H NMR: (400 MHz, CDCl3) δH 2.12 (2 H, quintet, J

- 10 - 6.65, CH2CH2CH2), 2.17 (3 H, s, CH3), 2.65 (2 H, t, J 7.05, CH2C=O), 3.45 (2H, t, J 6.26,

13 BrCH2); C NMR: (100 MHz, CDCl3) δC 26.38 (CH2CH2CH2), 30.10 (CH3), 33.31 (BrCH2),

41.46 (CH2C=O), 207.37 (C=O).

(E)-Ethyl 6-bromo-3-methylhex-2-enoate

Br O

(2)

In a 250 mL oven-dried round-bottom flask, sodium hydride (0.48g, 19.2 mmol) was suspended in anhydrous THF (40 mL) while stirring at 0 °C under N2 atmosphere as triethyl phosphonoacetate (3.8mL, 19.2mmol) and a solution of (1) (2.64 g, 16.0 mmol) in anhydrous

THF (20 mL) was added drop-wise via syringe. The reaction mixture was stirred for 14 hours at room temperature under N2. To the reaction mixture, water (80 mL) and diethyl ether (80 mL) were added, and the organic layer was separated from the aqueous layer through a 500 mL separation funnel. The aqueous layer was extracted with ether (2 × 60 mL), and the organic layers were combined and washed with water (2 × 40 mL) and then brine (40 mL). The washed organic layer was dried over anhydrous MgSO4, filtered and then concentrated under reduced pressure. After concentration, the yellow crude material was purified over flash chromatography on silica column with hexane and ethyl acetate (40:1) mobile phase, and gave

- 11 - both E and Z isomers of (2). Fractions were analyzed by thin-layer chromatography (TLC) developed in 40:1 hexane/ethyl acetate and visualized with UV light and KMnO4. The Z isomer was eluted first and was followed by the E isomer. Fractions containing each isomer were pooled and concentrated under reduced pressure, giving Z isomer of (2) as a pale yellow oil

1 (1.04g, 27.3%); Rf 0.32 (Hexane:ethyl acetate = 40:1); H NMR: (400 MHz, CDCl3) δH 1.27

(3 H, t, J 7.0, CH2CH3), 1.91 (3 H, d, J 1.5, CH3C=CH), 2.05 (2H, m, BrCH2CH2), 2.74 (2 H, t, J 8.0, BrCH2CH2CH3), 3.44 (3 H, t, J 6.8 BrCH2), 4.15 (2 H, 1, J 7.0, CH2CH3), 5.71 (1 H, d,

13 J 1.5, C=CH); C NMR: (100 MHz, CDCl3) δC 14.28 (CH2CH3), 18.65 (CH3C=CH), 31.38

(BrCH2CH2), 33.26 (BrCH2), 32.23 (BrCH2CH2CH2), 59.59 (CH2CH3), 117.17 (C=CH),

158.25 (C=CH), 166.17 (C=O) and E isomer of the ester as a pale yellow oil (2.58g, 67.6%);

1 Rf 0.27 (Hexane:ethyl acetate = 40:1); H NMR: (400 MHz, CDCl3) δH 1.28 (3 H, t, J 7.2,

CH2CH3), 2.04 (2 H, m, BrCH2CH2), 2.17 (3 H, d, J 1.2, CH3C=CH), 2.30 (2 H, t, J 7.8,

BrCH2CH2), 3.40 (2 H, t, J 6.8, BrCH2), 4.16 (2 H, q, J 7.0 CH2CH3), 5.70 (1 H, m, C=CH);

13 C NMR: (100 MHz, CDCl3) δC 14.28 (CH2CH3), 18.65 (CH3C=CH), 30.21 (BrCH2CH2),

32.61 (BrCH2), 38.96 (BrCH2CH2CH2), 59.59 (CH2CH3), 116.61 (C=CH), 157.51 (C=CH),

166.54 (C=O).

- 12 - (E)-6-bromo-3-methylhex-2-en-1-ol

Br OH

(3)

In a 500 mL oven-dried round-bottom flask, a solution of E isomer of (2) (3 g, 12.8 mmol) in anhydrous TFH (120 mL) was stirred at -78 °C in a dry ice/ethanol bath and under N2 atmosphere as diisobutylaluminium hydride (1 M solution in hexane, 31.0 mL, 30.8 mmol) was added drop wise for over 15 minutes via syringe. The reaction mixture was stirred at -78°C under N2 for 2 hours and then allowed to warm to 0 °C. The reaction completion was judged by

TLC analysis (Hexane:ethyl acetate = 2:1). To the reaction mixture, saturated potassium sodium tartrate solution (100 mL) and diethyl ether (100 mL) were added and stirred at room temperature for 30 minutes. The organic layer was separated from the aqueous layer through a

1000 mL separation funnel. The aqueous layer was extracted with ether (3 × 60 mL), and the organic layers were combined and washed with brine (60 mL), dried over anhydrous MgSO4, filtered and then concentrated under reduced pressure. After concentration, the yellow crude material was purified over flash chromatography on silica column with hexane and ethyl acetate

(2:1) mobile phase. Fractions were analyzed by TLC developed in 2:1 hexane/ethyl acetate and visualized with KMnO4. Fractions containing (3) were pooled and concentrated under reduced pressure, giving (3) as a light yellow oil (2.08 g, 84.9%); Rf 0.31 (Hexane:ethyl acetate = 2:1);

1 H NMR: (400 MHz, CDCl3) δH 1.68 (3 H, s, CH3C=CH), 1.99 (2 H, quintet, J 7.0,

BrCH2CH2), 2.05 (1 H, b, OH), 2.18 (2 H, t, J 7.0, BrCH2CH2CH2), 3.40 (2 H, t, J 7.0, BrCH2),

- 13 - 13 4.16 (2 H, d, J 7.0, CH2OH), 5.45 (1 H, m, C=CH); C NMR: (100 MHz, CDCl3) δC 16.19

(CH3C=CH), 30.63 (BrCH2CH2), 33.19 (BrCH2), 37.71 (BrCH2CH2CH2), 59.30 (CH2OH),

124.61 (C=CH), 137.77 (C=CH).

(E)-2-((6-bromo-3-methylhex-2-en-1-yl)oxy)tetrahydro-2H-pyran

Br O O

(4)

In a 250 mL oven-dried round-bottom flask, a solution of (3) (2.08 g, 10.76 mmol) and 3,4- dihydro-2H-pyran (1.98 mL, 21.54 mmol) in CH2Cl2 (60.0 mL) was stirred at 0 °C and under

N2 atmosphere as p-toluenesulfonic acid (0.103g, 0.55 mmol) was added quickly. The reaction mixture was stirred for 16 hours whilst warming to room temperature, diluted with diethyl ether

(60.0 mL) and then washed with a saturated NaHCO3 solution (40 mL). The aqueous layer was separated from the organic layer and extracted with ether (2 × 40 mL), and the organic layers were combined and washed with brine (45 mL), dried over anhydrous MgSO4, filtered and then concentrated under reduced pressure. The brown crude material was purified over flash chromatography on silica column with hexane and ethyl acetate (9:1) mobile phase. Fractions were analyzed by TLC developed in 9:1 hexane/ethyl acetate and visualized with KMnO4.

Fractions containing (4) were pooled and concentrated under reduced pressure, giving (4) as a

- 14 - 1 clear oil. (2.18 g, 73.8%); Rf 0.35 (Hexane:ethyl acetate = 9:1); H NMR: (400 MHz, CDCl3)

δH 1.60-1.85 (6 H, m, CH(CH2)3), 1.68 (3 H, s, CH3C=CH), 1.98 (2 H, quintet, J 7.0,

BrCH2CH2), 2.18 (2 H, t, J 7.0, BrCH2CH2CH2), 3.38 (2 H, t, J 7.0, BrCH2), 3.52 and 3.89 (2

H, m, (CH2)2CH2O), 4.02 and 4.24 (2 H, m, C=CHCH2O), 4.62 (1 H, m, OCHO), 5.41 (1 H,

13 m (CH2)2CH2O); C NMR: (100 MHz, CDCl3) δC 16.36 (BrCH2), 19.64, 25.50, and 30.68

((CH2)2CH2O), 30.73 (BrCH2CH2), 33.28 (BrCH2), 37.82 (BrCH2CH2CH2), 62.36

((CH2)3CH2O), 63.60 (CHCH2O), 97.99 (OCHO), 121.98 (CHCH2O), 138.17 (CH3C=CH).

(E)-2-((8-fluoro-7-(fluoromethyl)-3-methylocta-2,6-dien-1-yl)oxy)tetrahydro-2H-pyran

F O O

(6)

In a 50 mL oven-dried round-bottom flask, a solution of (4) (1.10 g, 3.97 mmol) in anhydrous acetonitrile (20 mL) was stirred under reflux at 85 °C and under N2 atmosphere as triphenylphosphine (1.57 g, 5.99 mmol) was added to the reaction mixture quickly. The reaction was stirred under reflux at 85 °C and under N2 for 14 hours. After cooling, reaction solvent was evaporated under reduced pressure to give a clear oily mixture. To the oily mixture, about 15 mL of anhydrous diethyl ether was added and triturated with a glass rod to remove excess

- 15 - triphenylphosphine. A white sticky residue was formed while adding ether, and the ether layer was removed. The remaining sticky residue was dried under reduced pressure for 5 hours to give the intermediate phosphonium salt as a viscous oil. The viscous oil was immediately dissolved in anhydrous THF (40 mL) and stirred at -78 °C under N2 atmosphere as lithium hexamethyldisilazide (1 M solution in THF, 4.0 mL, 4.0 mmol) was added to the reaction mixture drop-wise via a syringe over 10 minutes. Upon addition of lithium hexamethyldisilazide, the reaction mixture turned into dark orange from colorless and the viscous oil completely dissolved in the solvent. The mixture was stirred at -78 °C under N2 for

30 minutes, and then 1, 3-difluoro acetone (0.3 mL, 3.64 mmol) was added drop wise through a 500 µL glass syringe. Upon addition of 1, 3-difluoro acetone, the reaction mixture color immediately changed from dark orange to pale yellow. The whole reaction mixture was allowed to warm to -20 °C under N2 over 1 hour, and then maintained at -20 °C for another 2 hours.

After completion, the reaction was quenched with ether (40 mL) and water (40 mL). Aqueous was separated from the organic layer via a 250 mL separation funnel and then extracted with ether (3 × 30 mL). The organic layers were combined and washed with brine (30 mL), dried over anhydrous MgSO4, filtered and then concentrated under reduced pressure. The brown crude material was purified over flash chromatography on silica column with hexane and ethyl acetate (20:1) mobile phase. Fractions were analyzed by TLC developed in 20:1 hexane/ethyl acetate and visualized with KMnO4. Fractions containing (6) were pooled and concentrated under reduced pressure, giving (6) as a clear oil. (0.63g, 63%); Rf 0.27 (Hexane:ethyl acetate =

1 20:1); H NMR: (400 MHz, CDCl3) δH 1.56-1.84 (6H, m, (CH2)3CH2O), 1.68 (3 H, s,

CH3C=CH), 2.13 (2 H, t, J 7.6, FCH2C=CHCH2CH2), 2.32 (2 H, m, FCH2C=CHCH2), 3.52 and

- 16 - 3.89 (2 H, m, (CH2)3CH2O), 4.02 and 4.24 (2 H, m, C=CHCH2O), 4.62 (1 H, m, OCHO), 4.81

(2 H, d, JHF = 47.5, CH2F), 4.93 (2 H, d, JHF = 47.5, CH2F), 5.06 (2 H, d, JHF = 47.5, CH2F),

13 5.38 (1 H, dt, J 1.2, J 7.6, C=CHCH2O), 5.85 (1 H, m, CH2FC=CH); C NMR: (100 MHz,

CDCl3) δC 16.35 (CH3), 19.63, 25.50, 30.68 ((CH2)3CH2O), 25.79 (FCH2C=CHCH2), 38.80 (t,

JCF = 2.5, CH2FCCHCH2CH2), 62.35 ((CH2)3CH2O), 63.56 (CHCH2O), 76.86 (d, JCF = 161,

CH2F), 84.65 (d, JCF = 165, CH2F), 97.98 (OCHO), 121.82 (CHCH2O), 131.03 (t, JCF = 14,

19 CCH2F), 137.17 (t, JCF = 9, CH2FCCH), 138.52 (CH2CCH3). F NMR: (376 MHz,

D2O/ND4OD) δF -207.7 (1 F, t, JFH = 47.5 Hz, CH2F), -213.2 (1F, t, JFH = 47.5 Hz, CH2F)

(further FF and long-distance HF splitting leads to apparent quintets within each triplet)

8,9-Difluorogeraniol

F OH

(7)

In a 50 mL oven dried round-bottom flask, a solution of (6) (0.48 g, 1.75 mmol) in 100% ethanol

(20 mL) was stirred at 55°C as pyridinium p-toluenesulphonate (0.04 g, 0.17 mmol) was added.

The reaction mixture was stirred at 55 °C for 3 hours, and then concentrated under reduced pressure to give a pale yellow oil. Water (30 mL) and diethyl ether (20 mL) were added and organic layer was separated from the aqueous layer, which was then extracted with diethyl ether

- 17 - (3 × 10 mL). The organic layers were combined and washed with brine (10 mL), dried over anhydrous MgSO4, filtered and then concentrated under reduced pressure. The pale yellow crude material was purified over flash chromatography on silica column with hexane and ethyl acetate (4:1) mobile phase. Fractions were analyzed by TLC developed in 4:1 hexane/ethyl acetate and visualized with KMnO4. Fractions containing (7) were pooled and concentrated under reduced pressure, giving (7) as a clear oil. (0.32g, 95%); Rf 0.33 (Hexane:ethyl acetate =

1 4:1); H NMR: (400 MHz, CDCl3) δH 1.69 (3 H, s, CH3), 2.05 (1 H, s, OH), 2.13 (2 H, t, J 7.6,

C=CHCH2CH2), 2.32 (2 H, m, C=CHCH2CH2), 4.16 (2 H, t, J 5.6, CHCH2OH), 4.81 (2 H, d,

JHF = 47.7, CH2F), 4.93 (2 H, d, JHF = 47.7, CH2F), 5.05 (2 H, d, JHF = 47.7, CH2F), 5.43 (1 H,

13 dt, J 1.2, J 7.2, CHCH2OH), 5.85 (1 H, m, C=CHCH2CH2); C NMR: (100 MHz, CDCl3) δC

16.20 (CH3), 25.75 (C=CHCH2CH2), 38.62 (C=CHCH2CH2), 59.27 (CH2OH), 77.21 (d, JCF =

161, CH2F), 84.58 (d, JCF = 165, CH2F), 124.51 (CHCH2OH), 131.08 (t, JCF = 15, CCH2F),

19 137.05 (t, JCF = 9, CH2FC=CH), 137.99 (CH2CCH3); F NMR: (376 MHz, D2O/ND4OD) δF -

207.7 (1 F, t, JFH = 47.5 Hz, CH2F), -213.2 (1F, t, JFH = 47.5 Hz, CH2F) (further FF and long- distance HF splitting leads to apparent quintets within each triplet);

- 18 - 8,9-Difluorogeranyl Mesylate

F OMs

(8)

The mesylation of 8,9-difluorogeranial was adapted from a literature procedure previously described by Croteau et al. In a 100 mL oven dried round-bottom flask, (7) (340 mg, 1.81 mmol) and Et3N (280 mg, 2.72 mmol) in anhydrous dichloromethane (15 mL) was stirred at -10 °C and under N2 atmosphere as a solution of MsCl (250 mg, 2.18 mmol) in anhydrous dichloromethane (DCM, 10 mL) was added drop-wise through syringe over 30 minutes. Then the reaction mixture was moved to a 0 °C bath and stirred for one hour. Reaction was quenched with ice water (5 mL). The organic layer was separated, and the aqueous layer was extracted with DCM (2 × 10 mL). The organic layers were combined, washed with saturated NaHCO3

(10 mL) and brine (10 mL) and then dried over anhydrous MgSO4. The mixture was filtered and then concentrated under reduced pressure, keeping the vacuum water bath at 20 °C, to give the crude mesylate (460 mg) (8), which was used in the next step immediately.

- 19 - 8,9-Difluorolinalool

F OH

(9)

The solvolysis of (8) was conducted according to a literature procedure described by Croteau et al. 8,9-difluorogeranyl mesylate (8) (460 mg, 1.70 mmol) and 2, 4, 6-collidine (210 mg, 1.70 mmol) in 4:1 acetone-water (20 mL) was stirred at 60 °C for 3 hours. The reaction was monitored with TLC developed in 9:1 hexane/ethyl acetate. Once the reaction was completed, ice water (5 mL) was added to stop the reaction and the mixture was extracted with diethyl ether

(3 × 20 mL). The organic layers were combined and washed with 10% HCl (10 mL) and brine

(10 mL), dried over anhydrous MgSO4, filtered and then concentrated under reduced pressure.

The yellow crude material was purified over flash chromatography on silica column with hexane and ethyl acetate (9:1) mobile phase. Fractions were analyzed by TLC developed in 9:1 hexane/ethyl acetate and visualized with KMnO4. Fractions containing (9) were pooled and concentrated under reduced pressure, giving (9) as a yellow oil. (50 mg, 17%); Rf 0.31

1 (Hexane:ethyl acetate = 9:1); H NMR: (400 MHz, CDCl3) δH 1.309 (1 H, s, CH3), 1.391 (1 H, s, OH), 1.54-1.70 (2 H, m, CH3CCH2CH2), 2.15-2.32 (2 H, m, CH2CH2CH), 4.87 (2 H, d, JHF

= 47.4, CH2F), 5.00 (2 H, d, JHF = 47.2, CH2F), 5.10 (1 H, dd, J 10.6, H at C1), 5.23 (1 H, dd,

J 17.2, H at C1), 5.85-5.95 (app quartet, J 11.0 and J 10.6, H at C6 and C2); 19F NMR: (376

- 20 - MHz, D2O/ND4OD) δF -212.3 (1 F, t, JFH = 47.4 Hz, CH2F), -217r.1 (1F, t, JFH = 47.2 Hz, CH2F)

(further FF and long-distance HF splitting leads to apparent quintets within each triplet);

8,9-Difluorogeranyl Diphosphate (DFGPP)

8,9-Difluorolinalyl Diphosphate (DFLPP)

O O F O P P O

O O

(10)

O O F O P P O

F O O

(11)

Phosphorylation of 8,9-difluorogeraniol and 8,9-difluorolinalool to synthesize DFGPP and

DFLPP, respectively, was based on a literature procedure described by Keller and Thompson et al., and Morehouse et al. Each allylic alcohol (300 mg) and trichloroacetonitrile (5 mL, 50 mmol) were stirred at 37 °C as triethylammonium phosphate (TEAP, 5 mL) was added every 5 minutes for a total of 15 mL. The reaction mixture was purified over flash chromatography on silica column with isopropanol, ammonium hydroxide and water (12:5:1) mobile phase.

- 21 - Fractions were analyzed by TLC developed in the same mobile phase and visualized with

KMnO4. Fractions containing diphosphate product were pooled, concentrated under reduced pressure, flash-frozen in liquid nitrogen, and lyophilized to give both (10) and (11) a white powder.

1 DFGPP (80 mg, 38%); H NMR: (400 MH, D2O/ND4OD), δH 1.72 (3 H, s, CH3), 2.18 (2 H, t,

J = 7.3 Hz, H at C4), 2.32-2.42 (2 H, m, H at C5), 4.47 (2 H, app t, J = ~6.9 Hz, H at C1), 4.96

(2 H, d, JHF = 47.5 Hz, CH2F), 5.12 (2 H, d, JHF = 47.5 Hz, CH2F), 5.47 (1 H, app t, J = ~6.6

13 1 Hz, H at C2), 6.01-6.10 (1 H, m, H at C6); C{ H} NMR: (100 MHz, D2O/ND4OD) δC 18.41,

28.23 (1C, app t, JCF = ~2.3 Hz), 40.89 (1C, app t, JCP = ~2.6 Hz), 65.29 (1 C, d, JCP = 4.6 Hz),

82.02 (1C, d, JCF = 154.9 Hz), 89.09 (1C, d, JCF = 157.2 Hz), 123.66 (1 C, d, JCP = 8.4 Hz),

19 132.93 (1C, app t, JCF = ~14 Hz), 143.05 (1C, dd, JCF = 8.4 Hz), 144.08; F NMR: (376 MHz,

D2O/ND4OD) δF -207.7 (1 F, t, JFH = 47.5 Hz, CH2F), -213.2 (1F, t, JFH = 47.5 Hz, CH2F)

(further FF and long-distance HF splitting leads to apparent quintets within each triplet);

31 1 P{ H} NMR: (162 MHz, D2O/ND4OD) δP -6.7 (1 P, d, JPP = 22.5 Hz, P1), -10.5 (1 P, d, JPP =

22.5 Hz, P2).

Lyophilized DFLPP was further purified by anion exchange chromatography using a 1 cm × 8 cm column of DOWEX-1X2-400 strongly basic anion exchange resin, chloride form.

The resin was first equilibrated with 25 mM ammonium bicarbonate (pH = 8). 15 mg of DFLPP dissolved in 1 mL of 25 mM ammonium bicarbonate was then loaded onto the column and 40 mL of 25 mM ammonium bicarbonate was used to wash the column. Finally, 500 mM ammonium bicarbonate was used to elute DFLPP. Fractions were analyzed with TLC and visualized with KMnO4. Fractions containing DFLPP were pooled, flash-frozen in liquid nitrogen, and lyophilized to give (11) a white powder. (30g, 38%). DFLPP- 1H NMR: (400

- 22 - MHz, D2O/ND4OD), δH 1.58 (3 H, s, CH3), 1.76-1.96 (2 H, m, H at C4), 2.22-2.36 (2 H, m, H at C5), 4.97 (2 H, d, JHF = 47.9 Hz, CH2F), 5.14 (2 H, d, JHF = 47.5 Hz, CH2F), 5.16 (1 H, dd, J

= 1.2 and J = 10.4 Hz, H at C1), 5.27 (1 H, dd, J = 1.2 and J = 16.8 Hz, H at C1), 6.10 (2 H, app

13 1 quartet, J = 11.0 and J = 10.6 Hz, H at C6 and C2); C{ H} NMR: (100 MHz, D2O/ND4OD)

δC 20.76, 25.34 (1C, app d, JCF = ~5.5 Hz), 26.93 (1C, app t, JCF = ~3.0 Hz), 82.13 (1C, d, JCF

= 153.9 Hz), 84.17, 89.21 (1C, d, JCF = 157.2 Hz), 116.32, 145.67 (1C, d, JCF = 4.6 Hz), 147.81,

19 165.11; F NMR: (376 MHz, D2O/ND4OD) δF -201.2 (1 F, t, JFH = 47.7 Hz, CH2F), -216.1 (1F, t, JFH = 46.8 Hz, CH2F) (further FF and long-distance HF splitting leads to apparent quintets

31 1 within each triplet); P{ H} NMR: (162 MHz, D2O/ND4OD) δP -6.7 (1 P, d, JPP = 22.1 Hz, P1),

-14.6 (1 P, d, JPP = 21.6 Hz, P2).

- 23 - Enzymatic Activity Study

Protein Purification

Frozen cell pellets were thawed on ice and resuspended in 45 mL breaking buffer (50 mM

Tris, 100 mM NaCl, 20 mM imidazole, pH = 7.5) supplemented with five EDTA-Free Pierce protease inhibitor cocktail (La Roche). The cell suspension was sonicated with five 20-second pulses at approximately 75 Watt separated by 30-second rest period and then centrifuged at 16,

000g at 8°C for 30 minutes. The supernatant was filtered via a 0.22 µm filter and then loaded onto a 5 mL HiTrapFF Ni-Sepharose column (GE Healthcare Life Science). The column was washed with 40 mL wash buffer (50 mM Tris, 100 mM NaCl, 40 mM imidazole, pH = 7.5) at a rate of 1.5 mL per minute, and eluted using an 80 mL liner 40-500 mM imidazole gradient

(also in 50 mM Tris, 100 mM Nacl, pH 7.5) at a rate of 1.5 mL per minute. Fractions containing

(+)-LS were identified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE) and pooled, concentrated with an exchange of buffer (50 mM Tris, 100mM NaCl, pH

= 7.5) to remove imidazole via 50 kDa molecular weight cutoff Amicon Ultra Centrifuge Filter

(EMD Millipore), and stored at -80°C.

Kinetic Study

The enzymatic activity assay was conducted using the discontinuous single-vial assay described by O’Maille et al. with modification of using hexane for product extraction rather

- 24 - than ethyl acetate. In each single screw-up vial, 1 mL total volume of reaction mixture contained assay buffer (50 mM Tris, 100 mM NaCl, 10% (v/v) glycerol, pH = 8.0), purified (+)-LS, substrate and MnCl2 (600 µM). Reaction was initiated by addition of enzyme, gently mixed, immediately overlaid with 1 mL of hexane and allowed to proceed for various times. The reaction mixture was then vortexed for 20 seconds in order to terminate the reaction and extract terpene products into the hexane layer. Samples (500 µL) were taken from the hexane layer and examined by gas chromatography and mass spectrometry (GC-MS). GC peak area of each sample was integrated and compared with commercial (+)-limonene standard curve.

Gas Chromatography and Mass Spectrometry (GC-MS)

Enzymatic activity reactions were monitored with GC-MS (Agilent Technologies 7890A

GC System coupled with a 5975C VL MSD with a triple-axis detector) using 500µL of hexane extracts. Pulsed-splitless injection was used to inject 5µL samples onto an HP-5ms (5%- phenyl)-methylpolysiloxnae capillary GC column (Agilent Technologies, 30 m × 250 µm ×

0.25 µm) at an inlet temperature of 220 °C and run at constant pressure using helium as the carrier gas. Samples were initially held at an oven temperature gradient of 13 °C/min, followed by a linear temperature gradient of 50 °C/min to a final temperature of 240 °C, which was then held for 1 min. Retention times coupled with mass spectra were verified by comparing to the commercially available terpene standards.

- 25 - Fluorinated Terpene Product Structural Determination 2mM DFGPP and DFLPP were incubated with 100 µM (+)-LS, 30 mM MgCl2 with constant slow stirring on a magnetic stir plate for two days with 1.5 mL of deuterated benzene

(C6D6) overlaid. Reactions were vortexed and the samples centrifuged in a fixed-angle rotor for

5 minutes at 3,000 rpm to hasten phase separation. NMR was conducted on ~500 µL of the benzene layer using a 400 MHz Varian 400MR NMR magnet. 1D 1H and 19F NMR along with

2D gradient COSY and theoretical NMR prediction software were used to assign the resonances and identify the product of both reactions.

Crystallization

Crystallization trials were set by using the procedure described by Morehouse et al. 15 mg/mL (+)-LS in buffer (50 mM Tris, 100 mM NaCl, pH = 7.5) was mixed with mother liquor at 1:1 (v/v) ratio. Initial trials were performed using the sitting drop vapor diffusion method with Jena Bioscience sparse matrix crystallization screens (Jena Bioscience, Jena, Germany).

Drops were set with the aid of a Phoenix crystallization robot (Art Robbins Instruments). The hanging drop method was used for further optimizations. Crystals were grown in the prescnece of 12-16% PEG-8000, 100mM Tris (pH = 7.5-9.0), and 200-350 mM sodium tartrate at 20°C.

- 26 - Results and Discussion

Difluorinated Substrate Analogues Synthesis

Scheme 1. DFGPP and DFLPP Synthesis: Synthesis procedure adapted from Yu et al.24, for 8,9-difluorogeraniol synthesis; from Croteau et al.21, for 8,9-difluorolinalool synthesis; and from Morehouse et al.17, for DFGPP and DFLPP phosphorylation.

We designed two fluorinated substrate analogues to explore the (+)-LS cyclization

mechanism, especially focusing on the existence of the proposed universal intermediate LPP.

The whole synthesis for both DFGPP and DFLPP has been summarized in Scheme 1. The

DGPP synthesis includes six steps in total, including a Wittig olefination reaction to build in

the necessary stereochemistry around the C2-C3 bound (trans bromoester), and protection and

deprotection of the alcohol group. The fluorine substitutions on the terminal methyl groups is

achieved by using a Horner-Wadsworth-Emmons reaction with 1,3-difluoroacetone. The

- 27 - majority of the synthesis of DFLPP is similar to DFGPP, except the isomerization of the alcohol group on 8,9 difluorogeraniol to make 8,9-difluorolinalool through mesylation and solvolysis.

8,9-Difluorogeraniol

8,9-Difluorogeraniol, which is a precursor for both DFGPP and DFLPP, was synthesized according to the procedure of Yu et al.24 with slight modifications. The bromoketone (1), was synthesized from acetylbutyrolactone by HBr. The ketone then underwent a modified Wittig reaction (Horner-Wadsworth-Emmons reaction) with triethyl phosphonoacetate to give both cis and trans isomers of the bromoester (2). The trans bromoester was separated from the cis isomer by silica column chromatography, reduced by the ester reducing agent, DIBAL-H, resulting in the bromoalcohol (3). The alcohol group of (3) was protected with a tetrahydropyranyl (THP) protecting group, and then converted to a phosphonium bromide salt

(5) using triphenylphosphine. A second Wittig reaction was conducted with (5) and 1,3- difluoroacetone, putting one fluorine atom on each of the terminal methyl groups. The THP protecting group was then removed to give 8,9-difluorogeraniol (7), which was then either directly phosphorylated into DFGPP, or isomerized to 8,9-difluorolinalool for DFLPP synthesis.

- 28 - 8,9-Difluorolinalool

8,9-Difluorolinalool (9) was synthesized according to the procedure of Croteau et al.21 with slight modifications. 8,9-difluorogeraniol (7) was mesylated using mesyl chloride, giving

8,9-difluorogeranyl mesylate (8). The crude mesylate salt immediately underwent a solvolysis reaction by H2O, giving the novel chemical product 8,9-difluorolinalool (9). The solvolysis reaction yield of (9) was low (only 17%) because the reaction also produced two by-products, the starting material 8,9-difluorogeraniol and its geometric isomer 8,9-difluoronerol. This was because the nucleophile H2O had equal access when attacking C1 and C2 during solvolysis.

The purity and identity of 8,9-difluorogeraniol and 8,9-difluorolinalool were assessed by proton and fluorine NMR.

8,9-Difluorogeranyl Diphosphate and 8,9-Difluorolinalyl Diphosphate (DFGPP &

DFLPP)

Phosphorylation of 8,9-difluorogeraniol and 8,9-difluorolinalool was carried out using the phosphorylation procedure described by Morehouse et al.17 Each allylic alcohol was phosphorylated by reaction with trichloroacetonitrile and triethylammonium phosphate (TEAP) in a total 15-minute incubation. After silica column separation of the monophosphorylated, diphosphorylated, and triphosphorylated compounds, the diphosphate was lyophilized to dryness for 24 hours, and in separate reactions gave both DFGPP (10) (80 mg, 38%), and

DFLPP (11) a white powder. Lyophilized DFLPP was further purified by anion exchange chromatography using a procedure described previously by Morehouse et al. (30 mg, 30%).

- 29 - The purity and identity of DFGPP and DFLPP were assessed by proton, fluorine, phosphorus (Figure 5-8.), and carbon NMR.

DFGPP and DFLPP NMR Spectra and Interpretation

Purity and structural accuracy of the desired substrate analogues, DFGPP and DFLPP, were crucial and had to be confirmed before further enzymatic activity studies. As a result, we include the 1H, 19F, and 31P NMR spectra of DFGPP and DFLPP to show successful synthesis of both substrate analogues. We have also collected 13C NMR, and the spectra are included in the Appendix. Important features to note: 1) The successful incorporation of the fluorine atoms at positions C8 and C9 is confirmed by observation of the resonance for only a single methyl group in both compounds which implies the loss of the two other methyl group signals present in the non-fluorinated compounds. 2) Large J coupling is observed for peaks consistent with strong 2-bond coupling between the fluorine atoms and the attached protons at C8 and C9. 3)

This same large J coupling is also observed in the corresponding 19F NMR spectrum where the fluorine signals are mainly split into triplets by the neighboring protons with evidence of 4 and potentially 5-bond coupling causing additional finer splitting into apparent quintets. 4)

Diphosphorylation is evident from the presence of two separately observed doublets in the proton decoupled 31P spectrum resulting from phosphorus-phosphorus coupling. For DFGPP the proton coupled spectrum shows that one phosphorus atom is split further due to its ester linkage to the neighboring C1 which has two protons at 3 bonds distance. The coupled spectrum for DFLPP does not show this additional splitting.

- 30 - 1H – NMR for DFGPP and DFLPP

Figure 5. 1H-NMR of DFGPP: All protons on DFGPP are assigned to the corresponding peaks. Notice peak (e) and peak (f) are two doublets with JHF = 48, which is a result of 2-bond coupling to the fluorine atoms. Additionally, those two doublets are not overlapping with each other. (*Presaturation to remove the observed resonance for H2O in D2O at 4.78 ppm).

Figure 6. 1H-NMR of DFLPP: All protons on DFLPP are assigned to the corresponding peaks. Notice peak (d) and peak (e) are two doublets with JHF = 48, which is a result of the fluorine atoms. Additionally, those two doublets are not overlapping with each other. (*Presaturation to remove the observed

resonance for H2O in D2O at 4.78 ppm).

- 31 - 19F – NMR for DFGPP

19 Figure 7. F-NMR of DFGPP: Peaks show as two triplets as for two fluorine atoms each with JHF = 48. (*Splitting patterns and J values are similar with DFLPP. The exact DFLPP 19F spectrum can be found in Appendix).

31P – NMR for DFGPP

Figure 8. 31P-NMR of DFGPP (decoupled): Two doublets show the two phosphorus atoms on the diphosphate. (*Splitting patterns and J values are similar with DFLPP. The exact DFLPP 31P spectrum and the H-coupled spectrum for both analogues can be found in Appendix).

- 32 - Enzymatic Activity Study

(+)-LS Reaction with DFGPP and DFLPP We were firstly interested in seeing whether DFGPP and DFLPP were substrates for (+)-

LS, and we wanted to compare the product being made from both analogues. Finally, we tried to accumulate the reaction product in order to determine its chemical structure using NMR.

The reaction of (+)-LS with either DFGPP and DFLPP was incubated overnight using the single vial assay method described by O’Maille et al.25 (Figure 10) with slight modification following the procedure of Morehouse et al. Reaction was quenched the second day by vigorous mixing and the reaction product was extracted into the hexane layer, which was run directly on

GC/MS. According to the GC/MS, both DFGPP and DFLPP are substrates for (+)-LS.

Interestingly, both reaction products appeared to have the same retention time (7.6 minutes) over GC (Figure 10.), the same molecular weight, and fragmentation pattern over MS. The molecular weight of the both products was predicted to be 172 g/mol, and this was consistent with 8,9-difluorolimonene, but also consistent with all the other possible difluorinated and non- oxygenated monoterpenes.

Figure 9. Single Vial Method: Enzymatic reaction was performed in the aqueous layer, and overlaid with organic layer for product extraction. The extract was then measured on GC/ MS for analysis. Adapted from O’Maille, et al.25 (2004)

- 33 -

Figure 10. DFGPP Product GC, DFLPP Product GC and Standard Limonene GC: Both DFGPP and DFLPP were catalyzed by (+)-LS to produce a product that had the same retention time, which was also different from that of limonene.

In addition, LPP was synthesized from commercially available linalool according to the previously described phosphorylation procedure, and was tested on (+)-LS for the first time.

Previous studies have shown LPP being a substrate for (-)-LS and is converted to (-)- limonene.26 We incubated (+)-LS with synthesized LPP using the same single vial assay method described previously for an overnight reaction, and then quenched with According to the

GC/MS, LPP is substrate for (+)-LS and has the same retention time (Figure 11) and fragmentation pattern as commercially available (+)-limonene. The reaction of (+)-LS with LPP

Figure 11. LPP Product GC and Standard Limonene GC: LPP was catalyzed by (+)-LS to produce a product that had the same retention time with (+)-limonene, and also small amount of myrcene, linalool and α-terpineol.

- 34 - also produced small amount of myrcene, linalool and α-terpineol. We also ran a control experiment for DFGPP, DFLPP and LPP overnight incubation with (+)-LS, but without divalent metal ion. The hexane extract contained no product on the GC/MS.

Finally, we wanted to determine the chemical structure of the DFGPP and DFLPP product through NMR (Figure 12(a) and (b)). Overnight product accumulation reactions of DFGPP and

DFLPP with (+)-LS were performed and C6D6 was used for product extraction and as NMR solvent.

1 Figure 12 (a). H-NMR of the DFGPP and DFLPP Product in C6D6: The circle indicates a terminal double bound feature with the geminal proton and vinyl proton splitting pattern on the left, and the arrow indicates a methyl group structure in the product. 19 Figure 12 (b). F-NMR of the DFGPP and DFLPP Product in C6D6: The same splitting patterns between two fluorine atoms indicates the product to be acyclic.

From those experiment results, we can conclude that both DFGPP and DFLPP were substrates to (+)-LS. According to the DFGPP and DFLPP products’ retention time over GC,

- 35 - and the same molecular weight and fragmentation pattern from MS, (+)-LS makes the same product from DFGPP and DFLPP, and this was proved by the fact that the NMR of both products showed up with the same peaks.

Based on our analysis of the 1H and 19F NMR spectra for the DFGPP and DFLPP product produced by (+)-LS, we can say the product must be an acyclic monoterpene.

Tentatively, we believe this product to be 8,9-difluoro-β-ocimene (Figure 13), for the following reasons: Based on the 1H NMR, there was a terminal double bound feature with the geminal proton and vinyl proton splitting pattern around 5.0 ppm (circled in blue). The peak at 2.0 ppm appeared to be a singlet, and this indicates a methyl group in the product structure. In addition, the fluorine splitting showed that the proton on the terminal di-methyl group was not deprotonated, since both fluorine atoms appeared as triplets. This can only be true when they are both bonded to terminal di-methyl groups, which means that the product is not limonene which is deprotonated on one of the terminal methyl groups (Figure 14). Based on the information above, we hypothesized that the DFLPP product and DFGPP product might be 8,9- difluoro-β-ocimene.

Figure 13. 8,9-difluoro-β-ocimene: Hypothesized DFGPP and DFLPP product catalyzed by (+)-LS. (a) (b)

Figure 14 (a). Possible Product Fluorines Structure on C7: Since the NMR of the product fluorines showed up the same splitting pattern as the starting material DFGPP and DFLPP, we conclude that the proton on the terminal di-methyl group was not deprotonated. Figure 14 (b). Deprotonated Terminal Di-methyl Group: The terminal structure of fluorines if one proton on either terminal methyl groups was deprotonated. In this case, we would expect the 8,9-difluorolimonene (expected product) to have this structure.

- 36 - Due to low concentration of the product and the presence of impurities possibility derived from enzymatic reaction components. In addition, the product was extracted into C6D6, which is like other aromatic NMR solvents that can cause significant shifting of the resonances and further NMR analysis is require to confirm the identity of the product.

(+)-LS Kinetic Studies

After proving both DFGPP and DFLPP were substrates for (+)-LS, we also wanted to perform a kinetic study on the enzyme using both substrate analogues, and compare their kinetic activity profiles with the non-fluorinated substrates (GPP and LPP, respectively) to examine the fluorine atom’s effect on the enzyme’s catalytic activity.

According to Morehouse et al.17, (+)-LS from navel orange behaves according to

Michaelis-Menten enzyme kinetics when using GPP as the substrate. Here we are reporting the

(+)-LS kinetics data with DFGPP as the substrate by running the reaction under steady-state conditions and measuring the initial rates. We measured the product forming velocity while varying DFGPP concentrations over a 2 minutes, 4 minutes and 6 minutes’ reaction. A

Michaelis-Menten equation with R2 = 0.997 was fitted to the data:

!'() × [,] !(#) = ./ + [,]

- 37 - The Michaelis-Menten for (+)-LS with DFGPP was plotted (Figure 15), with the KM of

-1 20.50 ± 1.05 µM, and kcat of 0.00144 ± 0.00002 s . We have also conducted a similar study of

-1 GPP with (+)-LS (Figure 16), with the KM of 29.79 ± 7.83 µM, and kcat of 0.0905 ± 0.007 s in

order to compare the fluorines’ effect on the enzyme reaction rate. The KM of these two

substrates are very similar, indicating that putting the fluorines on the terminal di-methyl groups

might not likely affect the binding affinity. Comparing the kcat of these two studies, we conclude

that putting fluorine atoms on the terminal di-methyl groups slowed down the reaction by ~70

fold.

Figure 15. Michaelis-Menten Plot for Reaction of Figure 16. Michaelis-Menten Plot for Reaction of DFGPP and (+)-LS: the figure shows enzymatic GPP and (+)-LS: the figure shows enzymatic reaction velocity vs. substrate concentration. Each reaction velocity vs. substrate concentration. Each reaction contained 1 µM of (+)-LS, the indicated reaction contained 20 nM of (+)-LS, the indicated (10-300 µM) DFGPP concentrate and 600 µM (10-200 µM) GPP concentrate and 600 µM MnCl2. MnCl2. Data showed a KM of 20.50 ± 1.05 µM and a Data showed a KM of 29.79 ± 7.83 µM and a kcat of -1 -1 kcat of 0.00144 ± 0.00002 s 0.0905 ± 0.0070 s

Similar comparison reactions between DFLPP and LPP were also conducted (Figures 17

and 18). However, the data from the DFLPP reaction fit poorly to the Michaelis-Menten

-1 equation, with KM of 170.11 ± 60.72 µM, and kcat of 0.0592 ± 0.0142 s . The reaction of LPP

- 38 - and (+)-LS was also tested for the first time, but also did not fit very well to the Michaelis-

-1 Menten equation, with the KM of 50.75 ± 15.80 µM, and kcat of 0.758 ± 0.088 s . However, if only comparing the kcat, it showed that putting the fluorines slowed down the reaction by almost

13 fold. Based on the DFLPP and LPP Michaelis-Menten plot, it seems that 200 µM for both substrates was not saturating for the enzyme. However, the DFLPP yield from the chemical synthesis was so low that we were not able to get enough amount of the analogue for a full characterization at this time.

Figure 17. Michaelis-Menten Plot for Reaction of Figure 18. Michaelis-Menten Plot for Reaction of DFLPP and (+)-LS: the figure shows enzymatic LPP and (+)-LS: the figure shows enzymatic reaction velocity vs. substrate concentration. Each reaction velocity vs. substrate concentration. Each reaction contained 0.4 µM of (+)-LS, the indicated reaction contained 0.04 µM of (+)-LS, the indicated (10-200 µM) DFGPP concentrate and 600 µM (5-200 µM) DFGPP concentrate and 600 µM MnCl2. MnCl2. Data showed a KM of 170.11 ± 60.72 µM and Data showed a KM of 50.75 ± 15.80 µM and a kcat of -1 -1 a kcat of 0.0592 ± 0.0142 s . 0.758 ± 0.088 s .

Even though we were only able to roughly estimate how many folds the fluorines were able to slow down (+)-LS catalysis, we had to keep in mind that fluorines might have caused the reaction mechanism to go through another pathway because the DFGPP and DFLPP product

- 39 - showed as a non-expected acyclic product. Change of the reaction mechanism possibly caused another step to be partially rate-limiting to the original rate limiting step, which was the first ionization of the allylic diphosphate from GPP.

Another important point about making a non-expected acyclic product by using the fluorinated analogues is that, both analogues were able to prevent the ring closure and they were able to make the same product. Since the fluorines are only expected to affect the cyclization step, the result can possibly indicate that the DFGPP isomerization to DFLPP might still be possible, but the cyclization step was slowed down by fluorines so much that the reaction went through some other way after making DFLPP from DFGPP. In our hypothesis that the reaction product was 8,9-difluoro-β-ocimene, we believe the reaction went through a deprotonation on the C4 proton of 8,9-difluorolinalyl cation (Figure 19).

B H 3 2 4 5 1 6 7 8 9

F F F F 8,9-Difluorolinalyl Cation 8,9-difluoro-β-ocimene

Figure 19. 8,9-difluoro-β-ocimene Formation: hypothetically the fluorine atoms retard the cyclization step, and a deprotonation on 8,9-difluorolinalyl cation C4 happens instead and results in the 8,9-difluoro-β- ocimene.

If this is true, we might still be able to see DFLPP isomerized from DFGPP at the enzyme active site, and we can study the active site structural elements and compare to the enzyme bound with DFLPP. One interesting fact is, we independently tested the DFGPP as a substrate with another monoterpene synthase, 1,8-cineole synthase, and it also made the same

- 40 - difluorinated product just like (+)-LS with DFGPP. This indicates that the substrate analogue

DFGPP is universal, and it might affect the monoterpene synthase cyclization reaction in the same way.

Future Directions

Future studies of (+)-LS cyclization mechanism need to be continued in three directions:

1) Determine DFGPP/DFLPP product structure using NMR; 2) Repeat (+)-LS kinetics activity assay with LPP and DFLPP; 3) Co-crystallize both substrate analogues with apo-(+)-LS.

For the first direction, we hope to determine the correct chemical structure for

DFGPP/DFLPP product, and then we will be able to find a corresponding reaction pathway for the difluorinated product with either DFGPP or DFLPP as a substrate for (+)-LS. Future studies will involve extraction of the fluorinated product into other non-aromatic NMR solvents, such as CDCl3, since aromatic NMR solvent can cause resonance shifting. In addition, we can also utilize the two-dimensional NMR, the homonuclear correction spectrospcopy (COSY), which is able to identify the spins that are coupled to each other and tells the atoms that are connected to one another. For the second direction, we can repeat the kinetic study for LPP with (+)-LS, in order to achieve a better fit for the Michaelis-Menten equation. However, for DFLPP, which was only synthesized in a small quantity, will be impossible for a full characterization. This should not be a big problem since the reaction product was proved not to be 8,9- difluorolimonene, and we believe that the cyclization step was not observed with either DFGPP or DFLPP. A change in the reaction mechanism can lead to other steps being partially rate

- 41 - limiting and thus the kinetics study might not be as useful as our third future direction, which is to co-crystallize the analogues with apo-(+)-LS and to understand the structural elements involved in this enzyme’s cyclization mechanism.

- 42 - Appendix: Supplementary Figures

1. DFGPP Supplementary NMR

A1: DFGPP 19F-NMR: zoomed in to show pseudo-quintet splitting through long distance interactions

A2: DFGPP 31P-NMR (coupled): coupled spectrum, the doublet at -10 ppm is split into a doublet of triplets by the methylene protons at C1

A3: DFGPP 13C-NMR

- 43 -

2. DFLPP Supplementary NMR

19 A4: DFLPP F-NMR: Peaks show as two triplets as for two fluorine atoms each with JHF = 48

A5: DFLPP 31P{1H}-NMR: DFLPP decoupled phosphorous NMR.

A6: DFLPP 13C{1H}-NMR: (looks like low signal to noise, might be need to try on higher NMR with higher magnetic field.)

- 44 -

3. LPP Supplementary NMR

A7: LPP 1H-proton NMR: referenced to external TMSP and with presaturation to remove water peak at 4.78ppm

A8: DFGPP/DFLPP Product GC Overlapping with Commercially Available Ocimene GC: not an evidence for the product being difluoro-ocimene, but they do have similar retention time.

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