MIAMI UNIVERSITY-THE GRADUATE SCHOOL

CERTIFICATE FOR APPROVING THE DISSERTATION

We hereby approve the Dissertation

of

Lizhi Zhu

Candidate for the Degree:

Doctor of Philosophy

______Robert E. Minto, Director

______John R. Grunwell, Reader

______John F. Sebastian, Reader

______Ann E. Hagerman, Reader

______Richard E. Lee, Graduate School Representative

ABSTRACT

INVESTIGATING THE BIOSYNTHESIS OF POLYACETYLENES: SYNTHESIS OF DEUTERATED LINOLEIC ACIDS & MECHANISM STUDIES OF DMDS ADDITION TO 1,4-ENYNES

By Lizhi Zhu

A wide range of polyacetylenic natural products possess antimicrobial, antitumor, and insecticidal properties. The biosyntheses of these natural products are widely distributed among fungi, algae, marine sponges, and higher plants. As details of the biosyntheses of these intriguing compounds remains scarce, it remains important to develop molecular probes and analytical methods to study polyacetylene secondary metabolism.

An effective pathway to prepare selectively deuterium-labeled linoleic acids was developed. By this Pd-catalyzed method, deuterium can be easily introduced into the vinyl position providing deuterolinoleates with very high isotopic purity. This method also provides a general route for the construction of 1,4-diene derivatives with different chain lengths and 1,4-diene locations.

Linoleic acid derivatives (12-d, 13-d and 16,16,17,17,18,18,18-d7) were synthesized according to this method.

A stereoselective synthesis of methyl (14Z)- and (14E)-dehydrocrepenynate was achieved in five to six steps that employed Pd-catalyzed cross-coupling reactions to construct the double bonds between C14 and C15. Compared with earlier methods, the improved syntheses are more convenient (no spinning band distillations or GLC separation of diastereomers were necessary) and higher Z/E ratios were obtained. The overall percent yield for (14E)-isomer was 21% and 29% for the (14Z)-isomer.

The reaction between DMDS and 1,4-enynes in the presence of I2 was studied. 2,5-Disubstituted thiophene derivatives were produced as the main products under neutral and acidic conditions. The detailed mechanism of this reaction was studied. Current evidence is consistent with a mechanism that can be described as follows. Initially, electrophilic addition of a sulfenium ion to an alkene yields an episulfonium ion. The subsequent Wagner-Meerwein rearrangement leads to a cationic thietane intermediate through a ring expansion. This four-membered ring is opened by nucleophilic attack of iodide to give a MeSI adduct. Available protons activate the triple bond and promote the subsequent transformations to generate the final thiophene product and release MeI as a side product. The synthetic utility of this method was explored. The optimized reaction provides a mild synthetic route to 2,5-disubstituted thiophene derivatives.

INVESTIGATING THE BIOSYNTHESIS OF POLYACETYLENES:

SYNTHESIS OF DEUTERATEDLINOLEIC ACIDS & MECHANISM STUDIES

OF DMDS ADDITION TO 1,4-ENYNES

A DISSERTATION

Submitted to the Faculty of

Miami University in partial

fulfillment of the requirements

for the degree of

Doctor of Philosophy

Department of Chemistry & Biochemistry

by

Lizhi Zhu

Miami University

Oxford, Ohio

2003

Dissertation Director: Robert E. Minto

TABLE OF CONTENTS

CHAPTER PAGE

List of figures iv List of tables vi List of abbreviations & acronyms vii Acknowledgement viii

I Synthesis of regioselectively deuterated linoleic acids 1 1-1 Endiyne antibiotics 1 1-2 Naturally occurring polyacetylenes 5 1-3 Biosynthesis of polyacetylenic compounds 8 1-4 Synthesis of regioselectively labeled linoleic acids 13 1-5 Results and discussion 14 1-6 Conclusion 21 1-7 Experimental section 21 References 38

II Improved synthesis of methyl (14E)- and (14Z)-dehydrocrepenynate 47 2-1 Introduction 47 2-2 Results and discussion 49 2-3 Conclusion 51 2-4 Experimental section 51 References 59

III Mechanism of dimethyl disulfide addition to 1,4-enynes 61 3-1 Introduction 61 3-2 Results and discussion 63 3-2.1 Optimization of the reaction conditions 64 3-2.2 Effects of additives 67

ii

3-2.3 Proposed mechanisms for thiophene formation 70 3-3 Conclusion 73 3-4 Experimental section 74 3-4.1 General information 74 3-4.2 Materials 75 3-4.3 General procedure for making thiophene derivatives 75 3-4.4 Synthetic material 75 3-4.5 Experimental data 78 References 91

iii

LIST OF FIGURES

FIGURE PAGE

1 Members of enediyne antibiotics family 2 2 Cycloaromatization of enediynes and its utility in natural product synthesis 4 I 3 Mechanism of DNA cleavage by calicheamicin γ 1 6 4 Examples of linear biological active linear polyacetylenic natural products 7 5 Desaturase-like reaction catalyzed by an acetylenase 8 6 Expression of ELI12 in parsley induced by a fungal challenge 10 7 Proposed biosynthetic pathway of polyacetylenic compounds 11 8 Regioselectively labeled linoleic acids 13 9 Retrosynthesis analysis of vinyl deuterated linoleic acids 13 10 Synthesis of C1-C10 fragment 14 11 Synthesis of C11-C18 fragment 13a, 13b 15 12 Synthesis of C11-C18 fragment 13c 16 13 Proposed mechanism for Cu-catalyzed coupling reaction 18 14 Synthesis of regioselectively labeled linoleic acids 19 15 Crepenynate (30) and dehydrocrepenynate (31a, 31b) derivatives 47 16 Synthesis of methyl (14Z)-dehydrocrepenynate 31a 49 17 Synthesis of methyl (14E)-dehydrocrepenynate 31b 50 18 Dimethyl disulfide (DMDS) derivatization and its utilities 62 19 DMDS addition to 1,4-enyne derivatives 64 20 Effect of temperature on thiophene formation 65 21 Reaction of methyl 9-tridecynoate (49) with DMDS 69 22 Mechanism of DMDS addition to 1,4-eneyne compounds 70 23 GC-MS analysis of the reaction of cis-enynethiol ether 53 with HI at 60 °C 71 24 GC-MS analysis of the reaction of cis-enynethiol ether 54 with HI at 60 °C 72 25 Alternative mechanism through alkyne-allene rearrangement 73 26 Synthesis of methyl 9-tridecynoate 49 76 27 Synthesis of methyl (6Z)-tridecen-9-ynoate 45 76

iv

28 Synthesis of methyl cis-enyne thioether 53 77 29 Synthesis of methyl trans-enyne thioether 54 77 30 Synthesis of 1,4-enyne 46 77

v

LIST OF TABLES

TABLE PAGE

1 Results of copper catalyzed cross-coupling reaction 17 2 Optimization of the cross-coupling reaction for assembly of 1,4-diene unit 20 3 Solvent and concentration effects to the formation of thiophene derivatives 66 4 Effects of additives to DMDS derivatization of 45 67

vi

LIST OF ABBREVIATIONS & ACRONYMS

AcOH Acetic acid AcSH Thiolacetic acid DIAD Diisopropyl azodicarboxylate DIBAL-H Diisobutylaluminum hydride DMDS Dimethyl disulfide DMF N,N-Dimethylformamide DMA N,N-Dimethylacetamide DNA Deoxyribonucleic acid DPPF 1,1’-Bis(diphenylphosphino)ferrocene HMPA Hexamethylphosphoramide (hexamethylphosphoric triamide) KIE Kinetic isotope effect LAH Lithium aluminum hydride LDA Lithium diisopropylamide NAD(P)H Nicotinamide adenine dinucleotide phosphate, reduced NCS N-Chlorosuccinimide PC Phosphatidylcholine PE Phosphatidylethanolamine RBF Round bottom flask TBDMSCl tert-Butyldimethylsilyl chloride THF Tetrahydrofuran THP Tetrahydropyran (or tetrahydropyranyl) TLC Thin layer chromatography TMSCl Trimethylsilyl chloride

vii

ACKNOWLEDGEMENT

I want to thank Dr. Robert E. Minto for his kindness, guidance, and giving me the opportunity to consult on all of the problems. I appreciate him for providing me the chance to become a member of his research group. From him, I learned not only the knowledge of chemistry, also his attitude and enthusiasm toward the academic work. I would like to thank all of my committee members for their generosity and giving me advice. I thank the faculty in the department of chemistry and biochemistry at Miami University for the instruction. I appreciate the Department of Chemistry and Biochemistry of Miami University for providing me the opportunity to utilize the scientific instruments and facilities. I also would like to thank my colleagues in chemistry department for their kind help and generous suggestion. Finally, I wish to thank my parents and all of my family members for their strong supporting and being behind me all the time.

viii

Chapter I. Synthesis of Regioselectively Deuterated Linoleic Acids

Novel biologically active substances from nature often provide stimulation, challenges and opportunities for the scientific community. Naturally occurring acetylenes, as an example, have attracted wide interest during the past two decades. Even though acetylenic compounds have been studied by many research teams for more than a century, the most exciting results were obtained after 1980, through the discovery of a new antibiotic family, the so called enediyne antibiotics.1-6 Their phenomenal biological activities against selected cancer cells (calicheamicin I γ1 (4) is 1000 times more potent than adriamycin, a clinically useful antitumor antibiotic in murine tumor models2b) and their unusual molecular architecture elicited extensive research activities in chemical, biological and biomedical circles.7 As potential candidates for anticancer drugs, this family of compounds has been studied thoroughly. Several strategies exploiting modern synthetic technologies have led to the total synthesis of these compounds.8-15 Their fascinating modes of action have inspired the design of a number of novel analogs that mimic their chemical and biological functions. Continued studies will hopefully lead to the development of a second generation of designed enediyne antibiotics with improved potential over the natural products for treatment of cancer. On the other hand, the biosynthesis of these novel enediynes is a puzzle that still needs to be addressed.16

1-1. Enediyne antibiotics

The first member of the enediyne antibiotics reported was neocarzinostatin (1), which was disclosed in 1985.6 However, the full impact of this investigation did not occur until the isolations of the esperamicins (2) and calicheamicins (4) in 1987, events which aroused substantial interest by the chemical and biological communities. At the beginning, when neocarzinostatin’s structure was reported, the lack of X-ray crystallographic analysis left a few unresolved structural issues and nagging concerns over the unusual structure. However, after the fourth member of this family, dynemicin A (3), was reported, each of the unique structures became widely accepted.5 As a result of the structural questions, the enediynes were synthesized by different research groups all over the world.

1

O MeO HN OMe

O O OMe O HO O O Me O O NHCO2Me O OMe O

O HO H O

OH NHMe MeSSS O O O O HO MeS N HO O H O OH OH O i PrHN MeO

Neocarzinostatin Chromophore (1) Esperamicin A1 (2)

Me COOH NHCO Me OH O HN O 2 O OMe HO H R O OH

Dynemicin A (3) MeSSS O O O O I O S N HO H O O OMe OH O OMe O EtHN HO MeO MeO

I (-)-Calicheamicin γ1 (4)

Figure 1. Members of enediyne antibiotics family.

2

Surrounding this new family of compounds, new reactions were developed and novel strategies were designed aimed at their total synthesis.11 At the same time, a remarkable mechanism accounting for their phenomenal biological activities was proposed that recalled the earlier work of Bergman.17-19

In the early 1970s, Robert Bergman first studied the thermal cycloaromatization of an enediyne system ((Z)-1,5-diyn-3-ene) and demonstrated the existence of a highly reactive 1,4-benzenoid diradical intermediate (Figure 2a). This mechanism was later supported by Wong and Sondheimer through the facile cycloaromatization of a strained cyclic enediyne.20-21 When the reaction was carried out in THF-d8, deuterium was incorporated into the product, which clearly indicated the capability of the highly reactive diradical intermediate to abstract hydrogen atoms from solvent (Figure 2b). Later in 1990s, the ring closure was successfully used in the total synthesis of different natural products (Figure 2c) containing fused ring systems.22-23

With the natural products, the basic enediyne units were not capable of surviving under biological conditions; thus, their sophisticated structures played very important roles at this point. It was believed that the enediyne unit contained in the antitumor agents was embedded in a complex structural framework that allowed the targeted delivery of the warhead (enediyne unit) to the minor groove of a target cell’s DNA and stabilized the strained cycloenediyne rings.24 The marginal stability of the 9- and 10-membered rings is essential for the efficient triggering of the warhead at physiological temperature. Cycloaromatization resulted in the formation of 1,4- benzenoid diradical. This highly reactive diradical was then perfectly positioned to strip hydrogen atoms from sugar phosphate backbone of adjacent DNA strands, which would lead to the scission of the DNA double helix. The destruction of the cell’s genetic material finally leads to the target cell’s death. The mechanism of this process is shown in Figure 3 for calicheamicin I γ 1.

3

D H 200 °C D D 2 [H] D

t1/2 = 30 s D D D D H

a) System designed by Bergman to study the enediyne cycloaromatization.

Li TsO -10 to 25 °C + TsO THF-d8 Li

D

D

b) Observation by Wong and Sondheimer

MeO C 2 MeO2C CO2Me n n n

PhCl / 210 °C, 24 hr R R R

a: R = H, n = 1, 72% b: R = CH2OTBS, n = 1, 58% c: R = H, n = 2, 53%

c) Tandem Bergman / radical cyclization strategy for ring annulation.

Figure 2. Cycloaromatization of enediynes and its utility in natural product synthesis.

4

A nucleophile (e.g. glutathione) will attack the central sulfur atom of the trisulfide group, and generate a thiolate to intramolecularly attack the α,β-unsaturated ketone in the adjacent six- membered ring and give a cyclothioether (5). The thiolate attack rehybridizes the sp2 carbon at the attack point to sp3 carbon to initiate the Bergman cyclization. The benzenoid diradical generated from cycloaromatization is then able to abstract two proton atoms, one from the C5’ position of deoxycytidine (C) and the other one from the ribose position of the opposing strand. The DNA radicals generated from this reaction are then able to react with the molecular oxygen and finally lead to the DNA double strand cleavage.1, 2b, 25-26 Unfortunately, most of the natural enediyne compounds were highly toxic to the normal cells in test animals. Thus, derivatives of this family have been sought and some of the potential candidates already exhibited high potential activity against tumor with lower toxicity.27-33

1-2. Naturally occurring polyacetylenes

While the enediynes exemplify a branch of the naturally occurring acetylenic compounds, the polyacetylenes are another important group. Long before the discovery of the enediyne antibiotics, naturally occurring acetylenes were studied by chemists. In 1892, the first acetylenic compound, tariric acid (Figure 4), was isolated from seed oil of Picramnia tariri DC by Arnaud.34 However, he was unable to assign the correct structure of tariric acid until ten years later.35 At that time, the main difficulty in the identification of the acetylenes was a lack of proper structural analysis methods. In 1935, when the first natural diacetylene was isolated and characterized by Russian chemists,36 they tried to distill the lachnophyllum ester (Figure 4), which resulted in a violent explosion. In order to avoid other mishaps, they finally elucidated the structure by classical methods. Now, with the help of modern spectroscopic analysis, especially the utilization of NMR and MS, a few thousand natural acetylenes have been isolated and characterized. These compounds were found in 15 different families of higher plants as well as algae and micro-organisms. Most of the acetylenes, especially polyacetylenes, are thermally unstable and are usually extremely dangerous in the crystalline and liquid states.37

5

NHCO Me O 2 O NHCO2Me

1. Nucleophilic attack HO HO 2. Conjugate addition O O S sugar S sugar S 5 MeS Nu 4 P base Bergman cyclization O

P NHCO Me O NHCO2Me O 2

HO HO

O S O S sugar sugar P base O

P P base O HOO 1. O2

2. [H . ] P

Reduction

O base P O base cleavage O + H O P

P P

I Figure 3. Mechanism for DNA cleavage by calicheamicin γ1 (4).

6

With respect to natural product isolation, the absolute concentrations of acetylenes in different plants are extremely variable, from 10-6% to 1% of the fresh plant material.38 Furthermore, the acetylene content in individual plant parts can vary dramatically. To date, investigations addressing the degradation pathways of the natural acetylenes have been largely fruitless, because most radiolabeled acetylenes introduced into the plants were very rapidly transformed into highly polar compounds with undetermined structures.38

O O

MeO 3 MeO 9 Tariric acid Lachnophyllum ester OH

CH3 HO 6 HO OH Panaxytriol (7) CO H 2 Panax ginseng Minquartynoic acid (6) 7 Ochanostachys amentacea (root, China, Japan, Korea) 39 (tree stem, Malaysia) anticancer agent antitumor agent45 OH OH

5 5 OH OH Falcarindiol (8) Dehydrofalcarindiol (9) carrots, tomato, and eggplant Angelica tree 52 antifungal agent (tree leaves, Costa Rica) antitumor agent46 HO

Cicutoxin (10) OH Water hemlock (rhizomes, North America) 46 neurotoxin

Figure 4. Examples of biologically active linear polyacetylenic natural products.

7

Although the stereochemical challenges involved in the synthesis of the polyacetylenes are few, the fused carbon-carbon triple bonds make these compounds unstable and challenging to study. Compared with enediyne antibiotics, polyacetylenes have more diverse biological activities, even though they have much simpler frameworks. A few examples of natural acetylenes are shown in Figure 4.

Numerous biological activities have been shown for these compounds. For instance, panaxytriol (7, Figure 4), an apparently important contributor to the overall activity of red ginseng, was isolated in 1983.39-40 After the structure of panaxytriol was established,41 it was shown to have inhibitory activity against MK-1 cells (IC50 = 8.5 ng/mL) and to suppress the growth of B16 melanoma cells transplanted into mice.42 Moreover, it was highly effective in inhibiting cellular respiration and the energy balance in a human breast cancer cell line.43 In addition, it enhanced the cytotoxicity of mitomycin C against human gastric adenocarcinoma cell lines.44 Minquartynoic acid (6)45 and dehydrofalcarindiol (9)46 are potential antitumor agents, falcarindiol (8) is an antifungal agent,47-52 and cicutoxin (10)46 is a neurotoxin. Other acetylenes, such as acetylenic thiophenes, may be associated with the insecticidal and nematicidal properties of Tagetes species (e.g., marigold).53-54

1-3. Biosynthesis of polyacetylenic compounds

Recent evidence indicates that the biosynthesis of acetylenic products in plants generally involves the conversion of (Z)-alkenes in unsaturated fatty acids, such as linoleic acid, into internal acetylenes by functionally divergent desaturase enzymes.56 This transformation can be simplified as shown in Figure 5.

+ + NAD(P)H + O2 + 2H NAD(P) + 2H2O

O "Fe=[O]" R O R OR' n OR' acetylenase n

Figure 5. Desaturase-like reaction catalyzed by an acetylenase.

8

In the presence of cytochrome-b5, NADPH-cytochrome-b5 oxidoreductase and protons, an acetylenase consumes one NADPH and one oxygen molecule in the conversion of an unsaturated fatty acid (e.g., linoleate) into the corresponding acetylene (e.g., crepenynate) with the + 55b production of two molecules of H2O and one NADP .

Crepenynic acid, with an alkynyl group at C-12, is the first known metabolite on the pathway to most fatty acid-derived acetylenic natural products. Crepis rubra, C. foetida and C. alpina are small flowering plants capable of producing seed oils containing up to 60% crepenynic acid. A ∆12-desaturase-like gene, CREP-1 was isolated from C. alpina by Stymne and coworkers in 1998.56-57 Overexpressing this gene in Saccharomyces cerevisiae or the seeds of Arabidopsis thaliana resulted in the accumulation of crepenynic acid in their seeds up to 0.3% and 25% (w/w) of the total fatty acids, respectively.56 During the yeast expression of CREP-1, addition of linoleic acid to the culture medium was required because S. cerevisiae does not produce polyunsaturated fatty acids. Collectively, these results point to CREP-1 as the key enzyme in biosynthesis of crepenynic acid.

In 1998, a gene, designated as ELI12, was only expressed when parsley suspension cultures were infected by fungal elicitation.58, 59 Expression of this gene in S. cerevisiae did not lead to accumulation of linoleic acid in the transformed cells.60 Related experiments inferred that the ELI12 gene product might be associated with the production of polyunsaturated fatty acids, which are used in the maintenance of membranes following fungal pathogenesis or as substrates for the synthesis of signaling compounds such as jasmonic acid.61 However, recent investigation showed that the ELI12 gene product is also a fatty acid acetylenase or triple bond-forming enzyme.43 Expression of ELI12 gene in seeds of transgenic soybean was accompanied by accumulation of two novel fatty acids that comprise up to 3%-4% of the total fatty acids in the transgenic seeds. A hypothetical response of parsley toward the fungal challenge is illustrated in Figure 6.

9

Antifungal biological activity Attack Fungi Parsley

Limits the Biologically active extent of attack Expression of polyacetylenes Gene ELI12

O R O ELI12

Polyacetylene R n OR' n OR' biosynthesis

Figure 6. Expression of ELI12 in parsley induced by a fungal challenge.

The novel fatty acids isolated from the transgenic seeds were identified by comparison with isolated natural products from chanterelle mushroom and synthetic compounds.62 They were assigned to crepenynic acid and (14Z)-dehydrocrepenynic acid, respectively. Further investigation showed that ELI12-encoded fatty acid acetylenase is not only expressed in Apiaceae species (e.g., parsley), but also in members of Araliaceae (e.g., English ivy) and Asteraceae (e.g. sunflower and Calendula officinalis) families.43 However, most plants in these families do not accumulate the crepenynic acid and dehydrocrepenynic acid, which are expected from the transgenic expression of these enzymes.38 Nevertheless, members of Apiaceae, Araliaceae, and Asteraceae families produce linear polyacetylenic compounds that have strong biological activities.38 These compounds include falcarindiol (8) in Apiaceae and Araliaceae species, as well as dehydrofalcarinol (9) in Asteraceae species. Crepenynic and dehydrocrepenynic acids are intermediates in the biosynthetic pathway for these polyacetylenes, as shown by experiments in carrot tissue. According to previous investigations, a proposed biosynthetic pathway to polyacetylenic compounds is outlined in Figure 7.

10

Acetylenase I CO2R CO2R 7 CREP-1 homolog 7 Linoleate Crepenynate

Acetylenase I (dual function) Acetylenase II CO2R 7 or ∆ 14-desaturase (14Z)-Dehydrocrepenynate

Acetylenase III CO2R 7 CO2R 7

6 HO OH

OH

Panaxytriol (7)

Figure 7. Proposed biosynthetic pathway of polyacetylenic compounds.

Linoleic acid is used as the starting material that, during the first desaturation catalyzed by acetylenase I, is converted to crepenynic acid. This compound is the branch point of the primary metabolism and secondary metabolism pathway. Crepenynic acid then undergoes the second desaturation catalyzed by a ∆14-desaturase to introduce the cis- C=C double bond at C-14 affording (14Z)-dehydrocrepenynic acid. It should be noted that the generation of crepenynic and dehydrocrepenynic acids by this pathway has been observed through the expression of ELI12 in transgenic soybean seeds. However, it is still not clear whether both reactions were catalyzed by the same enzyme or not. An acetylenase II-catalyzed desaturation is presumed to introduce a triple bond into the C-14 position. A final round of desaturations, hydroxylations, and decarboxylation produces oxygenated polyacetylenes, such as falcarindiol and panaxytriol (7). These compounds may then be utilized as antifungal agents by the plant in defense of fungal attack.

11

However, the detailed mechanism of these desaturation and acetylenation reactions are still not well understood. In order to further decipher the transformation of C=C bond to C≡C bond, we undertook the synthesis of selected deuterated linoleic acid isotopomers. The applications for the deuterated linoleates include metabolic tracer and kinetic isotope studies of the linoleate to crepenynate transformation. Kinetic isotope effect studies will allow quantitation of the degree of bond-breaking at the transition state and provide a detailed view of the concertedness of the alkyne desaturation. Kinetic isotope effects (KIE) provide powerful insights into the detailed mechanism of desaturation.63 For alkene-generating desaturations, stepwise scission of carboxy- proximate C-H has been observed to occur first.63 If the C-H bond breaking is involved in the 63 rate-determining-step, a large isotope effect (kH/kD = 5-7.5) will be detected. On the other hand, if the C-H bond cleavage is not involved in the rate-determining-step, no primary kinetic isotope 63 2 2 effect will be detected (kH/kD = 1.0). Using [12- H]linoleic acid and [13- H]linoleic acid as substrates to study the enzyme-catalyzed acetylenation will give a clearer reaction model, which may be consistent with either stepwise removal of hydrogen atoms from C=C double bond starting at C-12 or concerted removal of both hydrogen atoms. However, performing these kinetic isotope effect experiments may require further development of a continuous acetylenase assay and substantial amounts of active acetylenase, which is outside the scope of this dissertation. In addition, these syntheses will also furnish substrates that may be used in a stable isotope-based enzyme assay. Also, the development of routes to polyunsaturated fatty acids with specific stable-isotopic labels will facilitate the study of membrane proteins in biologically relevant lipid bilayers composed of polyunsaturated phosphatidylcholine (PC) and phosphatidylethanolamines (PE). Model membranes currently used by most biophysicists contain medium- and long-chain saturated PC and PE derivatives. Thus, these synthetic membranes may not provide fluidities and intermolecular interactions experienced by a eukaryotic integral membrane protein in its native environment. To date, development of new bilayer environments has been hampered by both the cost and availability of labeled linoleate- containing PC and PE derivatives. Regiospecific deuterium substitution in polyunsaturated lipids provides new opportunities for the examination of dynamic processes in novel lipid bilayers and the study of protein-lipid interactions.

12

1-4. Synthesis of regioselectively labeled linoleic acids

A severe limitation for the study of unsaturated fatty acid metabolism is the lack of convenient approaches to synthesize regioselectively modified substrates at the vinylic positions. No syntheses of linoleic acids with deuterium labels at vinylic positions (e.g., 11a - c, 12) have been reported. Optimization of a Pd-catalyzed cross-coupling reaction, previously reported by Hutzinger and Oehlschlager, provided a convergent method for the stereospecific synthesis of isotopically labeled (Z,Z)-1,4-dienes.64a-b Through the syntheses described in this chapter, suitable lipid systems for each of the biochemical and biophysical studies described earlier are now accessible.

R' R DD CO H 2 CO2H 7 D3C D D 7 Linoleic acid: R = R' = H 11b: R = H, R' = D 11a: R = D, R' = H 11c: R = R' = D 12

Figure 8. Regioselectively labeled linoleic acids.

The present study targeted the syntheses of [12-2H]linoleic acid 11a, [13-2H]linoleic acid 11b, 2 and remotely labeled [16,16,17,17,18,18,18- H7]linoleic acid 12. The key step of the synthesis was achieved by a Pd(0)-catalyzed cross-coupling reaction between fragments 13 and 14. Numerous synthetic pathways have been developed to build the 1,4-diene units, such as semihydrogenation of 1,4-diyne or Wittig reaction.64c-d However, those methods do not provide a convenient way to regioselectively incorporate deuterium at the selected vinyl position. Even though the Wittig reaction is widely exploited in formation of C=C bonds with desired configuration (>95/5 E/Z ratio), a better E/Z ratio is desirable in our substrates.

Bu Sn R Pd (0) 3 + PG PG I 8 8 R' Cl C -C Fragment 14 Linoleic acid precursor C11-C18 Fragment 13 1 10 PG: Protecting group 13a: R = H, R' = C3H7 : Isotopic labelled position 13b: R = D, R' = C3H7 13c: R = H, R' = C3D7 Figure 9. Retrosynthesis analysis of vinyl deuterated linoleic acids.

13

As shown in the retrosynthesis analysis (Figure 9), the targeted linoleic acid derivatives only required changes in the C11-C18 fragment (13); fragment C1-C10 (14) was common to all desired products. Tributyltin group in fragment 3 was used as proton (H) or deuteron (D) mask; after the coupling reaction it could be easily replaced under acidic conditions by a proton or a deuteron.65-66 The stereoselective preconstruction of the vinyl groups in fragments 13 and 14 will ensure the 1,4-diene units have correct double bond configurations. By using the common fragment 14, the syntheses were simplified and required fewer synthetic steps. The flexibility of this convergent synthetic route has furnished a convenient way to prepare compounds with a variety of chain length and 1,4-diene locations. The described method also paved the way for making substrates for related studies, including its adaptation for preparing radiolabeled 1,4- diene derivatives.

1-5. Results and discussion

Fragment 14 was synthesized in five steps in high yield (Figure 10). Propargyl alcohol was

alkylated by treatment with LiNH2 (2.0 equiv.) in liquid NH3 at –33 °C followed by addition of 1-bromoheptane to yield 2-decyn-1-ol (15). The later was subjected to a Zip rearrangement in the t 67 presence of excess Li[NH(CH2)3NH2] and BuOK at 80 °C to give 9-decyn-1-ol (16). This primary alcohol was then protected as an acid-stable tert-butyldimethylsilyl (TBDMS) ether, which was required by the subsequent steps.68

Figure 10. Synthesis of C1-C10 fragment.

abOH OH c OH 7 77% 6 94% 98% 15 16

d OTBDMS e OTBDMS OTBDMS 7 I 8 7 92% I 95% 14 (62% overall yield) 17 18 C1-C10 fragment

t Key: (a) i. LiNH2 (2.0 eq), NH3 (l), ii. n-C7H15Br; (b) BuOK, LiHNC3H6NH2, 80 °C; (c) t n BuMe2SiCl, imidazole, CH2Cl2; (d) i. BuLi, -78 °C, ii. I2, -78 °C; (e) i. cyclohexene, BH3, n- pentane, 0 °C, ii. AcOH, HOC2H4NH2.

14

After iodination and borane reduction, the terminal alkyne was efficiently converted into (Z)- vinyl iodide 14, which has desired (Z)-configuration for cross-coupling reaction. The overall yield of 14 from propargyl alcohol is 62% and all of the reactions can be conducted on a multi- gram scale.

Starting with 1-heptyne, monodeuterated C11-C18 fragments (13a, 13b) were prepared in excellent yields and with high isotopic purity (Figure 11). Conversion of the terminal acetylene to ethyl 2-octynoate 19 by treatment of its lithium salt with ethyl chloroformate proceeded in 83% yield. Stannylcupration of compound 19 in THF followed by quenching of the reaction mixture at –78 °C with EtOH yielded ester 20a in excellent yield. However, if the reaction was quenched at 0 °C or room temperature, the main product was isolated from the reaction was the undesired (E)-isomer. It should be noted that all attempts to prepare the corresponding deuterated ester 20b using Bu3SnH and quenching the reaction with deuterated alcohol were unsatisfactory due to the low isotopic purity (60-70%-d) attained by this method. The reason for these results was likely the presence of other proton sources present in the reaction system.

Figure 11. Synthesis of C11-C18 fragment (13a, 13b). O b Bu3Sn H a OEt C4H9 97% CO2Et 83% C4H9 20a 19

Bu3Sn D c CO2Et 95% 20b

20a d Bu3Sn R e Bu3Sn R or 99% CH2OH 99% CH2Cl 20b 21a: R = H 13a: R = H 21b: R = D 13b: R = D C11-C18 fragment

Key: (a) i. BuLi (1.0 eq), -78 °C, ii. ClCO2Et, -20 °C to 10 °C; (b) i. Bu3SnLi, THF, -78 °C, ii. . CuBr SMe2, iii. ethyl ester, -78 °C, iv. EtOH, -78 °C to rt; (c) i. (Bu3Sn)2 (2.5 eq), BuLi (2.5 eq), -78 °C, ii. CuCN, -50 °C, iii. EtOD (excess), -78 °C; (d) DIBAL-H (2.5 eq), THF, 0 °C; (e) NCS, Me2S, CH2Cl2, 0 °C.

15

Alternatively, it was found that generating 2.5 equivalents of the lithiated stannane through n addition of BuLi to (Bu3Sn)2 for use in the stannylcupration and subsequently quenching the reaction mixture with a deuterated alcohol (typically MeOD or EtOD) furnished deuterated ester 20b containing >98%-d.69 Fragments 13a and 13b were readily obtained by reducing the esters 20a or 20b, respectively, with DIBAL-H and chlorinating the resulting alcohol with the N-

chlorosuccinimide (NCS)-SMe2 complex.

For the remotely deuterated fragment 13c, we started from 3-butyn-1-ol and commercially available 1-bromopropane-d7 (Figure 12). The terminal acetylene was protected with TMSCl and the crude propargylic alcohol was readily converted to bromide 23.

Figure 12. Synthesis of C11-C18 fragment (13c).

Br TMS OH a, b c TMS + C3D7MgBr C3D7 93% 89% 24 22 23

CO2Et Bu3Sn H d e f C D C3D7 CO2Et 3 7 52% 82% 25 from 24 26

Bu3Sn H g Bu3Sn H

C3D7 CH2OH 74% C3D7 CH2Cl 27 13c

Key: (a) i. i-PrMgCl (2.0 eq), THF, ii. TMSCl, iii. HCl (3.0 M); (b) Ph3P, Br2, pyridine, CH2Cl2; (c) i. Mg, THF, ii. CuCN (1.0 eq), LiCl (2.0 eq), iii. n-C3D7Br, THF; (d) i. KF, DMF, ii. BuLi, . THF, -20 °C, iii. ClCO2Et, -20 °C to 10 °C; (e) i. Bu3SnLi, THF, -78 °C, ii. CuBr SMe2, iii. ethyl ester, -78 °C, iv. EtOH, -78 °C to rt; (f) DIBAL-H (2.5 eq), THF, 0 °C; (g) NCS, Me2S, CH2Cl2, 0 °C.

However, when compound 23 is subjected to copper-catalyzed cross-coupling reaction to make compound 24, low percent yields of heptyne were observed. Coupling of unlabelled propyl magnesium bromide (1.0 eq) with 23 lead to desired coupling product 24 in 55% yield, and 40% of compound 23 was recovered after column purification. When the amount of Grignard reagent70 was increased to 1.5 eq, the coupling yield reached 69% and the addition of more

16

Grignard reagent did not benefit the reaction yield significantly (Entry 1, column 1 in Table 1). On the other hand, it was observed that by changing the coupling partner (Entry 2, column 2 in Table 1) to the saturated bromide an even lower coupling yield (39%) was obtained. At the same time, the self-coupling product of compound 23 was isolated 20% yield.

Table1. Results of copper catalyzed cross-coupling reaction. a

MgBr Entry C3H7MgBr C3D7MgBr C6H13MgBr TMS

Br b d d 1 TMS 55-69% N.D. 89% 81%

c 2 C3H7Br N.D. 39% N.D. N.D.

a. The reaction was performed in THF at rt with CuCN.2LiCl catalyst; b. 55% yield was observed with 1.0 eq Grignard reagent, and 69% yield was obtained with 1.5 eq of Grignard reagent; c. 1.0 eq of Grignard reagent was used, and similar result was obtained when only CuI was used as catalyst; d. 1.2 eq of Grignard reagent was supplied; N.D. Self-coupling experiments were not performed.

It was found that self-coupling of the Grignard reagent formed from 23 even happened before the addition of copper catalyst and bromopropane to the reaction. It seems that the bromobutyne (23) was attacked by the Grignard reagent generated in situ to give the self-coupling product. This result indicated that 23 would be useful as the bromide partner to couple with the propyl

Grignard reagent. Fortunately, when d7-propylmagnesium bromide (1.2 eq) was used to do the coupling with compound 23, desired product was afforded in 89% yield (Entry 1, column 3 in Table 1). The dramatic difference in yields was originally thought to be due to the isotope effect connected to the β-elimination of the organomagnesium reagent. In contrast, when the reaction was conducted using hexylmagnesium bromide and compound 23, the cross-coupling product was obtained in 81% yield (Entry 1, column 4 in Table 1). Any potential isotope effect bias seems to have disappeared in this case. Based on the results obtained, a possible mechanism is proposed in Figure 13. Grignard reagents formed in these reactions were capable of β- elimination of H(D)MgBr resulting in the formation of a terminal alkene. Similar phenomena were observed by Holm and Garst.70-72

17

Figure 13. Proposed mechanism for Cu-catalyzed coupling reaction.

(D)H H(D) MgBr n

Br

TMS n CuCN.2LiCl TMS H(D)

24: n = 0; 89% (d7) H(D) n = 0; 55% (d0) n = 3; 81% (d0) + H(D)MgBr n

The equilibrium between the terminal alkene and the Grignard reagent played a very important role in this reaction. When a short-chain bromide was used, the alkene formed from the β- elimination was very volatile and was emitted as gas, so the Grignard reagent was substantially consumed by this pathway. However, the same process with the longer chain alkylmagnesium bromide did not influence the reaction very much, because the alkene was retained in the solution. Coupling shifted the equilibrium back to generate the Grignard reagent thus increasing the overall yield. On the other hand, β-elimination of the deuterated compound is slower than that of the non-deuterated compounds as a result of a primary kinetic isotope effect, which is manifested by a shift in the equilibrium from alkene and magnesium hydride towards the Grignard reagent. Therefore, the yield of the coupling of a Grignard reagent with heavy isotopes in β-position was higher than that of nondeuterated compound.

With compound 24 in hand, the rest of the synthetic pathway is straightforward. After removal of

the TMS group with KF at 40 °C, a crude hexane solution of [d7]-heptyne was converted to ester 25 without purification. Following an identical procedure employed for the syntheses of 13a and 13b, fragment 13c was readily obtained.

18

The key cross-coupling of allylic chloride (13a, 13b, or 13c) with a vinylzinc reagent in the presence of a catalytic amount of Pd(PPh3)4 afforded the 1,4-dienes (Figure 14). A variety of catalysts and organometallic derivatives were explored in the optimization of the coupling reaction.

Figure 14. Synthesis of regioselectively labeled linoleic acids.

Bu Sn R Bu3Sn R 3 OTBDMS a + OTBDMS I R' R' CH2Cl 8 8

13a: R = H, R' = C3H7 Fragment 14 28a: R = H, R' = C3H7 13b: R = D, R' = C3H7 28b: R = D, R' = C3H7 13c: R = H, R' = C3D7 28c: R = H, R' = C3D7

"R R c "R R b OH CO2H R' R' 8 70-72% 8 29a: R = H, R" = D, R' = C3H7 11a: R = H, R" = D, R' = C3H7 29b: R = D, R" = H, R' = C3H7 11b: R = D, R" = H, R' = C3H7 29c: R = H, R" = H, R' = C3D7 12: R = H, R" = H, R' = C3D7 44% for two steps

t Key: (a) i. BuLi (2.0 eq), Et2O, -78 °C, ii. ZnBr2 (1.0 eq), 0 °C, iii. Pd(PPh3)4 (8% mol), tri-o- tolylphosphine (16% mol); (b) Dowex-50W, MeOD(H), 50 °C; (c) PDC, DMF/H2O.

64 Beginning with Hutzinger’s published method, we used Et2AlCl as the metal-lithium exchange reagent. However, cross-coupling yield of this reaction is poor. Through the addition of different

catalysts to the reaction, we found that Pd(PPh3)4 provided the best coupling outcome whereby a

29% yield was achieved. Pd(PPh3)2Cl2 and Ni(PPh3)2Cl2 did not catalyze this coupling reaction at all and unreacted allylic chloride was recovered from the failed reaction mixtures. On the other hand, by changing the metal-lithium exchange reagent in the presence of same catalyst,

Pd(PPh3)4, we found that zinc bromide (anhydrous ZnBr2 was used as a solution in anhydrous diethyl ether, 59% w/w) was a better metal-lithium exchange reagent for the coupling than

Et2AlCl. The reproducible isolated yield of the coupling product is 33% (ZnBr2) instead of 29%

(Et2AlCl). Moreover, ZrCp2Cl2 does not work for the coupling reaction. The results were summarized in Table 2.

19

Table 2. Optimization of the cross-coupling reaction for assembly of the 1,4-diene unit.

Entry Catalyst (5% mol) MXn Ligand (10% mol) Yield

1 Pd(PPh3)2Cl2 Et2AlCl / N.R.

2 Ni(PPh3)2Cl2 Et2AlCl / N.R.

a 3 Pd(PPh3)4 Et2AlCl / <29%

4 Pd(PPh3)4 ZrCp2Cl2 / N.R.

b 5 Pd(PPh3)4 ZnBr2 / 33%

b 6 Pd(PPh3)4 ZnBr2 Tri-o-tolylphosphine 44%

b 7 Pd(PPh3)4 ZnBr2 Tri-2-furylphosphine 18%

c 8 Pd(PPh3)4 ZnBr2 AsPh3 <10%

d 9 Pd(PPh3)4 ZnBr2 DPPF N.R.

e 10 Pd(dba)2 ZnBr2 Tri-o-tolylphosphine N.R.

a. Isolated yield after the first step; b. Isolated yield after deprotection; c. NMR yield; d. 1,1’- Bis(diphenylphosphino)ferrocene; e. Bis(dibenzylideneacetone)palladium.

Previous investigations on Pd(0)-catalyzed coupling reactions indicated that some additional ligands may benefit the cross-coupling reaction. Ligand exchange is usually rapid with palladium catalysts and modifying the ligands can result in the formation of easily dissociated catalyst species, which may enter the catalyst cycle.73 These ligands might benefit the coupling by increasing either the percent yield or the reaction selectivity. Our experimental results demonstrated that the coupling yield could be improved to 44% by adding tri-o-tolylphosphine

(16% mol) to the reaction system. However, other ligands, such as AsPh3, tri-2-furylphosphine and 1,1’-bis(diphenylphosphino)ferrocene (DPPF) did not provide satisfactory results. Ultimately, the best experimental results were obtained, as indicated in Entry 6 (Table 2), with the addition of tri-o-tolylphosphine (0.16 eq) to the coupling reaction.

20

The 1,4-diene is hard to purify after coupling reaction from the main side-product (10-(tert- butyldimethylsiloxy)-1-decene) of the Pd-catalyzed cross-coupling reaction. On the other hand, after deprotection, the high polarity of the dienyl alcohol required a more polar solvent for column chromatography. As a result, the best separation and percent yield of the coupling could be achieved at this stage. Accordingly, the 1,4-diene, without further purification, was treated with an acidic ion-exchange resin (Dowex-50W) to completely cleave the protecting groups. Subsequently, purification was performed through column chromatography to afford pure

linoleic alcohol. To selectively replace the Bu3Sn group with a deuteron, the Dowex resin was

pre-treated with D2O 3 times (5 min per time) and the entire reaction was conducted in MeOD. By this method, very high deuterium incorporation was achieved (>98% from 1H NMR and

HRMS results). The primary alcohol was finally oxidized with PDC in DMF/H2O to complete the synthesis.

1-6. Conclusion

In summary, we developed an effective way to make selectively deuterium-labeled linoleic acids. By this Pd-catalyzed method, deuterium was easily introduced into the vinyl position providing linoleates with very high isotopic purity. This method also provided a general route for the construction of 1,4-diene derivatives with different chain lengths and 1,4-diene locations. With the deuterium-labeled linoleic acids in hand, a solid-state 2H NMR study of phosphatidylcholine lipid bilayers prepared with mixed fatty acyl chains (saturated acyl chain at carbon 1 of glycerol backbone and unsaturated acyl chain at position 2) is currently underway.

1-7. Experimental section

General Experimental Information. All manipulations were performed in oven-dried glassware under a nitrogen atmosphere unless otherwise mentioned. All solvents were dried and were distilled freshly before use. Ether, THF, n-pentane and benzene were dried and deoxygenated by distillation under N2 from sodium-benzophenone ketyl. Methylene chloride was dried over CaH2; pyridine was dried over CaSO4; ethanol and methanol were dried with Mg metal and distilled before use. Standard work-up included washing with water, followed by

21

washing with brine twice, drying the organic phase over MgSO4, gravity filtration to remove

MgSO4 and concentrating in vacuo unless otherwise specified. Column chromatography was performed on silica gel (Natland International Corp. 200-400 mesh) using the indicated solvent. TLC analyses were carried out using precoated silica gel plates with aluminum backings (Whatman, Al Sil G/UV). Melting points were determined with a Gallenkamp melting point apparatus and were uncorrected. 1H and 13C NMR spectra were recorded on 200 or 300 MHz FT-NMR spectrometers (Bruker, Avance Series). IR spectra were determined as neat films (using NaCl plate) or KBr pellets on a Perkin-Elmer 1600 FT-IR spectrophotometer. High-resolution MS spectra were performed by the Chemistry Mass Spectrometry facility, Ohio State University, Columbus, OH.

Synthesis and spectra data

LiNH2, NH3 (l), DMSO OH OH 77% 6 15

Synthesis of 3-decyn-1-ol (15)74: To a 3000 mL round bottomed flask (RBF) equipped with mechanical stirrer and dry-ice condenser, NH3 was introduced at –78 °C. After liquid NH3 (400 . mL) was condensed, Fe(NO3)3 9H2O (0.35 g, 0.87 mmol) was added and a pale red solution was obtained in 5 min. To this red solution, lithium metal (4.0 g, 0.58 mol) was added in several portions over 20 min. The pale red solution turned deep blue within few minutes and copious amounts of gas were evolved. The mixture turned into gray slurry after stirring at –78 °C for 1.5 h. Propargyl alcohol (15.0 mL, 0.26 mol) was then added slowly through a drooping funnel over 15 min. Using the same dropping funnel, 1-bromoheptane (34.0 mL, 0.22 mol) was added in 15 min and followed by the addition of DMSO (180 mL) over 20 min. The reaction mixture was then allowed to warm to rt slowly. (Dry-ice bath was kept at hand to avoid violent NH3 evaporation.) All NH3 was evaporated after 3 h, and the resulting black slurry was stirred at rt for 1 h. The reaction was finally quenched by adding ice (~800 mL) and ether (100 mL). After

stirring at rt overnight, the layers were separated, and the aqueous layer was extracted with Et2O (5 × 150 mL). The combined organic layers were consequently washed with HCl (1 M, 200 mL),

22

saturated NaCl (200 mL), aqueous NaHCO3 (150 mL) and saturated NaCl (150 mL), and dried over MgSO4. After concentration with a rotary evaporator, the crude product was obtained as a red residue. It was purified by bulb-to-bulb vacuum distillation and the fraction boiling at 110- 120 °C/0.05 mm Hg was collected. The pure product was condensed as white solid at –78 °C, 1 which melted at rt to give a pale yellow oil (23.0 g, 77% yield). H NMR (CDCl3, 200 MHz) δ 4.23 (t, J = 1.8 Hz, 2H), 2.59 (s, 1H), 2.18 (tt, J = 6.5, 1.8 Hz, 2H), 1.72 (q, 2H), 1.48 (m, 2H), 13 1.31 (br, 6H), 0.86 (t, 3H); C NMR (CDCl3, 75 MHz) δ 87.08, 78.66, 51.83, 32.11, 29.22, 29.19, 29.00, 23.01, 19.13, 14.46.74

OH H2N(CH2)3NHLi OH 7 6 t BuOK, H2N(CH2)3NH2 15 94% 16

Synthesis of 9-decyn-1-ol (16): To a flask containing dry 1,3-diaminopropane (150 mL) and a magnetic stir bar was added lithium metal (3.0 g, 426 mmol) at rt. A deep blue solution was obtained in minutes. After stirring at rt for 2 h, the reaction mixture was warmed to 70 °C for 2 h yielded a white slurry. The mixture was allowed to cool to rt, and tBuOK (32.0 g, 285 mmol) was added slowly to give a orange slurry. To this suspension was added alcohol 15 (10.0 g, 65 mmol) over 15 min and the final deep orange mixture was stirred at rt for 1 h. The reaction was cooled to 0 °C and quenched with ice-cold water (150 mL). The layers were separated and the aqueous layer was extracted with hexanes (3 × 100 mL). The combined organic phases were washed with water (100 mL), HCl (1 M, 150 mL) and brine (100 mL), sequentially. After drying

over MgSO4, filtering, and concentrating with a rotary evaporator, a pale yellow oil was obtained. The crude product was purified by bulb-to-bulb distillation (0.25 mm Hg, 88 – 90 °C) to yield 1 colorless oil (9.4 g, 94% yield). H NMR (CDCl3, 200 MHz) δ 3.61 (t, J = 6.4 Hz, 2H), 2.16 (dt, J = 7.0 Hz, 2.6 Hz, 2H), 1.91 (t, J = 2.6 Hz, 1H), 1.51 (quint, J = 7.0 Hz, 2H), 1.23 (br, 10H); IR -1 74 (neat film) νmax 3362, 3309, 2932, 2857, 2117, 1465, 1056, 630 cm .

OTBDMS OH TBDMSCl, imidazole 7 7 CH2Cl2 98% 17 16

23

Synthesis of 1-(tert-butyldimethylsilyloxy)-9-decyne (17): To a solution of 9-decyn-1-ol 16

(9.3 g, 60 mmol) in dried CH2Cl2 was added imidazole (8.6 g, 126 mmol) and TBDMSCl (10.0 g, 66 mmol) at rt. A white slurry was formed immediately and it was stirred at rt for 3 h. The reaction was quenched by adding distilled water (200 mL) and the layers were separated. The

aqueous layer was extracted with CH2Cl2 (3 × 30 mL). The combined organic layers were

washed with brine (50 mL) and dried over MgSO4. After removing the solvent in vacuo, the crude product was heated under vacuum on rotary evaporator for 15 min at 60 °C. The final 1 product (15.8 g, 98% yield) was pure enough for subsequent reactions. H NMR (CDCl3, 200 MHz) δ 3.57 (t, J = 6.8 Hz, 2H), 2.16 (dt, J = 6.8 Hz, 2.4 Hz, 2H), 1.91 (t, J = 2.8 Hz, 1H), 1.48

(br, 12H), 0.87 (s, 9H), 0.02 (s, 6H); IR (neat film)νmax 3315, 2931, 2858, 2120, 1472, 1255, 1097, 836, 776, 630 cm-1.68

1. BuLi, THF, -78 °C OTBDMS OTBDMS 7 7 2. I2, THF I 92% 18 17

Synthesis of 10-iodo-1-(tert-butyldimethylsilyloxy)-9-decyne (18): To a solution of terminal alkyne 17 (2.7 g, 10 mmol) in THF (15 mL) was slowly added nBuLi (1.42 M, 7.8 mL) at –78 °C.

After 30 min, I2 (3.0 g, 11.7 mmol) was added dropwise as a solution in THF (15 mL). A white precipitate formed during the course of addition and the mixture became pink in color by the end of the addition. Stirring was continued at –78 °C for 15 min then the suspension was allowed warm to rt for 30 min. Aqueous Na2S2O3 (20%, 10 mL) was added to quench the reaction. Brine (15 mL) was added, the layers were separated, and the aqueous layer was extracted with hexanes

(3 × 20 mL). The combined organic phases were dried over MgSO4 and concentrated under vacuum. The crude product, obtained as pale yellow oil, was purified by flash column 1 chromatography (20:1 hexanes/EtOAc) to yield colorless oil (3.7 g, 95% yield). H NMR (CDCl3, 300 MHz) δ 3.58 (t, J = 6.6 Hz, 2H), 2.33 (t, J = 7.0 Hz, 2H), 1.48 (m, 4H), 1.34 (br, 8H), 0.87 (s, 13 9H), 0.03 (s, 6H); C NMR (CDCl3, 75 MHz) δ 95.23, 63.69, 33.24, 29.66, 29.45, 29.12, 28.87,

26.39, 26.15, 21.20, 18.87, -4.85, -7.30; IR (neat film) νmax 2930, 2856, 1463, 1255, 1096, 836, 775 cm-1.

24

1. cyclohexene (2.0 eq) BH , n-pentane OTBDMS OTBDMS 3 I 8 7 2, AcOH, HOC H NH I 2 4 2 95% 14 18

Synthesis of 10-iodo-1-(tert-butyldimethylsilyloxy)-9-decene (14): To a solution of . cyclohexene (2.0 mL, 19.7 mmol) in dried n-pentane (30 mL) was added Me2S BH3 (10 M, 1.0 mL, 10.0 mmol) at -78 °C. After 10 min at -78 °C, the reaction mixture was warmed up to rt for 1 h and a white slurry was obtained. Iodoalkyne 18 (4.0 g, 10.0 mmol) was added slowly at rt transforming the white slurry into clear yellow solution. After 3 h at rt, AcOH (1.5 mL) was added to stop the reaction. After 30 min, 2-aminoethanol (1.5 mL) was added to the reaction mixture resulting in the formation of a small quantity of white precipitate. After stirring at rt overnight (this is not necessary, but it may help to remove the precipitate completely), the pentane solution was decanted into a separatory funnel and washed with brine (3 × 25 mL). After the organic phase was dried over MgSO4 and filtered, the solvent was removed with a rotary evaporator. The crude product was purified by flash column chromatography (30:1 1 hexanes/EtOAc) to give pure product 14 (3.85 g, 95% yield). H NMR (CDCl3, 200 MHz) δ 6.15 (m, 2H), 3.58 (t, J = 6.4 Hz, 2H), 2.10 (q, J = 6.8 Hz, 2H), 1.48 (m, 4H), 1.28 (br, 8H), 0.87 (s, 13 9H), 0.03 (s, 6H); C NMR (CDCl3, 75 MHz) δ 141.85, 82.49, 63.70, 35.08, 33.27, 31.99, 29.73,

29.45, 28.33, 26.38, 23.05, 14.50, -4.86; IR (neat film) νmax 3071, 2929, 2856, 1463, 1255, 1102, 836, 775 cm-1.75

O 1. BuLi (1.0 eq), -78 °C OEt C4H9 2. ClCO2Et, -20 °C-10 °C C4H9 83% 19

Synthesis of ethyl 2-octynoate (19): To a solution of 1-heptyne (20.0 mL, 152 mmol) in dry THF (100 mL) was added nBuLi (1.28 M, 115 mL, 148 mmol) slowly at -78 °C. After stirring for 30 min, the mixture was transferred into a solution of ClCO2Et (16.5 g, 152 mmol) in THF (30 mL) slowly through cannula at -20 °C. Following the completion of the addition, the reaction mixture was stirred at -20 °C for 20 min and then warmed up to 10 °C for 1 h. The reaction

25

mixture was poured into saturated NH4Cl (500 mL) and stirred for few minutes. The layers were separated and the aqueous layer was extracted with ether (4 × 100 mL). The combined organic

phases were washed with water (100 mL). After drying over MgSO4 and filtering, the solution was concentrated with rotary evaporator to give the crude product. The pure product 19 was obtained in fractions at 78-79 °C/0.75 mm Hg by vacuum distillation as colorless oil (21.14 g, 1 83% yield). H NMR (CDCl3, 300 MHz) δ 4.20 (q, J = 7.2 Hz, 2H), 2.30 (t, J = 7.0 Hz, 2H), 1.56 (quint, J = 7.2 Hz, 2H), 1.33 (m, 4H), 1.28 (t, J = 7.0 Hz, 3H), 0.88 (t, J = 7.2 Hz, 3H); 13C

NMR (CDCl3, 75 MHz) δ 154.29, 89.89, 73.54, 62.14, 31.36, 27.62, 22.48, 19.03, 14.43, 14.24; -1 IR (neat film) νmax 2936, 2864, 2234, 1713, 1467, 1252, 1076, 753 cm .

0 °C, ether 4 Bu3SnCl + LiAlH4 4 Bu3SnH

Synthesis of tributyltin hydride: To a suspension of lithium aluminum hydride (4.6 g, 120

mmol) in dry ether (250 mL) was added Bu3SnCl (100 mL, 370 mmol) at 0 °C within 20 min. After 30 min, warmed up to rt for 3 h. The reaction mixture was poured into a 1000 mL RBF and the flask was cooled to 0 °C. Ice was added to quench the reaction very carefully until no more gas was given off. Brine (100 mL) was added and the layers were separated. If gel-like material formed, the mixture was filtered through a Celite pad. Aqueous phase was extracted with ether (5 × 100 mL) and the combined organic layers were washed with water (100 mL) and brine (150 mL). After the solution was dried over MgSO4 and filtered, it was concentrated under vacuum. Crude product was purified by vacuum distillation. The pure product (99.87 g, 93% yield) was collected in a fraction at 78-79 °C/0.075 mm Hg.

40-50 °C 2 Bu3SnH + (Bu3Sn)2O Bu3SnSnBu3 + H2O vacumm

Synthesis of bis(tributyl)ditin: Bu3SnH (39.6 g, 136 mmol) and (Bu3Sn)2O (40.55 g, 68 mmol) were placed in a RBF and warmed to 40 °C for 40 min. Then a Vigreux column was attached to the flask and the top of this was connected to an efficient vacuum pump. The mixture was heated under vacuum (0.05 mm Hg) at 40 °C for 10 h. Then, the crude product was distilled under

26

vacuum. The colorless fraction at 148-149 °C / 0.05 mm Hg contained the pure product (74.8 g, 95% yield).

O 1. Bu3SnLi, THF 2. Me S.CuBr 2 Bu3Sn H OEt 3. EtOH, -78 °C CO Et C4H9 2 97% 20a 19

Synthesis of ethyl (E)-3-tributylstannyl-2-octenoate (20a): To a solution of diisopropylamine (1.0 mL, 7.1 mmol) in dry THF (10 mL) at 0 °C was slowly added nBuLi (1.42 M in hexanes, 4.3 mL, 6.1 mmol). After 20 min, tributyltin hydride (Bu3SnH, 1.63 mL, 6.1 mmol) was added and the solution was stirred for 20 min. The pale yellow resultant was cooled to –78 °C and . Me2S CuBr complex (1.29 g, 6.3 mmol) was added. The solution turned dark yellow. After 30 min at –78 °C, the mixture became black then ethyl ester (0.75 g, 4.5 mmol) was added and the solution was stirred for 1.5 h. The reaction was quenched slowly with EtOH (1.0 mL, 17.4 mmol) at –78 °C, and the mixture was allowed to warm to rt within 3 h. Brine (30 mL) was then added to the mixture and a dark precipitate formed. The mixture was vacuum filtered through Celite to remove the solids and the filtrate was transferred into separatory funnel. The layers were separated and the aqueous layer was extracted with ether (2 × 15 mL). The combined organic phases were washed with brine and dried over MgSO4. After the solvent was removed with a rotary evaporator, the crude product was purified by flash column chromatography (50:1 1 hexanes/EtOAc) to afford a colorless oil 20a (2.03 g, 99% yield). H NMR (CDCl3, 300 MHz) δ

5.90 (t, J = 1.1 Hz, JSn-H = 64.4 Hz, 1H), 4.15 (q, J = 7.2 Hz, 2H), 2.83 (t, J = 6.6 Hz, JSn-H = 51.2 13 Hz, 2H), 1.47 (m, 8H), 1.29, (m, 18 H), 0.9 (m, 12H); C NMR (CDCl3, 75 MHz) δ 174.46 (JSn-

C = 429.6 Hz), 164.70, 128.03, 59.94, 35.71, 32.38, 29.78, 29.38, 27.75, 22.95, 14.73, 14.42,

14.02, 10.39 (JSn-C = 322.2 Hz); IR (neat film) νmax 2927, 2872, 1717, 1593, 1463, 1165, 1043 cm-1.

O (Bu3Sn)2CuLiCN (1.3 eq), Bu3Sn D OMe C4H9 THF, -10 °C, MeOD, 12 h CO2Me 97% 20b 19

27

Synthesis of ethyl (E)-3-tributylstannyl-2-octenonate-2-d (20b): To a solution of (Bu3Sn)2 (5.5 g, 9.5 mmol) in dried THF (8.0 mL) was added nBuLi (1.28 M in hexanes, 7.4 mL, 9.5 mmol) slowly at –78 °C. After 30 min the stannyllithium solution was transferred into a suspension of CuCN (0.45 g, 5.0 mmol) in dry THF (4.0 mL) through cannula. The mixture was warmed to –40 °C for 15 min to give a red orange solution, which was then cooled to –78 °C. MeOD (6.0 mL) was added providing a red solution. After 15 min, methyl ester 19 (0.46 g, 2.7 mmol) was added and the reaction mixture was warmed up to –40 °C for 20 min before the temperature was raised to –10 to –20 °C for 6 h. The reaction was quenched with brine (25 mL) at 0 °C, the layers were separated, and the aqueous layer was washed with ether (5 × 20 mL).

The combined organic layers were washed with water and brine. After drying over MgSO4 and filtering, the solution was concentrated under vacuum to give crude product. Purification by flash column chromatography (50:1 hexanes/EtOAc) gave pure 20b as colorless oil (1.23 g, 99% 1 yield). TLC (50:1 hexanes/EtOAc) Rf 0.30; H NMR (CDCl3, 300 MHz) δ 3.67 (s, 3H), 2.84, (t, 13 J = 7.1 Hz, JSn-H = 55.5 Hz, 2H), 1.47, (m, 8H), 1.31 (m, 18H), 0.86 (m, 12H); C NMR (CDCl3,

75 MHz) δ 175.11 (JSn-C = 430.5 Hz), 164.96, 127.43 (t, JD-C = 26.6 Hz), 51.12, 35.65, 32.36,

30.05, 29.37, 27.74 (JSn-C = 71.7 Hz), 23.04, 14.49, 14.01, 10.40 (JSn-C = 358.5 Hz); IR (neat film) -1 + νmax 2926, 2872, 1721, 1463, 1220, 1085 cm ; HRMS (Electrospray) m/z [M + Na ] calcd for + C21H43DO2SnNa 470.2161, found 470.2171.

Bu3Sn H 1. DIBAL-H (2.5 eq), THF Bu3Sn H

CO2Et 2. MeOH CH2OH 20a 99% 21a

Synthesis of (E)-3-tributylstannanyl-2-octen-1-ol (21a): To a solution of ethyl ester 20a (1.7 g, 3.7 mmol) in dry THF (40 mL) at –78 °C was added DIBAL-H (1.5 M in toluene, 6.0 mL, 9.0 mmol). The reaction was stirred for 2 h then warmed to rt and MeOH (5.0 mL) was added to quench the reaction. After 15 min, brine (20 mL) was added resulting a gel-like mixture. The gel-like material was filtrated through Celite and washed with ether. The layers were separated and the aqueous layer was extracted with ether (2 × 20 mL). Combined organic layers were

washed with water and brine and finally dried over MgSO4. After filtration the solvent was evaporated with a rotary evaporator to give crude product. Purification by flash column

28

chromatography (20:1, hexanes/EtOAc) yielded colorless oil 21a (1.54 g, 99% yield). 1H NMR

(CDCl3, 300 MHz) δ 5.71 (t, J = 8.9 Hz, JSn-H = 68.5 Hz, 1H), 4.23 (d, J = 4.8 Hz, 2H), 2.24 (t, J 13 = 6.0 Hz, JSn-H = 51.3 Hz, 2H), 1.46 (m, 8H), 1.28 (m, 18H), 0.85 (m, 12H); C NMR (CDCl3,

75 MHz) δ 149.05 (JSn-C = 394.4 Hz), 139.29, 59.41, 33.92, 32.15, 30.46, 29.51, 27.81 (JSn-C =

71.6 Hz), 22.96, 14.42, 14.05, 10.10 (JSn-C = 358.5 Hz); IR (neat film) νmax 3300, 2925, 2854, 1463, 1020, 865 cm-1.64b

Bu3Sn D 1. DIBAL-H (2.5 eq), THF Bu3Sn D

CO2Me 2. MeOH CH2OH 20b 98% 21b

Synthesis of (E)-3-tributylstannanyl-2-octen-1-ol-2-d (21b): Following a similar procedure described above for 21a, deuterated allylic alcohol 21b was obtained in 98% yield. 1H NMR

(CDCl3, 300 MHz) δ 4.20 (t, J = 4.5 Hz, 2H), 2.24 (t, J = 7.5 Hz, JSn-H = 49.5 Hz, 2H), 1.38 (m, 13 8H), 1.28 (m, 18H), 0.86 (m, 12H); C NMR (CDCl3, 75 MHz) δ 148.92 (JSn-C = 430.2 Hz),

138.89 (t, JD-C = 28.5 Hz), 59.33, 33.88, 32.15, 30.46, 29.51, 27.81 (JSn-C = 71.6 Hz), 22.96,

14.42, 14.05, 10.09 (JSn-C = 358.5 Hz); IR (neat film) νmax 3300, 2925, 2854, 1463, 1376, 1012 -1 + + cm ; HRMS (Electrospray) m/z [M + Na ] calcd for C20H41DOSnNa 442.2113, found 442.2110.

Bu3Sn H NCS, Me2S, CH2Cl2 Bu3Sn H

CH2OH 0 °C CH2Cl 99% 21a 13a

Synthesis of (E)-1-chloro-3-tributylstannyl-2-octene (13a): To a solution of N- chlorosuccinimide (1.77 g, 13.3 mmol) in methylene chloride (50 mL) at –15 °C was added dimethyl sulfide (1.1 mL, 14.4 mmol). The resulting white slurry was stirred at –15 °C for 20 min. Allylic alcohol 21a (4.62 g, 11.1 mmol) was added as a solution in methylene chloride (10 mL). The white slurry turned pale yellow. After stirring at 0 °C for 1.5 h, the reaction mixture was poured into cold (0 °C) brine (50 mL) and was stirred for 5 min. The layers were separated, and the aqueous layer was extracted with ether (3 × 50 mL). Combined organic phases were

dried over MgSO4 and filtered, then the solvent was removed with a rotary evaporator. Crude

29

product was purified by flash column chromatography (hexanes) to give 13a as pale yellow oil 1 (4.7 g, 97% yield). H NMR (CDCl3, 300 MHz) δ 5.71 (t, J = 7.2 Hz, JSn-H = 68.8 Hz, 1H), 4.09

(d, J = 7.2 Hz, 2H), 2.28 (t, J = 6.9 Hz, JSn-H = 50.5 Hz, 2H), 1.46 (m, 6H), 1.30 (m, 12H), 0.89 13 (m, 18H); C NMR (CDCl3, 75 MHz) δ 152.52 (JSn-C = 430.1 Hz), 134.89, 39.73, 33.40, 32.16,

30.20, 29.44, 27.77 (JSn-C = 71.6 Hz), 22.95, 14.41, 14.05, 10.18 (JSn-C = 358.6 Hz); IR (neat film) -1 64b νmax 2926, 2872, 1464, 689 cm .

Bu3Sn D NCS, Me2S, CH2Cl2 Bu3Sn D

CH2OH 0 °C CH2Cl 99% 21b 13b

Synthesis of (E)-1-chloro-3-tributylstannyl-2-octene-2-d (13b): Following the similar procedure described above, deuterated allylic chloride 13b was obtained in 99% yield. 1H NMR

(CDCl3, 300 MHz) δ 4.09 (t, J = 2 Hz, 2H), 2.29 (t, J = 7.8 Hz, JSn-H = 52.5 Hz, 2H), 1.46 (m, 13 6H), 1.28 (br, 12H), 0.86 (m, 18H); C NMR (CDCl3, 75 MHz) δ 152.32 (JSn-C = 430.6 Hz),

135.53 (t, JC-D = 27.0 Hz), 39.73, 33.36, 32.16, 30.20, 29.44, 27.77 (JSn-C = 71.6 Hz), 22.96,

14.41, 14.05, 10.16 (JSn-C = 358.6 Hz); IR (neat film) νmax 2957, 2926, 2855, 1464, 1377, 740 cm-1.

1, i-PrMgCl (2.0 eq) 2. TMSCl (2.0 eq) Br OH 3. HCl (3 M) TMS 4. Ph3P, Br2, CH2Cl2 22 23 93%

Synthesis of (4-bromo-1-butynyl)trimethylsilane (23): In a 250 mL RBF, Mg (4.9 g, 202 mmol), THF (10 mL) and a stir bar were placed. The first part of isopropyl chloride (1.7 g) was added, and stirrer was turned on. After the reaction was initiated, the remaining isopropyl chloride (14.2 g, 202 mmol total) was added as a solution in THF (80 mL). After addition, the gray suspension was stirred at rt for 1.5 h. To a suspension of 3-butyn-1-ol 22 (7.0 g, 100 mmol) in THF (20 mL) at 0 °C was added i-PrMgCl (prepared above) through cannula. The reaction was stirred at 0 °C for 1 h then warmed to rt for 1 h. Finally, TMSCl (22.1 g, 203 mmol) was

30

added. The obtained gray suspension was stirred at 0 °C for 1 h and warmed to rt overnight. The reaction was quenched by pouring into HCl (3 M, 800 mL) and stirred at rt for 3 h. Diethyl ether

(100 mL) was added and the layers were separated. The aqueous layer was extracted with Et2O (3 × 100 mL) and the combined organic phases were washed with HCl (3 M, 2 × 100 mL) and

brine (100 mL). After drying over MgSO4 and filtering, the solution was concentrated under vacuum to give yellow oil (14.1 g).

To a stirred solution of Ph3P (39.3 g, 150 mmol) in CH2Cl2 (200 mL) at 0 °C was added Br2 (7.7 mL, 149 mmol) slowly. The mixture was stirred for 1 h and crude alcohol (14.1 g, 100 mmol, obtained above) was added slowly, followed by addition of pyridine (12 mL, 149 mmol). After 1 h, the reaction mixture was warmed to rt for 5 h. Then the mixture was filtered through Celite, and the solid was washed with n-pentane (300 mL). After concentrating with a rotary evaporator, precipitated solids were removed by filtration and washed with n-pentane. This step was repeated until no more solids formed during concentration. The crude product was purified through a silica-gel pad (n-pentane) to yield colorless oil 23 (18.8 g, 93% yield, for two steps). 1H NMR 13 (CDCl3, 300 MHz) δ 3.40 (t, J = 7.5 Hz, 2H), 2.75 (t, J = 7.5 Hz, 2H), 0.13 (s, 9H); C NMR

(CDCl3, 75 MHz) δ 103.58, 87.40, 29.56, 24.69, 0.34; IR (neat film) νmax 2960, 2899, 2178, 1251, 1212, 844, 761, 681 cm-1.76

Br TMS CuCN.2LiCl TMS + C3D7MgBr C3D7 THF, 0 °C to rt 24 23 89%

Synthesis of 1-heptynyl-trimethylsilane-5,5,6,6,7,7,7-d7 (24): Magnesium (0.13 g, 5.4 mmol)

and dry THF (4.0 mL) were placed in an oven-dried RBF, n-propylbrimide-d7 (0.7 g, 5.4 mmol) was introduced slowly. After 1/3 of the bromide was added, the stirrer was turned on to initiate the Grignard reaction. The rest bromide was added over 20 min. The gray solution obtained was stirred at rt for 1 h. CuCN (0.024 g, 0.27 mmol), LiCl (0.022 g, 0.51 mmol, dried at 100 °C for 1 hr before use) and THF (5.0 mL) were placed in another RBF and stirring commenced. After few minutes at rt, all solid were dissolved to give a pale green solution. Bromide 23 (0.84 g, 4.1 mmol) was added to this green solution and the mixture was cooled to 0 °C. Grignard reagent

31

(prepared above) was added slowly (syringe pump addition at 0.2 mL/min) then the mixture

(dark or black) was warmed to rt for 3 days. The reaction was quenched with saturated NH4Cl solution (30 mL). The layers were separated and the aqueous layer was extracted with n-pentane (2 × 15 mL). The combined organic phases were washed with brine (40 mL) and dried over

MgSO4. After filtration the solvent was removed under vacuum. The crude product was purified on flash column chromatography (n-pentane) to yield pure compound 24 (0.49 g, 71% yield). 1H 13 NMR (CDCl3, 300 MHz) δ 2.18 (t, J = 7.5 Hz, 2H), 1.48 (t, J = 7.2 Hz, 2H), 0.12 (s, 9H); C

NMR (CDCl3, 75 MHz) δ 108.22, 84.58, 30.82 (m), 28.52, 23.04 (m), 20.19, 13.34 (m), 0.57; IR -1 (neat film) νmax 2961, 2217, 2176, 1250, 1056, 842, 760, 641 cm .

1. Bu3SnLi . 2. Me2S CuBr CO2Et Bu Sn H TMS 1. KF, DMF 3. EtOH, -78 °C 3 C D C3D7 CO2Et C3D7 2. BuLi 3 7 52% from 24 24 3. ClCO2Et 25 26

Synthesis of ethyl (E)-3-tributylstannyl-2-octenonate-6,6,7,7,8,8,8-d7 (26): Compound 24 (0.30 g, 1.7 mmol), KF dihydrate (0.25 g, 2.7 mmol) and DMF (2.0 mL) were placed in a plastic vial. After the mixture was stirred at 40 °C overnight, water (3.0 mL) was added followed by n- pentane (2.0 mL). The layers were separated and the aqueous layer was extracted with n-pentane (2 × 2 mL). Combined organic phases were washed with brine (2.0 mL) and dried over

anhydrous Na2SO4. After filtration through glass wool, the filtrate was cooled down to –78 °C and THF (5.0 mL) was added. nBuLi (1.24 M in hexanes, 1.38 mL, 1.71 mmol) was introduced through syringe. After 30 min at –78 °C, ethyl chloroformate (0.2 g, 1.84 mmol) was added and the reaction mixture was warmed up to 10 °C for 1.5 h. The reaction was quenched with saturated NH4Cl solution (30 mL). After general work-up procedure, crude product was obtained as pale yellow oil. Purification through flash chromatography (20:1, hexanes/EtOAc) yielded colorless oil (0.24 g) containing desired product 25.

To a solution of diisopropylamine (0.3 mL, 2.14 mmol) in dry THF (4.0 mL) at 0 °C was added nBuLi (1.24 M in hexanes, 1.4 mL, 1.74 mmol) slowly. After 20 min, tributyltin hydride

(Bu3SnH, 0.46 mL, 1.73 mmol) was added. Twenty min later, the pale yellow solution obtained

32

. was cooled to –78 °C, and Me2S CuBr complex (0.37 g, 1.79 mmol) was added affording a dark yellow solution. After 30 min at –78 °C, the mixture turned black, and ethyl ester 25 (mixture obtained above, 0.24 g, ca. 1.4 mmol) was added and the mixture was stirred for 1.5 h. Finally, the reaction was quenched with EtOH (0.5 mL, 8.7 mmol) at –78 °C. Then the mixture was warmed to rt over 3 h. Brine (10 mL) was added to the mixture forming some black precipitates. After filtering through Celite, the filtrate was transferred into a separatory funnel. The layers were separated and the aqueous layer was extracted with ether (2 × 10 mL). Combined organic

phases were washed with brine and dried over MgSO4. After filtering, the solvent was evaporated with a rotary evaporator. The crude product was purified through flash column chromatography (50:1, hexanes/EtOAc) to afford pure 26 (0.37 g, 52% yield, overall yield for 1 three steps from 24). H NMR (CDCl3, 200 MHz) δ 5.89 (t, J = 1 Hz, JSn-H = 66.8 Hz, 1H), 4.13 (q, J = 7.2 Hz, 2H), 2.82 (tt, J = 28.4, 8.2 Hz, 2H), 1.51 (m, 6H), 1.27 (m, 11H), 0.87 (m, 15H); 13 C NMR (CDCl3, 50 MHz) δ 174.31, 146.38, 127.61, 59.63, 32.61, 30.70, 29.05, 27.43, 24.32

(m), 22.72, 20.71 (m), 14.40, 13.72 (m), 10.02; IR (neat film) νmax 3009, 2957, 2927, 2854, 1717, -1 + + 1594, 1464, 1164, 870 cm ; HRMS (Electrospray) m/z [M + Na ] calcd for C22H37D7O2SnNa 490.2688, found 490.2692.

Bu Sn H NCS, Me S Bu Sn H Bu3Sn H DIBAL-H 3 2 3 C D CH OH 74% C D CH Cl C3D7 CO2Et 82% 3 7 2 3 7 2 26 27 13c

Synthesis of (E)-3-tributylstannyl-2-octen-1-ol-6,6,7,7,8,8,8-d7 (27): Following the same procedure for making 21a, allylic alcohol 27 was prepared from ethyl ester 26 in 82% yield, with 1 1 high deuterium incorporation (>98% by H NMR and HRMS). H NMR (CDCl3, 300 MHz) δ 5.70 (tt, J = 32.0, 7.1 Hz, 1H), 4.21 (t, J = 6.0 Hz, 2H), 2.23 (tt, J = 24.5, 7.1 Hz, 2H), 1.46 (m, 13 7H), 1.27 (m, 8H), 0.86 (m, 15H); C NMR (CDCl3, 75 MHz) δ 149.13, 139.25, 59.43, 33.89,

30.24, 31.10 (m), 29.51, 27.82, 21.79 (m), 14.06, 13.23 (m), 10.10; IR (neat film) νmax 3306, 2957, 2924, 2854, 2216, 1464, 1018, 663 cm-1. HRMS (Electrospray) m/z [M + Na+] calcd for + C20H35D7OSnNa 448.2582, found 448.2605.

33

Synthesis of (E)-1-chloro-3-tributylstannyl-2-octene-6,6,7,7,8,8,8-d7 (13c): Fragment 13c was prepared readily by following the same procedure for making allylic chloride 13a. Compound 13c was obtained in 74% yield, with high deuterium incorporation (>98% by 1H NMR, and 1 HRMS). H NMR (CDCl3, 300 MHz) δ 5.71 (tt, J = 31.0, 7.2 Hz, 1H), 4.08 (dt, J = 10.8, 4.8 Hz, 2H), 2.28 (tt, J = 25.4, 7.8 Hz, 2H), 1.46 (m, 6H), 1.30 (m, 8H), 0.89 (m, 15H); 13C NMR

(CDCl3, 75 MHz) δ 152.55, 134.87, 39.73, 33.36, 30.86 (m), 29.98, 29.44, 27.77, 22.84 (m), -1 18.05 (m), 14.05, 10.17; IR (neat film) νmax 3009, 2957, 2925, 2854, 2216, 1464, 1247, 668 cm ; + HRMS (EI) m/z [M ] calcd for C20H24D7ClSn 443.2346, found 443.2344.

Bu Sn R Bu3Sn R 3 OTBDMS Pd(0) + OTBDMS I R' R' CH2Cl 8 8

13a: R = H, R' = C3H7 Fragment 14 28a: R = H, R' = C3H7 13b: R = D, R' = C3H7 28b: R = D, R' = C3H7 13c: R = H, R' = C3D7 28c: R = H, R' = C3D7

"R R Dowex OH R' 8

29a: R = H, R" = D, R' = C3H7 29b: R = D, R" = H, R' = C3H7 29c: R = H, R" = H, R' = C3D7 44% for two steps

Synthesis of linoleoyl alcohols: To a solution of vinyl iodide (0.65 g, 1.6 mmol) in dry ether (4.0 mL), was added tBuLi (1.6 M in hexanes, 2.1 mL, 3.4 mmol) at -78 °C slowly. White precipitates formed and the mixture was stirred at -78 °C for 30 min. Zinc bromide was added as a solution in ether (59% w/w, 0.62 g, 1.6 mmol), then the reaction mixture was warmed up to 0 °C. The white precipitate that was initially formed dissolved and a pale yellow solution was

obtained. Allylic chloride 13 (0.69 g, 1.6 mmol), Pd(PPh3)4 (0.18 g, 0.16 mmol), tri-o- tolylphosphine (0.096 g, 0.3 mmol) and Et2O (5.0 mL) were pre-mixed in another RBF for 5 min then the flask was cooled down to 0 °C. The vinylzinc bromide solution was transferred to this mixture at 0 °C through a cannula. The reaction mixture was warmed to rt slowly and stirred

overnight. The reaction was quenched by pouring the mixture into saturated NH4Cl solution (15 mL). The layers were separated and the aqueous layer was extracted with ether (3 × 10 mL). The

combined organic layers were washed with water and brine. After drying over MgSO4, the

34

solution was concentrated under vacuum to give the crude product. The crude product was passed through a silica-gel pad (n-pentane) to remove polar impurities. The solvent was evaporated to give partially purified product 28 (including the desired product and a terminal olefin derived from 14), which was directly subjected to next reaction.

Dowex-50W (1.0 g) was vortexed with D2O three times (2.0 mL) for 5 min each, followed by two treatments with MeOD (1.5 mL). The deuterated Dowex-50W was placed in a RBF equipped with a magnetic stir bar. The coupling product, obtained above, was added followed by MeOD (2.0 mL) and the mixture was stirred for 12 h at 40 °C. The resin was removed by filtration, and the filtrate was concentrated under vacuum. The crude product was purified by flash chromatography (5:1 hexanes/EtOAc) to yield pure alcohol 29 (0.18 g, 44% yield, overall yield for two steps).

1 (Z,Z)-Octadeca-9,12-dien-1-ol-13-d (29a): H NMR (CDCl3, 300 MHz) δ 5.34 (m, 3H), 3.62 (t, J = 6.6 Hz, 2H), 2.75 (t, J = 6.6 Hz, 2H), 2.02 (m, 4H), 1.54 (q, J = 6.6 Hz, 2H), 1.41 (s, 1H), 13 1.28 (b, 16H), 0.87 (t, J = 6.8 Hz, 3H); C NMR (CDCl3, 75 MHz) δ 130.61, 130.26 (t, J = 19.1 Hz), 128.41, 128.33, 63.46, 33.19, 31.93, 30.05, 29.89, 29.79, 29.73, 29.63, 27.62, 27.24, 26.13, -1 26.00, 22.97, 14.46; IR (neat film) νmax 3339, 3010, 2927, 2855, 1466, 1057, 724 cm . HRMS + + (Electrospray) m/z [M + Na ] calcd for C18H33DONa 290.2560, found 290.2558.

1 (Z,Z)-Octadeca-9,12-dien-1-ol-12-d (29b): H NMR (CDCl3, 300 MHz) δ 5.37, (m, 3H), 3.62 (t, J = 6.6 Hz, 2H), 2.73 (d, J = 7.2 Hz, 2H), 2.01 (m, 4H), 1.54 (q, J = 6.6 Hz, 2H), 1.42 (s, 1H), 13 1.28 (br, 16H), 0.86 (t, 3H); C NMR (CDCl3, 75 MHz) δ 130.51, 130.47, 128.40, 127.99 (t, J = 23.3 Hz), 63.46, 33.19, 31.93, 30.05, 29.89, 29.79, 29.75, 29.64, 27.62, 26.13, 26.04, 25.93, -1 22.97, 14.46; IR (neat film) νmax 3337, 3010, 2927, 2855, 1466, 1055, 724 cm ; HRMS + + (Electrospray) m/z [M + Na ] calcd for C18H33DONa 290.2560, found 290.2577.

1 (Z,Z)-Octadeca-9,12-dien-1-ol-16,16,17,17,18,18,18-d7 (29c): H NMR (CDCl3, 500 MHz) δ 5.32, (m, 4H), 3.62 (t, J = 6.5 Hz, 2H), 2.75 (d, J = 7.2 Hz, 2H), 2.02 (m, 4H), 1.56 (m, 4H), 1.42 13 (s, 1H), 1.29 (br, 10H); C NMR (CDCl3, 125 MHz) δ 130.25, 130.13, 128.02, 127.92, 63.10,

35

32.81, 29.66, 29.51, 29.41, 29.25, 29.13, 27.23, 27.18, 25.74, 25.64, 24.73 (m), 22.69 (m), 14.64 -1 (m); IR (neat film) νmax 3336, 3009, 2927, 2854, 1466, 1060, 724 cm ;

"R R "R R PDC OH CO2H R' 8 R' 8 DMF/H2O 29a: R = H, R" = D, R' = C H 3 7 11a: R = H, R" = D, R' = C3H7 29b: R = D, R" = H, R' = C H 3 7 11b: R = D, R" = H, R' = C3H7 29c: R = H, R" = H, R' = C D 3 7 12: R = H, R" = H, R' = C3D7

Synthesis of deuterolinoleic acids: To a stirred mixture of [13-2H]linoleic alcohol 29a (0.11 g,

0.4 mmol) and DMF (5 mL) was added H2O (0.25 mL) followed by PDC (0.82 g, 2.2 mmol). The mixture was stirred in dark for 10 h at rt. Silica gel (1.0 g) was added to the mixture and the solid was removed by vacuum filtration through Celite. The solid was washed with ether (25 mL), combined filtrates were washed with H2O (2 × 25 mL), and the organic phases were dried over MgSO4. After filtration, the solution was concentrated under vacuum to afford crude product. Purification by flash column chromatography (3:1, hexanes/Et2O, with 0.5% of acetic 2 1 acid) yielded pure [13- H]linoleic acid 11a (0.08 g, 70% yield). H NMR (CDCl3, 300 MHz) δ 5.34 (m, 3H), 2.75 (t, J = 6.6 Hz, 2.0 H), 2.33 (t, J = 7.5 Hz, 2H), 2.02 (m, 4H), 1.61 (quint, J = 13 7.3 Hz, 4H), 1.27 (br, 10H), 0.86 (m, 3H); C NMR (CDCl3, 75 MHz) δ 179.92, 130.48 (t, JC-D = 27.3 Hz), 130.42, 128.48, 128.17, 34.19, 31.93, 29.97, 29.73, 29.53, 29.47, 29.42, 27.58, 27.50,

26.00, 25.06, 22.96, 14.45; IR (neat film) νmax 3383 (br), 3009, 2928, 2856, 1710, 1285, 933, 725 -1 + cm ; HRMS (EI) m/z [M ] calcd for C18H31DO2 281.2459, found 281.2473.

2 1 [12- H]Linoleic acid (11b): H NMR (CDCl3, 300 MHz) δ 5.34 (m, 3H), 2.74 (d, J = 6.0 Hz, 2H), 2.33 (t, J = 7.5 Hz, 2H), 2.02 (q, J = 7.0 Hz, 4H), 1.61 (quint, J = 7.5 Hz, 4H), 1.28 (br, 13 10H), 0.86 (m, 3H); C NMR (CDCl3, 75 MHz) δ 179.68, 130.48, 130.42, 128.46, 128.07 (t, JC-

D = 25.3 Hz), 34.29, 31.93, 29.98, 29.75, 29.63, 29.53, 29.47, 29.42, 27.58, 25.93, 25.06, 22.97, -1 14.46; IR (neat film) νmax 3404 (br), 3009, 2927, 2855, 1709, 1384, 933, 725 cm ; HRMS (EI) + m/z [M ] calcd for C18H31DO2 281.2459, found 281.2479.

2 1 [16,16,17,17,18,18,18- H7]Linoleic acid (12): H NMR (CDCl3, 500 MHz) δ 5.32 (m, 4H), 2.75 (d, J = 6.0 Hz, 2H), 2.33 (t, J = 7.5 Hz, 2H), 2.02 (q, J = 6.0 Hz, 4H), 1.61 (quint, J = 6.0 Hz,

36

13 4H), 1.31 (br, 8H); C NMR (CDCl3, 125 MHz) δ 179.66, 130.24, 130.04, 128.08, 127.91, 33.97, 31.54, 29.59, 29.36, 29.15, 29.08, 29.04, 27.22, 27.19, 25.64, 24.67 (m), 22.59 (m), 14.08 -1 (m); IR (neat film) νmax 3400 (br), 3010, 2925, 2853, 1710, 1384, 933, 726 cm .

37

References

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core of neocarzinostatin chromophore” J. Am. Chem. Soc. 1991, 113, 694-695. 10. Myers, A. G.; Alauddin, M. M.; Fuhry, M. A. M.; Dragovich, P. S.; Finney, N. S.; Harrington, P. M. “Versatile precursors for the synthesis of enynes and enediynes” Tetrahedron Lett. 1989, 30, 6997-7000. 11. a) Nicolaou, K. C.; Groneberg, R. D.; Miyazaki, T.; Stylianides, N. A.; Schulze, T. J.; Stahl, I W. “Total synthesis of the oligosaccharide fragment of calicheamicin γ1 ” J. Am. Chem. Soc. 1990, 112, 8193-8195. b) Halcomb, R. L.; Wittman, M. D.; Olson, S. H.; Danishefsky, S. J.; Golik, J.; Wong, H.; Vyas, D. “The synthesis of the core trisaccharide of esperamicin: corroboration of the proposed structure for its rearrangement product and stabilization of the core trisaccharide domain” J. Am. Chem. Soc. 1991, 113, 5080-5082. c) Wittman, M. D.; Halcomb, R. L.; Danishefsky, S. J.; Golik, J.; Vyas, D. “A route to glycals in the allal and gulal series: synthesis of the thiosugar of esperamicin A1” J. Org. Chem. 1990, 55, 1979-1981. d) Nicolaou, K. C.; Smith, A. L. “Molecular design, chemical synthesis, and biological action of enediynes” Acc. Chem. Res. 1992, 25, 497-503. e) Clive, D. L. J.; Bo, Y.; Tao, Y.; Daigneault, S.; Wu, Y.-J.; Meignan, G. “Synthesis of (±)- calicheamicinone by two methods” J. Am. Chem. Soc. 1998, 120, 10332-10349. 12. Groneberg, R. D.; Miyazaki, T.; Stylianides, N. A.; Schulze, T. J.; Stahl, W.; Schreiner, E. P.; Suzuki, T.; Iwabuchi, Y.; Smith, A. L.; Nicolaou, K. C. “Total synthesis of calicheamicin I γ1 1. Synthesis of the oligosaccharide fragment” J. Am. Chem. Soc. 1993, 115, 7593-7611. 13. Nicolaou, K. C.; Groneberg, R. D. “Novel strategy for the construction of the I oligosaccharide fragment of calichemicin γ1 . Synthesis of the ABC skeleton” J. Am. Chem. Soc. 1990, 112, 4085-4086. 14. Nicolaou, K. C.; Groneberg, R. D.; Stylianides, N. A.; Miyazaki, T. “Synthesis of the C, D I and E ring systems of the calicheamicin γ1 oligosaccharide” J. Chem. Soc. Chem. Commun. 1990, 18, 1275-1277. 15. Nicolaou, K. C.; Ebata, T.; Stylianides, N. A.; Groneberg, R. D.; Carrol, P. J. “4-Hydroxy-5- iodo-2,3-dimethoxy-6-methylbenzoic acid methyl ester. The aromatic portion of I calichemicin-γ1 . Synthesis, x-ray structure analysis, and properties” Angew. Chem. Int. Ed. Engl. 1988, 271, 1097-1099.

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31. Boger, D. L.; Zhou, J. “CDPI3-enediyne and CDPI3-EDTA conjugates: a new class of DNA cleaving agents” J. Org. Chem. 1993, 58, 3018-3024. 32. Maeda, H. “Neocarzinostatin in cancer chemotherapy (review)” Anticancer Res. 1981, 1, 175-186. 33. Schmitt, D. A.; Kisanuki, K.; Kimura, S.; Oka, K.; Pollard, R. B.; Maeda, H.; Suzuki, F. “Antitumor activity of orally administered SMANCS, a polymer-conjugated protein drug, in mice bearing various murine tumors” Anticancer Res. 1992, 12, 2219-2224. 34. Arnaud, A. Bull. Soc. Chim. France 1892, 3, 233. 35. Arnaud, A. Bull. Soc. Chim. France 1902, 3, 489. 36. a) Willams, W. W.; Smirnow, W. S.; Golmow, W. P. Zhur. Obschei. Khimij 1935, 5, 1195.

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b) Willams, W. W.; Smirnow, W. S.; Golmow, W. P. Chem. Zbl. 1936, I, 3347. 37. Cahoon, E. B.; Schnurr, J. A.; Huffman, E. A.; Minto, R. E. “Fungal responsive fatty acid acetylenases occur widely in evolutionarily distant plant families” Plant J. 2003, 34, 671- 683. 38. Bohlmann, F.; Burkhardt, T.; Zdero, C. “Naturally Occurring Acetylenes” 1973, Academic Press, New York. 39. Saita, T.; Matsunaga, H.; Yamamoto, H.; Nagumo, F.; Fujito, H.; Mori, M.; Katano, M. “A highly sensitive enzyme-linked immunosorbent assay (ELISA) for antitumor polyacetylenic alcohol, panaxytriol” Biol. Pharm. Bull. 1994, 17, 798-802. 40. Kitagawa, I.; Yoshikawa, M.; Yoshihara, M.; Hayashi, T.; Taniyama, T. “Chemical studies of crude drugs (1). Constituents of Ginseng radix rubra” Yakugaku Zasshi. J. Pharm. Soc. Jap. 1983, 103, 612-622. 41. Kitagawa, I.; Taniyama, T.; Shibuya, H.; Noda, T.; Yoshikawa, M. “Chemical studies on crude drug processing. V. On the constituents of ginseng radix rubra (2): comparison of the constituents of white ginseng and red ginseng prepared from the same panax ginseng root” Yakugaku Zasshi. J. Pharm. Soc. Jap. 1987, 107, 495-505. 42. Katano, M.; Yamamoto, H.; Matsunaga, H.; Mori, M.; Takata, K.; Nakamura, M. “Cell growth inhibitory substance isolated from panax ginseng root: panaxytriol” Gan To Kagaku Ryoho 1990, 17, 1045-1049. 43. Matsunaga, H.; Saita, T.; Nagumo, F.; Mori, M.; Katano, M. “A possible mechanism for the cytotoxicity of a polyacetylenic alcohol, panaxytriol: inhibition of mitochondrial respiration” Cancer Chemother. Pharmacol. 1995, 35, 291-296. 44. Matsunaga, H.; Katano, M.; Saita, T.; Yamamoto, H.; Mori, M. “Potentiation of cytotoxicity of mitomycin C by a polyacetylenic alcohol, panaxytriol” Cancer Chemother. Pharmaco. 1994, 33, 291-297. 45. Ito, A.; Cui, B.; Chavez, D.; Chai, H.-B.; Shin, Y. G.; Kawanishi, K.; Kardono, L. B. S.; Riswan, S.; Farnsworth, N. R.; Cordell, G. A.; Pezzuto, J. M.; Kinghorn, A. D. “Cytotoxic polyacetylenes from the twigs of Ochanostachys amentacea” J. Nat. Prod. 2001, 64, 246- 248. 46. Wittstock, U.; Hadacek, F.; Wurz, G.; Teuscher, E.; Greger, H. “Polyacetylenes from water hemlock, Cicuta virosa” Planta Med. 1995, 61, 439-445.

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47. Garrod, B.; Lewis, B. G. “Location of the antifungal compound falcarindiol in carrot root tissue” Trans. Br. Mycol. Soc. 1979, 72, 515-517. 48. Villegas, M.; Vargas, D.; Msonthi, J. D.; Marston, A.; Hostettmann, K. “Isolation of the antifungal compounds falcarindiol and sarisan from Heteromorpha trifoliata” Planta Med. 1988, 54, 36-37. 49. Olsson, K.; Svensson, R. “The influence of polyacetylenes on the susceptibility of carrots to storage diseases” J. Phytopath. 1996, 144, 441-447. 50. Eckenbach, U.; Lampman, R. L.; Seigler, D. S.; Ebinger, J.; Novak, R. J. “Mosquitocidal activity of acetylenic compounds from Cryptotaenia canadensis” J. Chem. Ecol. 1999, 25, 1885-1893. 51. Muir, A. D.; Majak, W. “Quantitative determination of 3-nitropropionic acid and 3- nitropropanol in plasma by HPLC” Toxicol. Lett. 1984, 20, 133-6. 52. Nakano, Y.; Matsunaga, H.; Saita, T.; Mori, M.; Katano, M.; Okabe, H. “Antiproliferative constituents in Umbelliferae plants II. Screening for polyacetylenes in some Umbelliferae plants, and isolation of panaxynol and falcarindiol from the root of Heracleum moellendorffii” Biol. Pharmaceut. Bull. 1998, 21, 257-261. 53. Jacobs, J. J.; Arroo, R. R.; De Koning, E. A.; Klunder, A. J.; Croes, A. F.; Wullems, G. J. “Isolation and characterization of mutants of thiophene synthesis in Tagetes erecta” Plant Physiol. 1995, 107, 807-814. 54. Towers, G. H. N.; Page, J. E.; Hudson, J. B. “Light-mediated biological activities of natural products from plants and fungi” Curr. Org. Chem. 1997, 1, 395-414. 55. a) Battey, J. F.; Schmid, K. M.; Ohlrogge, J. B. “Genetic engineering for plant oils: potential and limitations” Trends Biotech. 1989, 7, 122-126. b) Mazliak, P. “Desaturation processes in fatty acid and acyl lipid biosynthesis” J. Plant Physiol. 1994, 143, 399-406. 56. Lee, M.; Lenman, M.; Banas, A.; Bafor, M.; Singh, S.; Schweizer, M.; Nilsson, R.; Liljenberg, C.; Dahlqvist, A.; Gummeson, P. O.; Sjodahl, S.; Green, A.; Stymne, S. “Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation” Science 1998, 280, 915-918. 57. Kohn, G.; Hartmann, E.; Stymne, S.; Beutelmann, P. “Biosynthesis of acetylenic fatty acids in the moss Ceratodon purpureus” J. Plant Physiol. 1994, 144, 265-271.

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58. Somssich, I. E.; Bollmann, J.; Hahlbrock, K.; Kombrink, E.; Schulz, W. “Differential early activation of defense-related genes in elicitor-treated parsley cells” Plant Mol. Biol. 1989, 12, 227-234. 59. Kirsch, C.; Takamiya-Wik, M.; Schmelzer, E.; Hahlbrock, K.; Somssich, I. E. “A novel regulatory element involved in rapid activation of parsley ELI7 gene family members by fungal elicitor or pathogen infection” Mol. Plant Path. 2000, 1, 243-251. 60. Kirsch, C.; Hahlbrock, K.; Somssich, I. E. “Rapid and transient induction of a parsley microsomal ∆12 fatty acid desaturase mRNA by fungal elicitor” Plant Physiol. 1997, 115, 283-289. 61. a) Kirsch, C.; Takamiya-Wik, M.; Reinold, S.; Hahlbrock, K.; Somssich, I. E. “Rapid, transient, and highly localized induction of plastidial ω-3 fatty acid desaturase mRNA at fungal infection sites in Petroselinum crispum” Proc. Natl. Acad. Sci. USA 1997, 94, 2079- 2084. b) Reymond, P.; Farmer, E. E. “Jasmonate and salicylate as global signals for defense gene expression” Curr. Opin. Plant Biol. 1998, 1, 404-411. 62. Zhu, L.; Minto, R. E. “Improved syntheses of methyl (14E)- and (14Z)-dehydrocrepenynate: key intermediates in plant and fungal polyacetylene biosynthesis” Tetrahedron Lett. 2001, 42, 3803-3805. 63. a) Buist, P. H.; Behrouzian, B. “Use of deuterium kinetic isotope effects to probe the cryptoregiochemistry of ∆9 desaturation” J. Am. Chem. Soc. 1996, 118, 6295-6296. b) Savile, C. K.; Reed, D. W.; Meesapyodsuk, D.; Covello, P. S.; Buist, P. H. “Crytoregiochemisry of a Brassica napus fatty acid desaturase (FAD3): a kinetic isotope effect study” J. Chem. Soc. Perkin Trans. 1 2001, 9, 1116-1121 c) Reed, D. W.; Savile, C. K.; Qiu, X.; Buist, P. H.; Covello, P. S. “Mechanism of 1,4- dehydrogenation catalyzed by a fatty acid (1,4)-desaturase of Calendula officinalis” Eur. J. Biochem. 2002, 269, 5024-5029. 64. a) Hutzinger, M. W.; Oehlschlager, A. C. “Stereoselective synthesis of 1,4-dienes. Application to the preparation of insect pheromones (3Z,6Z)-dodeca-3,6-dien-1-ol and (4E,7Z)-trideca-4,7-dienyl acetate” J. Org. Chem. 1995, 60, 4595-4601. b) Singer, R. D.; Hutzinger, M. W.; Oehlschlager, A. C. “Additions of copper cyanide (CuCN)-derived stannylcuprates to terminal alkynes: a comparative spectroscopic and

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chemical study” J. Org. Chem. 1991, 56, 4933-4938. c) Yoon, N. M.; Park, K. B. Lee, H. J.; Choi, J. “The semihydrogenation of acetylenes over Pd catalyst on BER in the presence of CsI” Tetrahedron Lett. 1996, 37, 8527-8528. d) Schlosser, M. “Stereochemistry of the Wittig reaction” Top. Stereochem. 1970, 5, 1-30. 65. a) Piers, E.; Wong, T.; Ellis, K. A. “Use of lithium (trimethylstannyl)(cyano)cuprate for the conversion of alkyl 2-alkynoates into alkyl (Z)- and (E)-3-(trimethylstannyl)-2-alkenoates” Can. J. Chem. 1992, 70, 2058-2064. b) Nielsen, T. E.; Cubillo de Dios, M. A.; Tanner, D. “Highly stereoselective addition of stannylcuprates to alkynones” J. Org. Chem. 2002, 67, 7309-7313. c) Nielsen, T. E.; Tanner, D. “Stereoselective synthesis of (E)-β-tributylstannyl-α,β- unsaturated ketones: construction of a key intermediate for the total synthesis of zoanthamine” J. Org. Chem. 2002, 67, 6366-6371. d) Piers, E.; McEachern, E. J.; Romero, M. A. “Copper chloride-catalyzed and hydrochloric acid-mediated chemoselective protodestannylations of alkyl (Z)- or (E)-2,3- bis(trimethylstannyl)-2-alkenoates. Stereoselective preparation of alkyl (E)- and (Z)-3- trimethylstannyl-2-alkenoates” J. Org. Chem. 1997, 62, 6034-6040. 66. Ensley, H. E.; Buescher, R. R.; Lee, K. “Reaction of organotin hydrides with acetylenic alcohols” J. Org. Chem. 1982, 47, 404-408. 67. Abrams, S. R.; Shaw, A. C. “Triple-bond isomerizations: 2- to 9-decyn-1-ol” Org. Syn. 1988, 66, 127-131. 68. Kalivretenos, A.; Stille, J. K.; Hegedus, L. S. “Synthesis of β-resorcylic macrolides via organopalladium chemistry. Application to the total synthesis of (S)-zearalenone” J. Org. Chem. 1991, 56, 2883-2894. 69. Neumann, W. P.; Schneider, B. “A simple synthesis of organic di- and polystannanes” Angew. Chem. Int. Ed. Engl. 1964, 3, 751-752. 70. Knochel, P.; Rozema, M. J.; Tucher, C. E. “Preparation of highly functionalized reagents” from “Organocopper Reagents: A Practical Approach” Taylor, R. J. K. Ed.; Oxford University Press, 1994, pp. 85-95. 71. Holm, T.; Crossland, I. “Mechanistic features of the reactions of organomagnesium compounds” from “Grignard Reagents: New Developments” Richey, H. G. Jr. Ed.; Wiley, 2000.

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72. Garst, J. F.; Ungvary, F. “Mechanisms of Grignard reagent formation” from “Grignard Reagents: New Developments” Richey, H. G. Jr. Ed.; Wiley, 2000. 73. Farina, V.; Krishnan, B. “Large rate accelerations in the Stille reaction with tri-2- furylphosphine and triphenylarsine as palladium ligands: mechanistic and synthetic implications” J. Am. Chem. Soc. 1991, 113, 9585-9595. 74. Yadav, J. S.; Deshpande, P. K.; Sharma, G. V. M. “Stereoselective synthesis of (S)-13- hydroxyoctadeca-(9Z,11E)-di- and -(9Z,11E,15Z)-trienoic acids: Self defensive substances against rice blast disease” Tetrahedron 1992, 48, 4465-4474. 75. Makabe, H.; Tanaka, A.; Oritani, T. “Total synthesis of solamin and reticulatacin” J. Chem. Soc., Perkin Trans. 1. 1994, 14, 1975-1981. 76. Negishi, E.; Boardman, L. D.; Sawada, H.; Bagheri, V.; Stoll, A. T.; Tour, J. M.; Rand, C. L. “Metal promoted cyclization. 18. Novel cycloalkylation reactions of (ω-halo-1-alkenyl)metal derivatives. Synthetic scope and mechanism” J. Am. Chem. Soc. 1988, 110, 5383-5396.

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Chapter II. Improved syntheses of methyl (14E)- and (14Z)- dehydrocrepenynate

2-1. Introduction

Secondary metabolism commencing from polyunsaturated fatty acids leads to many biologically active natural products such as the polyacetylenes and the prostaglandins. As shown in Figure 6 (Chapter I), crepenynate and dehydrocrepenynate play important roles in the biosynthesis of polyacetylenic compounds. The formation of crepenynic and dehydrocrepenynic acids constitutes the departure between primary and secondary metabolism for the biosynthesis of many polyacetylenic natural products.1

R' O O R RO "RO 7 7

R = Me, Methyl crepenynate 30 Methyl dehydrocrepenynate R = H, Crepenynic acid 31a: R = H, R' = C3H7, R" = Me 31b: R = C3H7, R' = H, R" = Me Dehydrocrepenynic acid R = R" = H, R = C3H7

Figure 15. Crepenynate (30) and dehydrocrepenynate (31a, 31b) derivatives.

Crepenynic acid is widely produced by plants, particularly those of the families Umbelliferae and Compositae, Basidiomycetes (which include the gilled fungi), and certain bryophytes. It has been reported that crepenynic acid is potentially a toxic constituent in Ixiolaena brevicompta (a plant responsible for causing acute muscular degeneration in sheep in western New South Wales and Queensland).2 Biological activity studies have shown that crepenynic acid has significant inhibitory effects on prostanoid synthesis in sheep and rats at concentrations of 40-45 µM.3-4 In contrast, the biological activity of the dehydrocrepenynates 31a and 31b is essentially unknown.5

Few organisms appear to accumulate substantial amounts of the early acetylenic metabolites 30 and 31b. Dehydrocrepenynic acid was present in trace amounts in the fungus Tricholoma

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grammopodium examined by Bu’Lock and Smith.6 Powell and co-workers subsequently examined Afzelia cuanzensis seed oil as a better source of this acid.7 The common edible fungi Cantharellus cibarius (golden chanterelle) and Craterellus cornucopiodes (horn of plenty) accumulate up to 66% (14Z)-dehydrocrepenynate-containing triacylglycerols in their fruiting bodies.8 Nevertheless, the susceptibility of 31b to decomposition during isolation procedures makes the purification of 31b from natural sources inconvenient. Alternatively, chemical preparation can be used to provide both isomers conveniently in gram scale. Meanwhile, a recent investigation revealed that gene ELI12, only expressed when parsley was challenged by fungal species, could be expressed in transgenic soybean leading to the accumulation of crepenynic acid and dehydrocrepenynic acid to 3% - 4% of total fatty acids in the seeds.9a These isolated fatty acid derivatives were identified through comparison with chemical synthetic compounds reported in this chapter.9b Further investigation also showed that homologs of the ELI12-encoded fatty acid acetylenase are not only expressed in Apiaceae species (parsley), but also in members of Araliaceae and Asteraceae families. However, most plants of these families do not accumulate the crepenynic acid and dehydrocrepenynic acid, which are the direct products from the transgenic expression of these enzymes. It has, nevertheless, been shown that members of Apiaceae, Araliaceae, and Asteraceae families produce linear polyacetylenic compounds, which possess strong biological activities. These compounds include carotatoxin, falcarindiol, and dehydrofalcarinol. Crepenynic and dehydrocrepenynic acids are believed to be key intermediates in the biosynthetic pathway of these polyacetylenes. In addition, current existing syntheses for the 14C-labeled isomers of 31 have been used in experiments to probe the conversion of 31b to more highly unsaturated polyacetylenes in certain Basidiomycetes.10 In the reported example, much lower incorporations of 31a compared to 31b were found, pointing to the (14Z)-compound as the biologically relevant isomer.

Unfortunately, the reported syntheses have several drawbacks including difficult distillations to purify crucial diastereomeric intermediates, low diastereoselectivity and yields, and poor adaptability for other acetylenic regioisomers. To prepare substrates 31a and 31b for studies of fatty acid desaturating enzymes and to examine the microbicidal activity of these isomers, we have developed an improved synthesis of (E)- and (Z)-31.

48

2-2. Results and discussion

The synthesis of methyl (14E)-dehydrocrepenynate 31a began with the iodination of tetrahydro- 2H-pyran-2-yl (THP)-protected 3-butyn-1-ol 32 with n-butyllithium and iodine to afford the iodoalkyne 33 (Figure 16). (E)-Dicyclohexyl-1-pentenylborane 34, formed in situ by adding 1- pentyne to dicyclohexylborane, was cross-coupled with 33 by a Suzuki reaction.11 This Pd- catalyzed coupling reaction stereospecifically produced the (E)-enynyl THP ether 35 in 89% yield. After treatment with triphenylphosphine dibromide, compound 35 was converted directly 12-13 to bromide 36. Heating 36 in a sealed tube with PPh3 and methanol produced the corresponding phosphonium salt 37. Compound 37 smoothly underwent a Wittig reaction with methyl 9-oxononanate 38 in ether at room temperature to give the desired methyl (14E)- dehydrocrepenynate 31a.10

Figure 16. Synthesis of methyl (14E)-dehydrocrepenynate (31a).

OTHP abOTHP c 92% I 89% THPO 60% 32 33 35

de 72% 59% Br BrPh3P 36 37

O O O B MeO 7 MeO 7 H 31a 34 38

n Key. (a) i. BuLi (1.0 eq), Et2O, -78 °C, ii. I2, Et2O; (b) i. Pd(PPh3)4, benzene, ii. 34, iii. EtONa, EtOH, iv. NaOH, H2O2; (c) PPh3, Br2, CH2Cl2, pyridine, 0 °C to rt; (d) PPh3, MeCN, 95 °C; (e) i. n BuLi, Et2O, rt, ii. 38; iii. HCl (1 M).

NaN(SiMe3)2 and NaH were also tested as bases to generate the ylide for this reaction. BuLi, which used in earlier syntheses, gave the best yield after purification (59% isolated yield, 85/15 Z/E by GC), whereas NaH in THF gave the best Z/E ratio (33% isolated yield, 97/3 Z/E by GC).

49

When NaN(SiMe3)2 was used to generate the ylide in ether prior to adding the aldehyde, no product was obtained. However, adding the disilylamide to the mixture of 37 and the aldehyde 38 in THF provided 31b in 30% isolated yield (Z:E > 96:4, by GC).

For the synthesis of methyl (14Z)-dehydrocrepenynate 31b, a Pd-catalyzed Sonagashira coupling reaction was used to introduce the cis-alkene at C14 (Figure 16). Iodination of 1-pentyne (39) gave 1-iodo-1-pentyne 40 in excellent yield and the crude product was directly subjected to a stereospecific hydroboration without any further purification to generate (Z)-1-iodo-1-pentene 41 in 80% yield.14 A Pd-catalyzed cross-coupling reaction furnished the THP-protected (Z)-enynol 42 in 90% yield.15 Under the same conditions that were used for the (E)-isomer, 42 was brominated to form 43 in 86% yield. Bromoenyne 39 was readily converted to the white crystals of phosphonium salt 44. Finally, the synthesis of 31b was completed using a Wittig reaction to connect 44 with aldehyde 38 at room temperature. Good stereoselectivity (95.5/4.5 Z/E by GC) was achieved using BuLi to generate the ylide. When NaH and NaN(SiMe3)2 were used for this reaction in THF, yields dropped to 30% (97/3 Z/E) and 29% (96/4 Z/E), respectively.

Figure 17. Synthesis of methyl (14Z)-dehydrocrepenynate.

ab c I 92% I 80% 90% 39 40 41

d e THPO 86% Br 87% 42 43

f O 59% BrPh3P MeO 7 44 31b

n Key. (a) i. BuLi (1.0 eq), Et2O, -78 °C, ii. I2, Et2O; (b) i. cyclohexene (2.0 eq), BH3 (1.0 eq), n- pentane, -78 °C, ii. AcOH, ethanolamine; (c) i. Pd(PPh3)4, benzene, rt, ii. 32, CuI, Et3N, 30 h, rt; n (d) PPh3, Br2, CH2Cl2, pyridine, 0 °C to rt; (e) PPh3, MeOH, 100 °C; (f) i. BuLi, Et2O, rt, ii. 38, iii. HCl (1 M).

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During the course of these studies, we found that the (E)-isomers 35 and 36 were surprisingly less stable than their corresponding (Z)-isomers 42 and 43. The (Z)-isomers 42 and 43 were stored as pure liquids at 4 °C for 2 months which resulted in no significant changes as detected by 1H NMR. After storage as neat liquids at 4 °C for 3 weeks, the (E)-isomers (35, 56) had partially isomerized to the (Z)-isomers (25% by 1H NMR).

Disk diffusion assays were used to test the bacteriostatic activity of the dehydrocrepenynate isomers against Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922). Levels up to 810 µg/disk for each isomer had negligible activity. In the case of P. aeurginosa, changes in the growth characteristics resulting in yellowish pigmentation and a flattened bacterial lawn surrounding the disk were observed for 31a at loadings above 27 µg/disk.

2-3. Conclusion

In summary, a stereoselective synthesis of methyl (14Z)- and (14E)-dehydrocrepenynate was achieved in five to six steps that employed a Pd-catalyzed cross-coupling reactions to construct the double bonds between C14 and C15. Compared with the earlier methods, the improved syntheses are more convenient (no spinning band distillations or GLC separation of diastereomers were necessary) and higher Z/E ratios were obtained. The overall percent yield for (14E)-isomer was 20.8% and 29.2% for the (14Z)-isomer.

2-4. Experimental Section

General Experimental Information. All manipulations were performed in oven-dried glassware under a nitrogen atmosphere unless otherwise mentioned. All solvents and reagents were dried using standard procedures and were distilled freshly before using. Ether, THF, n- pentane and benzene were dried and deoxygenated by distillation under N2 from sodium-

benzophenone ketyl. Methylene chloride was dried over CaH2; pyridine was dried over CaSO4; ethanol and methanol were dried with Mg metal and distilled before use. The standard work-up

51

included washing with water and followed by brine twice, then the organic phase was dried over

MgSO4, gravity filtered and concentrated in vacuo.

Column chromatography was performed on silica gel (Natland International Corp. 200-400 mesh) using the indicated solvent. TLC analyses were carried out using aluminum precoated silica gel plates (Whatman, Al Sil G/UV). Melting points were determined with a Gallenkamp melting point apparatus and were uncorrected. 1H and 13C NMR spectra were recorded on 200 or 300 MHz FT-NMR spectrometers (Bruker, Avance 200). IR spectra were determined as neat films (using NaCl plate) or as KBr pellets on a Perkin-Elmer 1600 FT-IR spectrophotometer. High- resolution MS spectra were performed by the Chemistry Mass Spectrometry facility, Ohio State University, Columbus, OH.

Synthesis and spectra data

1. BuLi, Et2O, -78 °C OTHP 2. I2, Et2O OTHP 92% I 32 33

Synthesis of 2-(4-iodo-3-butynyloxy)tetrahydropyran (33): To a stirred solution of compound

32 (2.76 g, 18.0 mmol) in Et2O (20 mL), chilled to –78 °C was added n-BuLi (12.6 mL, 17.9 mmol). The mixture was stirred at –78 °C for 1 h then iodine (4.54 g, 17.9 mmol in 40 mL Et2O) was added. The red solution was stirred at –78 °C for 10 min and warmed to room temperature for 1 h, which gave a clear yellow solution. The reaction was quenched with aqueous Na2S2O3

(20%, 30 mL) followed by brine (30 mL) and extracted with Et2O (3 × 20 mL). Following the standard work-up procedure, the crude product was obtained and purified by flash column (5:1 1 hexanes/EtOAc) yielding 33 as colorless oil (4.62 g, 92% yield). H NMR (CDCl3, 200 MHz) δ 4.61 (t, J = 3.5 Hz, 1H), 3.82 (m, 2H), 3.51 (m, 2H), 2.64 (t, J = 7.0 Hz, 2H), 1.51 (m, 6H); 13C

NMR (CDCl3, 50 MHz) δ 98.72, 91.43, 65.41, 62.19, 30.49, 25.38, 22.20, 19.33, -5.58; IR (neat -1 film) νmax 2942, 2872, 2217, 1122, 1032 cm .

52

1. Pd(PPh3)4, benzene OTHP 2. 34 3. EtONa, EtOH I THPO 4. NaOH, H O 33 2 2 35 89% Synthesis of (E)-1-(2-tetrahydropyranyloxy)-5-nonen-3-yne (35): To a solution of 33 (2.75 g, 9.8 mmol) in benzene (15 mL) at 0 °C was added tetrakis(triphenylphosphine)-palladium (0.11 g, 0.095 mmol). The mixture was stirred at 0 °C for 1 h, then (E)-1-pentenyldicyclohexenylborane 34 (Prepared by adding borane-methyl sulfide complex (1.0 mL, 10 mmol) to the solution of cyclohexene (2.0 mL, 20 mmol) at –78 °C, then warming the solution to room temperature for 40 min, resulted in a white suspension. After the addition of 1-pentyne (0.94 mL, 9.5 mmol), the mixture was stirred at room temperature for 1h.) was added followed by EtONa (1.31 g, 19.3 mmol in 10 mL EtOH). The final mixture was heated to reflux overnight. The reaction was quenched with aqueous NaOH (2 M, 1.0 mL) followed 30 min later by H2O2 (50%, 1.0 mL) at 0 °C. Water (20 mL) was added and the reaction mixture was extracted with hexanes (3 × 10 mL). Following the standard work-up procedure, the crude product was obtained and purified by flash column chromatography (10:2 hexanes/EtOAc) yielding compound 35 (1.94 g, 89% yield). 1H

NMR (CDCl3, 300 MHz) δ 6.04 (dt, J = 15.8, 7.1 Hz, 1H), 5.42 (dm, J = 15.8 Hz, 1H), 4.63 (t, J = 3.1 Hz, 1H), 3.83 (m, 2H), 3.55 (m, 2H), 2.58 (td, J = 7.2, 2.0 Hz, 2H), 2.03 (qd, J = 7.1, 0.9 13 Hz, 2H), 1.51 (m, 8H), 0.87 (t, J = 7.3 Hz, 3H); C NMR (CDCl3, 75 MHz) δ 143.71, 109.73, 98.73, 85.05, 80.15, 65.83, 62.20, 34.99, 30.55, 25.42, 21.98, 20.81, 19.41, 13.57; IR (neat film) -1 + νmax 3020, 2935, 2872, 1455, 1122, 1034 cm ; HRMS (EI) m/z M calcd for C14H22O2 222.1621, found 222.1620.

Ph3P, Br2, CH2Cl2

THPO pyridine, 0 °C to rt Br 60% 35 36

Synthesis of (E)-1-bromo-5-nonen-3-yne (36): To a stirred solution of Ph3P (1.56 g, 5.96 mmol) in methylene chloride (20 mL) at 0 °C was added bromine (0.3 mL, 5. 8 mmol). The yellow solution obtained was stirred for 1 h then compound 35 (0.88 g, 3.9 mmol) and pyridine (0.48 mL, 5.9 mmol) were added sequentially. The mixture was stirred for 1 h and then warmed to rt

53

for 5 h. The solvent was removed in vacuo. The obtained white crystals were washed with n- pentane (25 mL) and removed by filtration. After concentrating with a rotary evaporator, the crude yellow oil was purified by flash column chromatography (petroleum ether) to yield 36 1 (0.48 g, 60% yield). H NMR (CDCl3, 200 MHz) δ 6.10 (dt, J = 15.9, 7.1 Hz, 1H), 5.42 (dm, J = 15.8 Hz, 1H), 3.42 (t, J = 7.3 Hz, 2H), 2.83 (td, J = 7.3, 2.0 Hz, 2H), 2.04 (qd, J = 7.1 Hz, 1.4 Hz, 13 2H), 1.39 (m, J = 7.5 Hz, 2H), 0.88 (t, J = 7.2 Hz, 3H); C NMR (CDCl3, 50 MHz) δ 144.63,

109.28, 84.76, 81.12, 35.03, 29.69, 23.84, 21.89, 13.59; IR (neat film) νmax 3021, 2960, 2871, -1 + 1716, 1270, 1212 cm ; HRMS (EI) m/z M calcd for C9H13Br 200.0201, found 200.0196.

PPh3, MeCN 95 °C Br BrPh3P 72% 36 37

Synthesis of (E)-5-nonen-3-yn-1-yltriphenylphosphonium bromide (37): Compound 36 (0.30 g, 1.5 mmol), triphenylphosphine (0.53 g, 2.0 mmol) and methanol (12 mL) were placed into a

sealed tube. After three freeze-thaw cycles in liquid N2, the mixture was heated to 110 °C for 20 h. The solvent was removed in vacuo and the brown oil was washed with ether (3 × 5 mL). 1 Cooling the residue to 0 °C yielded yellow crystals 37 (0.51 g, 72% yield). H NMR (CDCl3, 200 MHz) δ 7.75 (m, 15H), 5.61 (dt, J = 16.0, 7.0 Hz, 1H), 4.95 (d, J = 16.0 Hz, 1H), 4.13 (quint, J = 6.6 Hz, 2H), 2.93 (dm, J = 22.4 Hz, 2H), 1,90 (q, J = 7.0 Hz, 2H), 1.30 (sextet, J = 7.4 Hz, 2H), 0.83 (t, J = 7.3 Hz, 3H).

O 1. BuLi, Et2O 2. 38 MeO BrPh3P 7 3. HCl 31a 37 59%

Synthesis of methyl (14E)-dehydrocrepenynate (31a): To a stirred suspension of compound

37 (0.46 g, 1.0 mmol) in Et2O (5.0 mL) was added n-BuLi (0.70 mL, 1.0 mmol) at room temperature. The suspension turned to dark red and was stirred for 45 min. A solution of 38 (0.19

g, 1.0 mmol) in Et2O (1.5 mL) was added. The mixture was stirred at rt for 1.5 h then heated to

54

50 °C for 0.5 h. The solid that precipitated was removed by filtration and rinsed with Et2O (2 × 15 mL). The combined filtrates were washed with HCl (1 M, 2 × 15 mL). Following the standard work-up procedure, the crude product was obtained and purified by flash column 1 chromatography (10:1 hexanes/EtOAc) to yield 31a (0.25 g, 59% yield). H NMR (CDCl3, 200 MHz) δ 6.04 (td, J = 15.7, 7.1 Hz, 1H), 5.41 (m, 3H), 3.64 (s, 3H), 3.01 (m, 2H), 2.28 (t, J = 7.3 13 Hz, 2H), 2.0 (m, 2H), 1.59 (m, 2H), 1.28 (m, 10H), 0.87 (t, J = 7.3 Hz, 3H); C NMR (CDCl3, 50 MHz) δ 174.76, 143.97, 132.10, 124.61, 110.22, 87.10, 79.33, 51.90, 35.46, 34.51, 29.69,

29.52, 29.46 (2C), 27.52, 25.35, 22.44, 18.17, 14.05; IR (neat film) νmax 3020, 2926, 2855, 1743, -1 + 1463, 1170 cm ; HRMS (EI) m/z M calcd for C19H30O2 290.2247, found 290.2249.

1. BuLi, Et2O, -78 °C 2. I2, Et2O 92% I 39 40

16 Synthesis of 1-iodo-1-pentyne (40) : To a solution of 1-pentyne 39 (3.65 g, 53.6 mmol) in Et2O (40 mL), stirred at –78 °C was added n-BuLi (37.0 mL, 52.5 mmol) over 10 min. The mixture was stirred at –78 °C for 1 h. Iodine (13.02 g, 51.2 mmol) in Et2O (80 mL) was added over 30 min resulting in a red solution that was stirred at –78 °C for 1.5 h and 0 °C for another 1.5 h. The reaction was quenched with aqueous Na2S2O3 (20%, 40 mL) followed by brine (60 mL) and extracted with hexanes (3 × 30 mL) followed by the standard work-up. The crude product 40, a 1 faint yellow oil, was used without further purification (9.15 g, 92% yield). H NMR (CDCl3, 200 MHz) δ 2.32 (t, J = 7.0 Hz, 2H), 1.51 (sextet, J = 7.2 Hz, 2H), 0.96 (t, J = 7.2 Hz, 3H); 13C NMR

(CDCl3, 50 MHz) δ 95.11, 23.20, 22.41, 13.85, -7.04; IR (neat film) νmax 2962, 2870, 2185, 1461 cm-1.

1. cyclohexene, BH3, n-pentane, -78 °C I I 2. AcOH, ethanolamine 40 41 80%

Synthesis of (Z)-1-iodo-1-pentene (41)16-17: To a solution of cyclohexene (2.8 mL, 27.0 mmol) in n-pentane (30 mL) at –78 °C was added borane-methyl sulfide complex (1.4 mL, 14.0 mmol).

55

The solution was allowed to warm to rt to give a white suspension within 40 min. To this suspension was added 1-iodo-1-pentyne (2.6 g, 13.4 mmol) and the mixture was stirred for 2 h. The reaction was quenched with glacial acetic acid (2.0 mL) followed 30 min later by the addition of ethanolamine (2.0 mL). The reaction mixture was stirred for 6 h prior to extracting with water and brine. Following the standard work-up, the resulting yellow oil was purified by distillation (133-135 °C) to obtain (Z)-vinyl iodine 41 (2.1 g, 80% yield) as faint yellow oil. 1H

NMR (CDCl3, 200 MHz) δ 6.16 (m, 2H), 2.10 (m, 2H), 1.44 (sextet, J = 7.3 Hz, 2H), 0.93 (t, J = 13 7.4 Hz, 3H); C NMR (CDCl3, 50 MHz) δ 141.24, 82.28, 36.64, 21.27, 13.65; IR (neat film) -1 νmax 3067, 2959, 2870, 1610, 1459, 1286 cm .

1. Pd(PPh3)4, benzene I 2. 32, CuI, Et3N, 30 h THPO 41 42 90%

Synthesis of (Z)-1-(2-tetrahydro-2H-pyranyloxy)-5-nonen-3-yne (42): To a solution of (Z)-1- iodo-1-pentene 41 (3.07 g, 15.7 mmol) in benzene (30 mL), stirred at room temperature, was added tetrakis(triphenylphosphine)palladium (0.24 g, 0.2 mmol). The mixture was stirred for 1 h. A solution of 2-(3-butynyloxy)tetrahydro-2H-pyran 32 (2.37 g, 15.4 mmol) in triethylamine (16 mL) was added followed by addition of copper iodide (0.53 g, 2.8 mmol). A mild exothermic reaction occurred and the temperature reached ~30 °C. After 30 h at rt, the mixture was poured into saturated NH4Cl (50 mL) and extracted with Et2O (3 × 20 mL). Following the standard work-up, the crude product was obtained and purified by flash column chromatography (10:2 1 hexanes/EtOAc) to yield 42 (3.08 g, 90% yield). H NMR (CDCl3, 200 MHz) δ 5.81 (dt, J = 7.3, 10.7 Hz, 1H), 5.41 (dm, J = 10.7 Hz, 1H), 4.65 (t, J = 3.3 Hz, 1H), 3.83 (m, 2H), 3.55 (m, 2H), 2.63 (dt, J = 7.1, 2.1 Hz, 2H), 2.24 (qd, J = 7.2, 1.3 Hz, 2H), 1.55 (m, 8H), 0.90 (t, J = 7.3 Hz, 13 3H); C NMR (CDCl3, 50 MHz) δ 142.92, 109.21, 98.73, 90.80, 78.27, 65.91, 62.14, 32.07,

30.56, 25.43, 22.11, 21.02, 19.36, 13.71; IR (neat film) νmax 3032, 2939, 2872, 1455, 1122, 1034 -1 + cm ; HRMS (EI) m/z M calcd for C14H22O2 222.1619, found 222.1629.

Ph3P, Br2, CH2Cl2 THPO pyridine, 0 °C to rt Br 42 43 86%

56

Synthesis of (Z)-1-bromo-5-nonen-3-yne (43): To a stirred solution of Ph3P (1.97 g, 7.5 mmol) in dry methylene chloride (20 mL) at 0 °C was added bromine (0.38 mL, 7.4 mmol). The yellow solution obtained was stirred for 1 h and 42 (1.10 g, 5.0 mmol) was added to the reaction mixture followed by pyridine (0.60 mL, 7.4 mmol). The mixture was stirred for 1 h, warmed to rt for 5 h, and the solvent was subsequently removed in vacuo. The obtained white crystals were washed with n-pentane (30 mL), and removed by filtration. After concentrated under vacuum, the crude yellow oil was purified by flash column chromatography (petroleum ether) to yield 43 (0.86 g, 1 86% yield). H NMR (CDCl3, 200 MHz) δ 5.88 (dt, J = 10.7, 7.4 Hz, 1H), 5.41 (dm, J = 10.8 Hz, 1H), 3.45 (t, J = 7.3 Hz, 2H), 2.89 (td, J = 7.3, 2.0 Hz, 2H), 2.25 (qd, J = 7.3, 1.3 Hz, 2H), 1.41 13 (m, J = 7.4 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H); C NMR (CDCl3, 50 MHz) δ 144.37, 109.21,

90.83, 79.78, 32.62, 30.30, 24.45, 22.52, 14.18; IR (neat film) νmax 3021, 2960, 1455, 1270, 1210 -1 + cm ; HRMS (EI) m/z [M ] calcd for C9H13Br 200.0200, found 200.0203.

PPh3, MeOH BrPh P Br 100 °C 3 43 87% 44

Synthesis of (Z)-5-nonen-3-ynyltriphenylphosphonium bromide (44): Compound 53 (0.50 g, 2.5 mmol), triphenylphosphine (0.85 g, 3.3 mmol) and methanol (15 mL) were placed into a

sealed tube. After three freeze-thaw cycles in liquid N2, the mixture was heated to 110 °C for 20 h. The solvent was removed in vacuo and the faint yellow oil was washed with ether (2 × 5 mL). The residue was cooled down to 0 °C to give white crystals 44 (1.0 g, 87% yield). m.p. 141.2- 1 142 °C; H NMR (CDCl3, 200 MHz,) δ 7.73 (m, 15H), 5.67 (dt, J = 10.8, 7.5 Hz, 1H), 4.90 (dm, J = 10.8 Hz, 1H), 4.15, (m, 2H), 3.01 (dm, J = 22.4 Hz, 2H), 1.98 (m, 2H), 1.29 (sextet, J = 7.4 Hz, 2H), 0.83 (t, J = 7.3 Hz, 3H).

O 1. BuLi, Et2O MeO BrPh3P 2. 38 7 44 3. HCl 31b 59%

57

Synthesis of methyl (14Z)-dehydrocrepenynate (31b): To a stirred suspension of compound 44

(0.20 g, 0.43 mmol) in Et2O (3.0 mL) at rt was added n-BuLi (0.30 mL, 0.43 mmol). The suspension turned to dark red and was stirred for 45 min. A solution of 38 (0.080 g, 0.42 mmol) in Et2O (1.0 mL) was added. The mixture was stirred at rt for 1.5 h then heated to 50 °C for 0.5 h.

The solid was removed by filtration and rinsed with Et2O (2 × 10 mL). The combined filtrates were washed with HCl (1 M, 2 × 15 mL). Following the standard work-up procedure, the crude product was obtained and purified by flash column chromatography (10:1 hexanes/EtOAc) to 1 yield 31b (0.074 g, 59% yield). H NMR (CDCl3, 200 MHz) δ 5.80 (dt, J = 10.7, 7.3 Hz, 1H), 5.43 (m, 3H), 3.64 (s, 3H), 3.07 (t, J = 2.4 Hz, 2H), 2.28 (t, J = 7.3 Hz, 2H), 2.24 (qd, J = 7.4, 1.2 Hz, 2H), 2.03 (q, J = 5.8 Hz, 2H), 1.59 (m, 2H), 1.28 (m, 10H), 0.90 (t, J = 7.3 Hz, 3H); 13C

NMR (CDCl3, 50 MHz) δ 174.28, 142.69, 131.62, 124.29, 109.30 (2 carbons), 92.35, 51.43,

34.07, 32.06, 29.25, 29.07, 29.01, 27.09, 24.91, 22.71, 22.13, 17.93, 13.73; IR (neat film) νmax -1 + 3020, 2928, 2856, 2214, 1742, 1462 cm ; HRMS (EI) m/z [M ] calcd for C19H30O2 290.2246, found 290.2250.

58

References

1. a) Heywood, V. H.; Harborne, J. B.; Turner, B. L. In The Biology and Chemistry of the Compositae; Academic Press: London, 1977; Vol. 1, Chapters 10, 13 and 14, pp. 284-334 and 384-432. b) Bohlmann, F.; Weber, D. “Polyacetylenic compounds. 219. Biogenesis of C17- enediynedienes” Chem. Ber. 1973, 106, 3020-3025. 2. Ford, G. L.; Fogerty, A. C.; Walker, K. H. “Crepenynic acid and muscle breakdown” Prog. Lipid Res. 1986, 25, 263-267. 3. Croft, K. D.; Beilin, L. J.; Ford, G. L. “Differential inhibition of thromboxane B2 and leukotriene B4 biosynthesis by two naturally occurring acetylenic fatty acids” Biochim. Biophys. Acta. 1987, 921, 621-624. 4. Nugteren, D. H.; Christ-Hazelhof, E. “Naturally occurring conjugated octadecatrienoic acids are strong inhibitors of prostaglandin biosynthesis” Prostaglandins 1987, 33, 403-417. 5. Barley, G. C.; Graf, U.; Higham, C. A.; Jarrah, M. Y.; Jones, E. R. H.; O'Neill, I.; Tachikawa, R.; Thaller, V.; Turner, J. L. “Natural acetylenes. Part 61. Fungal polyacetylenes and the crepenynate pathway: the biosynthesis of some C9-C14 polyacetylenes in fungal cultures” J. Chem. Res. Syn. 1987, 7, 232-233. 6. Bu'Lock, J. D.; Smith, G. N. “Origin of naturally-occurring acetylenes” J. Chem. Soc. (C) 1967, 5, 332-336. 7. Gunstone, F. D.; Kilgast, D.; Powell, R. G.; Taylor, G. M. “Afzelia cuanzensis seed oil: a source of crepenynic and 14,15-dehydrocrepenynic acid” Chem. Commun. 1967, 6, 295-296. 8. Magnus, V.; Lacan, G.; Aplin, R. T.; Thaller, V. “Glycerol tridehydrocrepenynate from the basidiomycete Craterellus cornucopioides” Phytochemistry 1989, 28, 3047-3050. 9. a) Somssich, I. E.; Bollman, J.; Hahlbrock, K.; Kombrink, E.; Schulz, W. “Differential early activation of defense-related genes in elicitor-treated parsley cells” Plant Mol. Biol. Reporter 1989, 12, 227-234. b) Cahoon, E. B.; Schnurr, J. A.; Huffman, E. A.; Minto, R. E. “Fungal responsive fatty acid acetylenases occur widely in evolutionarily distant plant families” Plant J. 2003, 34, 671- 683. 10. Farrell, I. W.; Higham, C. A.; Jones, E. R. H.; Thaller, V. “Natural acetylenes. Part 62.

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Fungal polyacetylenes and the crepenynate pathway: experiments relevant to the biogenesis of the acetylenic bond” J. Chem. Res. Syn. 1987, 7, 234-235. 11. Miyaura, N.; Yamada, K.; Suzuki, A. “A new stereospecific cross-coupling by the palladium-catalyzed reaction of 1-alkenylboranes with 1-alkenyl or 1-alkynyl halides” Tetrahedron Lett. 1979, 36, 3437-3440. 12. a) Jones, E. R. H.; Bradshaw, R. W.; Day, A. C.; Page, C. B.; Thaller, V.; Hodge, R. A. V. “Natural acetylenes. XXXII. Synthesis of crepenynic acid (9-octadecen-12-ynoic acid)” J. Chem. Soc. (C) 1971, 6, 1156-1158. b) Bradshaw, R. W.; Day, A. C.; Jones, E. R. H.; Page, C. B.; Thaller, V. “Synthesis of crepenynic acid” Chem. Commun. 1967, 20, 1055-1056. 13. Sonnet, P. E. “Direct conversion of an alcohol tetrahydropyranyl ether to a bromide, chloride, methyl ether, nitrile or trifluoroacetate” Syn. Commun. 1976, 6, 21-26. 14. Brown, H. C.; Blue, C. D.; Nelson, D. J.; Bhat, N. G. “Vinylic organoboranes. 12. Synthesis of (Z)-1-halo-1-alkenes via hydroboration of 1-halo-1-alkynes followed by protonolysis” J. Org. Chem. 1989, 54, 6064-6067. 15. Ratovelomana, V.; Linstrumelle, G. “New synthesis of the sex pheromone of the Egyptian cotton leafworm, Spodoptera littoralis” Syn. Commun. 1981, 11, 917-923. 16. Brown, H. C.; Blue, C. D.; Nelson, D. J.; Bhat, N. G. “Vinylic organoboranes. 12. Synthesis of (Z)-1-halo-1-alkenes via hydroboration of 1-halo-1-alkynes followed by protonolysis” J. Org. Chem. 1989, 54, 6064-6067. 17. Cabezas, J. A.; Oehlschlager, A. C. “Stereospecific synthesis of (E,Z) and (Z,Z)-hexadeca- 10,12-dienal. Sex pheromone components of Diaphania hyalinata” Synthesis 1999, 1, 107- 111.

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Chapter III Mechanism of Dimethyl Disulfide Addition to 1,4-Enynes

3-1. Introduction

Characterization of unsaturated fatty acids by GC-MS methods requires the formation of a derivative to prevent the migration of the C=C double bond during electron impact ionization.1 Dimethyl disulfide (DMDS) derivatization of unsaturated fatty acid esters and amides (especially for monoenoic examples) is a widely used method for the mass-spectrometric determination of C=C double bond position.2 The mass spectra of the DMDS adducts of monoenoic acid give distinct molecular ions, which contain the requisite structural information for locating the C=C double bond. Under the reaction conditions, dimethyl disulfide will add to the C=C double bond to generate the DMDS adducts. These adducts are suitable for GC-MS analysis without isolation or purification.3-4 The subsequent ionization (MS fragmentation) gives two intense fragment ions, because the cleavage tends to occur between the carbons that originally constituted the double bond. The stability of these fragment ions results in very intense mass spectrum peaks, which serve to locate the unsaturated position (Figure 18a). Dienoic fatty esters usually present more difficulties than monoenes with the DMDS procedure. If the double bonds are interrupted with more than four carbon atoms, independent derivatization of the alkenes occurs. Unfortunately, fatty acids with this unsaturation pattern are a relatively rare occurrence in nature. When the double bonds are closer together, (such as in methyl linoleate, alkene units interrupted by one methylene group) a complex product mixture is formed (Figure 18b).5-7 Derivatization reactions at higher temperatures (60 °C) and longer reaction period (40 h) result in cyclizations to give complicated heterocyclic compounds, such as thietane, tetrahydrothiophene and tetrahydrothiopyran derivatives (4-, 5-, and 6-membered rings).5 In order to simplify the derivatization chemistry, the analytical approach can be modified by partially hydrogenating the C=C double bonds of pure polyunsaturated fatty acids derivatives to generate monoenoates prior to the DMDS reaction (Figure 18c).1d The resulting mixture of monoenoic compounds is used for the DMDS derivatization. The GC-MS analysis of the adducts will give four distinct fragment ions in two resolved chromatographic peaks. These ions provide the necessary structural information for locating the C=C bonds. Thus, this method can be expanded to locate the C=C bonds in polyunsaturated fatty acids.3e When Carballeira and coworkers studied the direct

61

DMDS derivatization of ethyl (9E, 12E)- and (9Z, 12Z)-9,12-octadecadienoate, they found that each configurational isomer displayed a unique GC trace pattern for the 4-, 5-, and 6-membered ring cyclization products obtained. Thus they were able to determine the stereochemistry of C=C double bond by this method as well.6 However, in order to make this method a useful tool to determine unknown sample, a good GC-MS spectrum library would be required.

+/ DMDS MeS SMe GC-MS SMe SMe CO Me + 2 CO Me CO Me R n 2 +/ 2 I2, C-C6H12 R nnR

a) Dimethyl disulfide derivatization of monoenoic acid methyl ester.

MeS SMe SMe SMe

R R' R S R' S DMDS, I2 R R' cyclohexane 35-60 °C SMe SMe S R' S R R R'

SMe SMe

b) Derivatization of dienoic fatty acid derivatives results in complicated product mixtures.

H2 (1.0 eq) CO Me RCOMe R 2 2 + RCOMe nnPt n 2

DMDS derivatization

MeS SMe MeS SMe RCO2Me + n RCn O2Me GC-MS fragmentation

SMe SMe SMe SMe + ++ R +/ +/ CO2Me +/ +/ CO Me n R n 2

c) Partially hydrogenation of dienoic fatty acid derivatives expands the utility of this method.

Figure 18. Dimethyl disulfide (DMDS) derivatization and its utilities.

62

For 1,3-dienoic compounds, Diels-Alder adducts, prepared by reacting the diene with 4-methyl- 1,2,4-triazoline-3,5-dione, MTAD, were employed to lock the conjugated C=C double bonds. Consequently, GC-MS analysis was able to locate the positions of the double bonds.8-10 However, no research concerning DMDS derivatization of fatty acid derivatives containing 1,4-enyne units has been reported to date.

An aim of the research described in this chapter is to expand the standard DMDS method to acetylenic compounds, as these compounds are often biologically important and partially characterizable by GC-MS methods. For instance, crepenynic acid is a very interesting constituent of certain seed oils.12 Crepenynate and its close relative, dehydrocrepenynate, serve as the biosynthetic progenitors to a large family of polyacetylenes.13 It would be very useful to have a convenient quantification method to analyze this family of compounds. Therefore, we initially set out to explore the possible use of the DMDS method for esters of acetylenic fatty acids, as exemplified by crepenynic acid (30). Transesterification of the fatty acids (usually 5%

H2SO4 in methanol) before GC-MS analysis is necessary to get a satisfactory GC elution profile.1a,b We unexpectedly isolated a thiophene derivative from the DMDS derivatization of fatty acids containing 1,4-eneyne units. In the course of our studies, the mechanism for the formation of the thiophene derivatives (1,5-disubstituted thiophene) through DMDS derivatization was explored and the results are discussed in this chapter.

3-2. Results and discussion

Transesterification of Crepis alpina seed oil in methanol containing 5% H2SO4 provided methyl crepenynate. After column purification, the pure methyl crepenynate (30) was subjected to the DMDS reaction conditions. By analyzing the reaction mixture by GC-MS, a fraction with m/z =

324 was found as the major product. Quenching the reaction with aqueous Na2S2O3 solution and purifying the crude product by column chromatography, the main GC fraction was identified as a 2,5-disubstituted thiophene derivative 47a (Figure 19). The cyclization has the net effect of regioselectively inserting a sulfur atom between the C9 and C12. Encouraged by the results obtained with methyl crepenynate, methyl (6Z)-tridecen-9-ynoate (45) was synthesized as a

63

model compound to study the reaction in detail. Conducting the DMDS derivatization with 45, thiophene derivative 47b was isolated as the major product from the reaction mixture (Figure 19).

DMDS, I2 MeS R m R + R m solvent S I n 60 °C, 5 h n m n

30: R = CO2Me, n = 7, m = 3 47a: R = CO2Me, n = 7, m = 3 48a: R = CO2Me, n = 7, m = 3 45: R = CO2Me, n = 4, m = 1 47b: R = CO2Me, n = 4, m = 1 48b: R = CO2Me, n = 4, m = 1 48c: R = CH , n = 4, m = 1 46: R = CH3, n = 4, m = 1 47c: R = CH3, n = 4, m = 1 3

Figure 19. DMDS addition to 1,4-enyne derivatives.

3-2.1 Optimization of the reaction conditions

According to the previous reports,1-3 the DMDS derivatization reaction for analytical purposes was conducted in cyclohexane with the presence of large excess of DMDS. Although substantial variability is tolerated, the general conditions utilized for this reaction included 650 eq of DMDS, 2 eq of iodine and adjusting the ester concentration to 1 mg/mL with cyclohexane. However, the solubility of iodine in cyclohexane is low. Therefore it is impossible to conduct the reaction at higher concentrations. Thus, a better solvent system was necessary to achieve our goal of developing a synthetically useful method. Among the solvents we employed, methanol was the best candidate for performing this reaction at high concentration. The concentration of the methyl ester can be increased to 10 mg/mL by reducing the amount of methanol used without any detectable solubility problems. When the reaction was conducted in MeOH at various temperatures, a temperature dependence for this reaction was observed. Using methyl nonanoate as internal standard, the relative rate of thiophene formation were determined. The best yield was achieved at 60 °C after 5 hours (Figure 20). Performing the reaction at lower temperature also provided the thiophene, however, the yield was low and a longer reaction time was required. It should be noted, according to the previous work, DMDS adduct formation is favored at lower reaction temperature and longer reaction period.1

64

Figure 20. Effect of temperature on thiophene formation.

0.6

0.5

ene 0.4

thioph 0.3 60 °C 0.2 50 °C

Yield of 40 °C 0.1

0 012345 Reaction time (h)

Conditions: Ester 45 reacted with 650 eq. DMDS and 2 eq iodine in MeOH. Yield of thiophene derivatives based on a methyl nonanoate internal standard. Reaction conditions explored for this reaction are summarized in Table 3. Interestingly, it was found that this reaction was not effected by water (up to 10 eq in cyclohexane, more water resulted in immiscible phases). Thiophene derivatives were generated with a yield comparable to that of reagent-grade cyclohexane as solvent. This makes the reaction a good candidate for preparing thiophene derivatives under mild conditions. Furthermore, when D2O (10 eq) was used with enyne 45, a deuterated thiophene derivative 47b (d0: d1: d2: d3 = 21: 44: 28: 5.5) was isolated (Table 3, entry 2). Elucidated by 2H NMR, the primary location of deuterium occurs at a single α-position of the thiophene ring, corresponding to the methyl-proxmate α-carbon. This was also confirmed by comparison with the nondeuterated thiophene product. These results indicate that a proton source is involved in this reaction and may benefit the generation of thiophene derivatives. Meanwhile, a competitive amount of H+ is available in the reaction system versus D2O. In the presence of aprotic solvent, such as DMA or DMF, no thiophene derivative was detected by GC-MS analysis (Table 3, entry 3 and 4).

65

Table 3. Solvent and concentration effects to the formation of thiophene 47b. a Entry Enyne Conc. Solvent DMDS I2 Yield 1 1 mg/mL cyclohexane 650 eq 2 eq 52% b b 2 1 mg/mL Cyclohexane & D2O 650 eq 2 eq 59% 3 1 mg/mL DMA 650 eq 2 eq <1% c 4 1 mg/mL DMF 650 eq 2 eq 0% c 5 3.3 mg/mL neat 650 eq 2 eq 32% c 6 1 mg/mL MeOD 650 eq 2 eq 62% b 7 1 mg/mL MeOH 650 eq 2 eq 55% b 8 1 mg/mL MeOH 10 eq 3 eq 63% c 9 10 mg/mL MeOH 10 eq 3 eq 56% b

a. Reactions were performed at 60 °C for 5 hours; b. Isolated yields after column chromatography; c. Yields of 47b obtained from GC-MS analysis.

Conducting the reaction in neat DMDS (Table 3, entry 5) thiophene product was also observed, however, the GC-MS spectra indicated the reaction mixtures were more complicated than cases with added solvent. Performing the reaction in MeOH, thiophene product was isolated in 55%

yield (Table 3, entry 7). When the reaction was performed in MeOD, high level deuteration (d0: d1: d2: d3 = 0: 13: 84: 4) was observed (Table 3, entry 6). In addition, the amount of iodine exhibited a strong influence on this reaction. Two equivalents of iodine were necessary to drive this reaction to completion. By reducing the quantity of iodine to 1 eq, a mixture of thiophene (47) and MeSI adduct (48) was obtained. However, the amount of DMDS could be reduced (to 10 eq) without significant influence to the yield of thiophene (Table 3, entry 8 and 9). In order to drive the reaction to completion and reduce the reaction time, 3 eq of iodine were used in these cases. In addition, by reducing the amount of DMDS, GC analysis indicated that the product mixtures were simplified. By increasing the concentration of ester to 10 mg/mL, the viability of this method a potential synthetic strategy for preparing thiophene derivatives was improved. Under these optimized conditions (Table 3, entry 9), thiophene derivative was isolated through column chromatography in 56% yield.

66

3-2.2 Effects of additives

Based on the optimized conditions, additional investigation revealed that several additives demonstrated strong influences on this reaction (Table 4). In the presence of acidic additives (Table 4, entry 1 and 2), thiophene derivative 47b was detected by GC-MS analysis in similar yields to the standard conditions. When benzoic acid and acetic acid (10 equiv. to the methyl ester) were applied to the reaction system, thiophene was detected by GC-MS in 54 and 59% yield, respectively. However, basic additives (Table 4, entries 4-6) terminated the reaction. Triethylamine halted the reaction and starting material was recovered (Table 4, entry 4) after 5 h at 60 °C. When pyridine or K2CO3 and 18-crown-6 (Table 4, entry 5 and 6) were added, a new compound was found as the major product from the GC-MS analysis, as well as trace amount of thiophene product.

Table 4 Effects of additives to DMDS derivatization of 45. Entry Additive Thiophene yielda 1 Benzoic acid (10 eq) 53% 2 AcOH (10 eq) 59% 3 Butylated hydroxytoluene (BHT, 10 eq) 54% b 4 Et3N (10 eq) No reaction 5 Pyridine (10 eq) Adduct 48bc c 6 K2CO3/18-crown-6 (3 eq) Adduct 48b c, d 7 K2CO3/18-crown-6 (3 eq) in MeOD Adduct 48b 8 LiI (3 eq) <10%e a. GC-MS yield; b. starting material was recovered; c. compound 48b was formed and starting material was recovered; d. no d-incorporation observed in compound 48b; e. reaction is very slow, lengthy reaction times still give mixture of 47b and 48b.

This new compound gave parent ion with m/z = 396, which is corresponding to the MeSI addition product of the methyl ester. In order to identify its structure, a large-scale reaction was undertaken and separation and purification of the apparent adduct was performed. After washing

the reaction mixture with aqueous Na2S2O3 solution and column purification, compound 48b was obtained. 1H NMR, 13C NMR and high resolution MS analysis are consistent with addition to the

67

triple bond instead of the C=C double bond. Surprisingly, only one regioisomer was apparently produced from the reaction.

NOE difference experiments were used to deduce the structure of compound 48b as shown below. When the protons on the methylene group (HB) were selectively irradiated, the NOE enhancements (interaction a and b) of the vinyl proton (HA) signal and the methyl group (HC) signal were observed (enhancement a and b). NOE enhancements b and c were observed by irradiating the protons (HC) on the methyl group attached to the sulfur atom. On the other hand, when HB was irradiated, no NOE effects were observed on signal of HD. These results confirmed the construction of the tetrasubstituted C=C double bond is E-configuration (as shown). The observation of NOE enhancement b, also provides evidence for the regiochemistry of addition in product 48b.

a H HA HB b MeO2C CH C I S 3

HD c 48b

However, the reason for generating 48b as sole isomer is not very clear. We have not been able to provide a reasonable explanation for this result so far. In addition, repeating this reaction with methyl 9-tridecenoate (49) under the standard conditions, the MeSI adduct (50) and diiodide product (51) were isolated from the reaction mixture (Figure 21). However, in the presence of 1 K2CO3 and crown ether, only MeSI adducts (50) were detected by GC-MS analysis. The H and 13C{1H} NMR data of compound 50 displayed that both isomers were generated (in 1:1 ratio), however, their NMR spectra (300 MHz) are very similar to each other the only striking difference could be found at the terminal methyl group from 1H NMR. These data implied, the additional C=C double bond in compound 45 may have a significant regiochemical influence in the production of 48b.

68

O DMDS (I)MeS O I + MeO MeO C MeO 7 I2, MeOH 2 4 I(SMe) 7 I Methyl 9-tridecenoate 49 50 51

Figure 21. Methyl 9-tridecynoate (49) reacts with DMDS.

Performing the reaction in MeOD with the presence of K2CO3 and crown ether (Table 4 entry 7), no deuterium incorporation in adduct 48b was observed. The only chemistry involved under these conditions is the anti-addition to the triple bond, so it is reasonable that no deuterium exchange was observed. In general, the reaction was sluggish in present of K2CO3, and remaining starting material could be recovered after reaction. However, when adding HI to this halted reaction mixture (e.g. entry 6 in Table 4) to consume K2CO3, thiophene was generated immediately, and at the same time the adduct 48b disappeared. However, when the isolated compound 48b was treated with HI in methanol for 3 hours, only trace amount of thiophene was detected by GC-MS. In addition, when compound 48b was treated with iodine and DMDS under the standard reaction conditions for 3.5 hours, thiophene was detected by GC-MS as major product and all of the MeSI adduct 48b was consumed. From these data, it seems that basic conditions favor the formation of the MeSI adducts and the starting enyne is in equilibrium with it. The MeSI adduct is apparently not an intermediate in producing thiophene, but the equilibrium between it and 1,4-enyne (e.g. 30, 45 or 46) allowed its eventual transformation to thiophene (e.g. 47a-c) through 1,4-enyne in the presence of an acid catalyst.

Butylated hydroxytoluene (BHT), a free radical scavenger, did not have a significant influence on the reaction (Table 4, entry 3), which is inconsistent with a free radical mechanism. In this case, thiophene product was found in the reaction mixture in 54% yield by GC-MS. Lithium iodide (Table 4, entry 8) did not improve the reaction, with only trace amount of thiophene being detected after 5 hours at 60 °C. Longer reaction times finally led to the mixture of 47b and 48b. At this circumstance, the competition between forming 47b and 48b is the major chemistry involved.

69

3-2.3 Proposed mechanisms for thiophene formation

Methyl iodide was detected by GC-MS analysis by injecting an aliquot of the reaction mixture during the course of the reaction run under standard conditions. However, after heating the mixture of DMDS and methanol or DMDS and iodine or the mixture of all three compounds at 60 °C for 5 hours, no methyl iodide was found by GC-MS analysis in any case. This indicated that MeI is a side product generated from the conversion of enynes to thiophenes. It is likely that the nucleophilic attack by the iodide to a methyl group occurs during the reaction.

I I , DMDS R' 2 R' Base MeS R' R I R R I 60 °C I2, DMDS 52 45 48 MeSI

CH3 R' S MeI 1,2-H-shift R H3C a c S b S R' R' R I H R' (D) R 56 55 R SMe 53, or 54 H+

R MeS MeI -HI R S R' R S R' R' + I H (D)H H(D) H CH3 H (D) - 57 I 58 Thiophene 47

Figure 22. Mechanism of DMDS addition to 1,4-enyne compounds.

According to the results obtained in previous sections, the most reasonable mechanisms for the derivatization reaction are proposed (Figure 22). Under the reaction conditions, iodine will react with DMDS first to produce MeSI, which then will react with C=C double bond to form episulfonium ion. From the episulfonium, there are three different pathways (Figure 22, pathway a, b and c) that can be followed to generate the thiophene product. They are all consistent with

70

our experimental results. Ring expansion through the 1,2-H shift followed by the attack of iodide could form the MeSI addition product 57, which then cyclizes finally leading to thiophene product (Figure 21, pathway a). Alternatively, the episulfonium ion could undergo deprotonation to form methyl enynethiol ether (53 or 54) and, following an acid-catalyzed cyclization, will give thiophene products (Figure 21, pathway b). Another pathway involves the formation of episulfide 55 by expulsion of methyl iodide. The episulfide then undergoes cyclization to build the thiophene ring (Figure 21, pathway c). The reaction was studied thoroughly by synthesizing possible intermediates and trying to cause thiophene production from these intermediates.

Enynethioether (both cis- and trans-isomers 53 and 54) analogs are expected for the pathway b in Figure 22. However, only the cis- isomer (53) was converted into thiophene product when treated with HI (Figure 23). After heating with HI for 5 hours, trans- isomer was not consumed, and no thiophene derivatives were detected by GC-MS analysis (Figure 24). This may be because the ring formation requires the cis- C=C double bond, which will provide a close enough approach for the sulfur atom to attack the triple bond. No isomerization between cis- and trans- isomers was observed under these conditions.

Abundance

Abundance TIC: 2.D TIC: 3.D 3200000 7.35 5.95 3000000 400000

2800000 C5H11 2600000 350000 4 S

2400000 SMe

2200000 300000

2000000 250000 1800000

1600000 200000 1400000 C5H11 1200000 150000 SMe 1000000 7.29 800000 100000

600000

400000 50000 8. 8.5581 9.92 11.40 15.14 200000 3. 44 4. 4.0938 6. 94 7. 588.20 12.19 14.76 8.84 0 0 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Time--> Time--> cis-Methyl thioether with HI in MeOH cis-Methyl thioether in MeOH for 4 hours for 4 hours, thiophene formed

Figure 23. GC-MS analysis of cis-enynethiol ether 53 reacted with HI at 60 °C.

71

Abundance Abundance TIC: 5.D 9.14 TIC: 4.D 2.2e+07 1.6e+07 9.08

2e+07 SMe 1.4e+07 SMe

1.8e+07 C5H11 C5H11 1.2e+07 1.6e+07

1.4e+07 1e+07

1.2e+07 8000000 1e+07

6000000 8000000

6000000 4000000

4000000 2000000 2000000 12.38 8.18 8.18 7.36 5.12 8 .9.6 810.9.268705 0 0 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 Time--> Tim e-->

trans-Methyl thioether with HI in MeOH trans-Methyl thioether in MeOH for 4 hours for 4 hours

Figure 24. GC-MS analysis of trans-enynethiol ether 54 reacted with HI at 60 °C.

However, when these two isomers were subjected to the standard DMDS-I2 reaction conditions, both isomers were converted into an unknown product. Unfortunately, we could not identify this compound, but GC-MS analysis indicated this new compound has different retention time and MS spectrum from thiophene product. It should be noted that no accumulation of either isomer was detected under the DMDS derivatization conditions.

Thiirane derivative (55) of ethyl crepenynate was synthesized to test the possibility of the

thiirane pathway (pathway c in Figure 22). Under standard DMDS-I2 conditions, no thiophene product was produced from thiirane compound, nor did the addition of HI facilitate thiophene formation.

According to these data, both pathway b and c in Figure 22 are not supported by the information collected. Considering the above results and literature precedents, the mechanism of the DMDS reaction is described as follows (pathway a, in Figure 22). An episulfonium ion initially formed at the alkene functionality through the reaction with MeSI. However, at the same time, equilibria

between starting material 45 and I2-adduct 52 and 48 are also established. Productively, a

72

Wagner-Meerwein shift provides intermediate 56. Under acidic conditions, intermediate 56 will be converted to 57 readily, and the latter will eliminate HI to form intermediate 58. Finally intermediate 58 is converted to the thiophene derivative 47 through the isomerization of the diene and expulsion of MeI. It should be noted that a MeSI adduct consistent with the 1,2- hydride shift product (57) was observed by GC-MS, however it was too labile to allow isolation and full characterization. Alternatively, a second possible mechanism involves the acid-catalyzed cyclization of the alkene-MeSI adduct onto an allenic intermediate (shown as Figure 25).

H(D)+ R R' R MeSI MeS R C C H (D)H R' (D)H R'

MeI H(D)+ R' H R S R' R S R S R' - CH3 H(D) I H(D)

Figure 25. Alternative mechanism through alkyne-allene rearrangement.

Finally, the utility of this reaction as a synthetic method was explored by reacting various enynes with DMDS under the optimized reaction conditions. We have found that, while general for 1,4- enynes, this reaction is sensitive to both functional groups and the length of the methylene tether between the ester and alkene moieties (data not shown). Synthetically, separation of the alkene and ester by more than four carbons typically leads to moderate to high yields of 2,5- dialkylthiophenes.

3-3. Conclusions

In summary, the reaction between DMDS and 1,4-enyne in the presence of I2 was studied. 2,5- Disubstituted thiophene derivatives were produced as the main product under neutral and acidic conditions. The detailed mechanism of this reaction was studied. Current evidence is consistent with a mechanism that can be described as follows. Initially, electrophilic addition of a sulfenium ion to an alkene yields an episulfonium ion. The subsequent Wagner-Meerwein rearrangement

73

leads to a cationic thietane intermediate through a ring expansion. This four-membered ring is opened by nucleophilic attack of iodide to give a MeSI adduct. Available protons activate the triple bond and promote the subsequent transformations to generate the final thiophene product and release MeI as a side product. A second mechanism that involves an alkyne-allene rearrangement remains as an alternative reaction pathway. The synthetic utilities of this method were explored. The optimized reaction provides a mild route to 2,5-disubstituted thiophene derivatives from 1,4-enynes bearing alkyl substitutes.

3-4. Experimental section

3-4.1 General Information

GC-MS was performed on HP-5890 II with a DB5 column, the temperature program used for resolution of DMDS derivatization compounds began at 70 °C and ramped to 150 °C (10 °C/minute). 1H and 2H NMR spectra were recorded with a Bruker 300 MHz NMR 13 1 spectrometer( C{ H} NMR spectra at 75.5 MHz), where the signal for residual CHCl3 in the

CDCl3 solvent (δ 7.24 ppm) was used as internal standard. FT-IR spectra were obtained as neat films (NaCl plate) or KBr pellets on a Perkin-Elmer 1600 FT-IR spectrophotometer. Flash column chromatography was performed on silica gel (Natland International Corp. 200-400 mesh) using the indicated solvent. TLC analyses were carried out on aluminum pre-coated silica gel plates (Whatman, Al Sil G/UV) with the indicated solvent. High-resolution MS spectra were performed by the Chemistry Mass Spectrometry facility, Ohio State University, Columbus, OH. All manipulations were performed in oven-dried glassware under nitrogen atmosphere unless other mentioned. All solvents and reagents were dried and distilled freshly before using. Ether,

THF, n-pentane, dioxane and benzene were dried and deoxygenated by distillation under N2 from sodium-benzophenone ketyl. Methylene chloride was dried over CaH2; pyridine was dried

over CaSO4; ethanol and methanol were dried with Mg metal and distilled before use; acetone was dried over 4Å sieves and distilled before use.

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3-4.2 Materials

DMDS and 18-crown-6 were purchased from Aldrich and used without purification.

Cyclohexane was used as purchased from Baxter (HPLC grade). CH3OD were purchased from Isotec and used without further purification. Methyl crepenynate was transesterified by refluxing Crepis alpina seed oil (generously provided by Dr. Richard Adlof, USDA, Peoria, IL) with 5%

H2SO4 methanol solution for 2 hours and followed by flash column chromatography (20:1 hexanes/EtOAc) purification.

3-4.3 General procedure for making thiophene derivatives

Unless stated otherwise the following procedure was used for DMDS derivatization experiment. To a solution of an enyne methyl ester and DMDS in methanol, iodine was added as a solution in methanol (20 mg/mL) and the concentration of ester was adjusted to 10 mg/mL with methanol.

The mixture was stirred in the dark under N2 atmosphere at 60 °C for 5 h. The reaction was

quenched with aqueous Na2S2O3 solution (20%, w/w) followed by the addition of ether. The layers were separated and the aqueous phase was extracted with ether three times. Combined organic phases were dried over MgSO4, and concentrated with a rotary evaporator. The crude product was purified by flash column chromatography (20:1 petroleum ether/ethyl ether, Rf 0.4). Solvent optimization experiments were conducted with indicated solvents instead of methanol. Additives and inhibitors were added to the reaction system in the indicated amounts (based on methyl ester) and the reaction was carried out following the general procedure.

3-4.4 Synthetic material

Methyl 9-tridecynoate was prepared according to the pathway shown in Figure 25. 1,7- diioheptane was obtained from 1,7-dibromoheptane through halogen exchange in acetone. After reaction with the lithium salt of 1-pentyne, compound 59 was produced and was transformed into nitrile 60. After hydrolysis in aqueous NaOH, the acid was afforded and it was converted to methyl ester 49 by reacting with diazomethane at room temperature. The desired ester was produced from 1-pentyne in 15% overall yield.

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Figure 26. Synthesis of methyl 9-tridecynoate (49).

Br Br abI I c + I 7 7 2 7 59 d e

NC HO2C MeO2C 7 7 7 60 49

Key: (a) NaI, acetone, 98%; (b) LiNH2, dioxane, reflux, overnight; (c) NaCN, EtOH, reflux, 12 h; (d) NaOH, EtOH/H2O, reflux; (e) Diazald, NaOH (6 M), 15% from 1-pentyne.

The model compound (45) was prepared according to the concise pathway in Figure 26. . Brominating 3-heptyn-1-ol with Ph3P Br2 gave 1-bromo-3-heptyne (61) quantitatively. Treatment of 61 with Ph3P in methanol at 100 °C afforded compound 62 in 88% yield. Through a Wittig reaction, methyl (6Z)-tridecen-9-ynote (47) was obtained within three steps in good yield. When compound 45 was subjected to the standard reaction conditions, thiophene derivative 47b was isolated as the main product (Figure 19).

Figure 27. Synthesis of methyl (6Z)-tridecen-9-ynote (45).

OH abBr PPh3Br c 98% 88% 70% 61 62

MeO

O 45

Key. (a) PPh3, Br2, CH2Cl2, 0 °C; (b) PPh3, MeOH, 100 °C, 24 h; (c) MeO2C(CH2)4CHO, NaH, THF/HMPA (10:1).

The possible intermediates (53 and 54), found on hypothetical pathway b of the reaction mechanism, were synthesized. The synthetic pathway of both isomers, 53 (cis-, Figure 27) and 54 (trans-, Figure 28), are shown below. The syntheses of these two compounds are straightforward starting from commercially available 1-octyn-3-ol (63). Protecting the hydroxyl

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group with TBDMSCl, compound 64 was used as the common starting point for both compounds. The key step for building the conjugated eneyne unit was achieved through a palladium-catalyzed cross-coupling reaction.

Figure 28. Synthesis of methyl cis-enynthiol ether (53).

OH OTBDMS ab, cC5H11 I d, e 92% 83% OTBDMS 66% 63 64 65

C H C5H11 f 5 11 g, h C5H11 OH 85% S 53% SMe

66 67 O 53

Key. (a) TBDMSCl, imidazole, CH2Cl2 (b) i. BuLi, Et2O, -78 °C; ii. I2, Et2O (c) cyclohexene, BH3 (d) i. Pd(PPh3)4; ii. 1-pentyne; iii. Et3N, CuI (e) TBAF, THF (f) PPh3, DIAD, AcSH (g) LAH, THF, 0 °C (h) MeI, LiOH.

Figure 29. Synthesis of methyl trans-enynthiol ether (54).

OTBDMS OH OTBDMS abc C5H11 C5H11 50% 98% 85% 64 68 69

SAc SH SMe d e C5H11 C5H11 C5H11 98% 55% 70 71 54

Key. (a) i. cyclohexene, BH3, n-pentane, 1-iodopentyne; ii. Pd(PPh3)4; iii. NaOEt/EtOH (b) TBAF, THF (c) PPh3, DIAD, AcSH (d) LAH, THF, 0 °C (e) MeI, LiOH.

Figure 30. Synthesis of enyne 59.

PPh3Br O a + H 62 46 Key. (a) NaH, THF/HMPA (10:1), rt, 24 h, 63%.

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The cross-coupling furnished both isomers through either Suzuki or modified Heck reactions. After deprotection, the secondary alcohols (66 and 69) were converted into thiolacetic acid ester (67 and 70) through a Mitsunobu reaction, which also introduced sulfur atom into these molecules. LAH reduction successfully converted the ester to a secondary thiol (71, cis-isomer was not isolated), which is readily transformed into methyl thioether (53 and 54) by using MeI and LiOH. These methyl thioethers were used after purification by flash column chromatography. In order to compare with the possible thiophene generated from these methyl thioethers, compound 46 was synthesized and used as a standard compound. The thiophene generated from 46 and from 53 or 54 share the same structure.

3-4.5 Experimental Data

Br Br NaI, Acetone I I 7 98% 7

Synthesis of 1,7- diiodoheptane 1: To a solution of NaI (19.0 g, 126.8 mmol) in dry acetone (250 mL) at rt was added 1,7-dibromoheptane (10.0 g, 36.8 mmol) over 5 min. A yellow precipitate formed immediately. The obtained yellow slurry was then stirred for 5 h. Solids were removed by filtration and the filtered was concentrated in vacuo. To the yellow solid obtained was added water (30 mL) followed by extraction with ether (5 × 25 mL). The combined organic phases were dried over MgSO4 and filtrated. The solvent was evaporated with a rotary evaporator. The crude product was purified by flash column chromatography (n-pentane) to yield 1 colorless oil (13.6 g, 98% yield). H NMR (CDCl3, 300 MHz) δ 3.16 (t, J = 7.0 Hz, 4H), 1.80 13 (quint, J = 7.0 HZ, 4H), 1.37 (m, 6H); C NMR (CDCl3, 75 MHz) δ 33.73, 30.67, 27.85, 7.50; -1 IR (neat film) νmax 2929, 2853, 1461, 1425, 1210, 1177, 720 cm .

I I 7 NC MeO2C 7 7 60 49

1 Synthesis of 9-tridecynoic acid (49) : To a suspension of LiNH2 (0.095 g, 4.2 mmol) in dry 1,4- dioxane (10 mL) at rt was added 1-pentyne (0.28 g, 4.1 mmol) as a solution in 1,4-dioxane (5.0

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mL) under N2. Then the mixture was warmed to reflux for 8 h. To the cloudy lithioalkyne suspension, 1,7-diiodoheptane (1.5 g, 4.1 mmol) was added as a solution in 1,4-dioxane (5.0 mL). The mixture was refluxed overnight, then cooled to rt. The reaction was quenched by pouring the mixture into water (100 mL). Hexanes (30 mL) were added. The layers were separated and the aqueous layer was extracted with hexanes (3× 30 mL). The combined layers were washed with water (50 mL) and brine (50 mL), then dried over MgSO4. After filtering, the solvent was removed in vacuo. The crude product 59, a yellow oil, was directly used for next step without purification.

To the solution of 1-iodo-9-heptadecyne 59 (ca. 4.1 mmol, crude product from the first step) in EtOH (95%, 15 mL) was added KCN (1.0 g, 15.4 mmol) in one portion. The mixture was then refluxed overnight. After cooling to rt, the reaction mixture was poured into cold brine (50 mL) and followed by addition of hexanes (20 mL). The layers were separated and the aqueous layer was extracted with hexanes (4 × 10 mL). The combined organic phases were washed with water

(30 mL) and brine (30 mL). After drying over MgSO4 and filtration, solvent was removed with a rotary evaporator. The crude nitrile 60 was readily used for next step without purification. Cyanide 60 obtained from previous reaction was dissolved in EtOH (100%, 10 mL) and NaOH (1.0 g, 25.0 mmol) was introduced as a solution in water (10 mL). The reaction mixture was warmed to reflux overnight. After cooling to rt, the mixture was acidified with concentrated HCl (pH 1.0). Water (30 mL) was added, followed by addition of ether (20 mL). The layers were separated and the aqueous layer was extracted with ether (5 × 15 mL). The combined organic

layers were washed with water (40 mL) and brine (40 mL). After drying over MgSO4 and filtration, solvent was removed under vacuum to get a pale yellow residue. The crude acid was used without purification for next step.

Esterification was performed in micro diazomethane generator. The crude product (c.a. 0.6 mmol) was dissolved in ether (2.0 mL) and placed into the outside tube of the generator. Diazald (0.10 g, 0.5 mmol) was placed in the inside tube, then the generator was sealed. NaOH (6 M, 2.0 mL) was then added through a syringe slowly. After the addition, the reaction was kept at rt for 10 h. Repeating the same procedure three times to drive the reaction to completion and then the reaction mixture was transferred into a RBF. The solvent was removed in vacuo. The crude

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product obtained was purified by column chromatography (hexanes/EtOAc 20:1) toy give a 1 colorless oil 49 (0.14 g, 15% overall yield). H NMR (CDCl3, 300 MHz) δ 3.64 (s, 3H), 2.28 (t, J 13 = 7.5 Hz, 2H), 2.10 (m, 4H), 1.23-1.60 (m, 16H), 0.94 (t, J = 7.2 Hz, 3H); C NMR (CDCl3, 75 MHz) δ 174.68, 80.65, 80.52, 51.82, 34.48, 29.46, 29.43, 29.16, 29.01, 25.30, 22.94, 21.16, -1 19.10, 13.86; IR (neat film) νmax 2933, 2858, 1742, 1435, 1171 cm .

Methyl (E)-10-iodo-9-(methylthio)tridec-9-enoate, methyl (E)-9-iodo-10-(methylthio)tridec- 9-enoate (50) and methyl (E)-9,10-diiodotridec-9-enoate (51): Compound 49 (0.10 g, 0.4 mmol), DMDS (0.43 g, 4.5 mmol), iodine (0.34 g, 1.3 mmol) and methanol (10 mL) were placed in a 25 mL RBF. The flask was sealed and warmed to 60 °C for 5 h. Aqueous NaS2O3 (25%, 15 mL) was added to the reaction mixture and ether (15 mL) was added. Separating layers and the aqueous layer was extracted with ether (3 × 15 mL). Combined organic phases were dried over

MgSO4 and concentrated in vacuo to give crude product as a pale yellow oil. This crude product was run through column (30:1 hexanes/EtOAc) and two main fractions were collected. The first

fraction (larger Rf value) is MeSI adduct 50 (40 mg) and the second fraction (lower Rf value) is a mixture of compound 50 and 51. It was then purified through HPLC to separate those two compounds and give compound 50 (20 mg) and compound 51 (25 mg).

Methyl (E)-10-iodo-9-(methylthio)tridec-9-enoate and methyl (E)-9-iodo-10-(methylthio)- tridec-9-enoate (50): From the 1H NMR and 13C NMR data, this fraction is a mixture of two 1 1 compounds, even though they have identical Rf values and very similar H NMR spectrum. H

NMR (CDCl3, 300 MHz) δ 3.64 (s, 3H), 2.81 (t, J = 7.0 Hz, 2H), 2.47 (t, J = 6.8 Hz, 2H), 2.27 (t, J = 6.5 Hz, 2H), 2.18 (s, 3H), 1.53 (m, 8H), 1.31 (m, 6H), 0.94 (t, J = 7.2 Hz, 1.5 H), 0.88 (t, J = 13 7.2 Hz, 1.5H); C NMR (CDCl3, 75 MHz) δ 174.69, 137.74, 137.40, 107.13, 107.08, 51.84, 45.15, 43.35, 42.89, 41.09, 34.50, 29.53, 29.45, 29.32, 28.55, 28.14, 25.32, 22.95, 21.62, 17.12, -1 17.06, 13.97, 13.21; IR (neat film) νmax 2920, 2943, 1740, 1160 cm . H-H COSY and HETCOR spectrum of this mixture are available.

1 Methyl (E)-9,10-diiodotridec-9-enoate (51): H NMR (CDCl3, 300 MHz) δ 3.65 (s, 3H), 2.65 (m, 4H), 2.29 (t, J = 6.5 Hz, 2H), 1.59 (m, 8H), 1.31 (m, 6H), 0.93 (t, J = 7.2 Hz, 3 H); 13C NMR

(CDCl3, 75 MHz) δ 174.66, 102.45, 102.20, 52.90, 51.86,51.24, 34.49, 29.47, 29.42, 28.48,

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-1 28.44, 25.31, 22.01, 13.19; IR (neat film) νmax 2928, 2854, 1740, 1160 cm . H-H COSY and HETCOR spectrum of this mixture are available.

OH PPh3, Br2, 0 °C Br

CH2Cl2 61 98%

Synthesis of 1-bromo-3-heptyne (61): To a solution of Ph3P (3.15 g, 12.0 mmol) in dry CH2Cl2

(25 mL) at 0 °C was added Br2 (0.6 mL, 11.9 mmol) slowly. The bright yellow suspension obtained was stirred for 1 h then 3-heptyn-1-ol (0.88 g, 7.8 mmol) was introduced through syringe, followed immediately by addition of pyridine (0.9 mL, 11.2 mmol). After stirring for 1 h, the reaction mixture was warmed to rt for 5 h. The solvent was removed with a rotary evaporator and white solids were obtained. The white crystals was washed with n-pentane (2 × 25 mL) and removed by filtration. The clear filtrate collected was concentrated in vacuo to give crude product. Purification by flash column chromatography (petroleum ether) afforded pure bromide 1 61 (1.2 g, 88% yield). H NMR (CDCl3, 300 MHz) δ 3.40 (t, J = 7.4 Hz, 2H), 2.69 (tt, J = 7.4, 2.2 Hz, 2H), 2.11 (tt, J = 7.2, 2.2 Hz, 2H), 1.49 (sextet, J = 7.2 Hz, 2H), 0.95 (t, J = 7.3 Hz, 3H); 13 C NMR (CDCl3, 75 MHz) δ 82.91, 77.42, 30.80, 23.75, 22.62, 21.07, 13.83; IR (neat film) νmax 2965, 2935, 2873, 2211, 1772, 1212, 641 cm-1.

1. PPh , MeOH, 100 °C Br 3 MeO

2. MeO2(CH2)4CHO 61 NaH, THF/HMPA O 45

Synthesis of methyl (6Z)-tridecen-9-ynote (45): A solution of PPh3 (1.72 g, 6.6 mmol) and 1- bromo-3-heptyne in dry methanol (7.0 mL) was placed in a sealed-tube. After three freeze-thaw-

freeze cycles in liquid N2, the tube was sealed and heated to 100 °C for 12 h. After cooling, the solvent was removed with a rotary evaporator. The thick residue obtained was washed with dry ethyl ether (3 × 10 mL). Finally, the oily material was dried under vacuum (0.05 mm Hg) at rt for 8 h to afford pale yellow crystals 62 (2.42 g, 88% yield).

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To a suspension of 62 (0.91 g, 2.1 mmol) in dry ethyl ether (10 mL), n-BuLi (1.42 M, 1.5 mL, 2.1 mmol) was added at rt. The yellow suspension turned into brown at once and the mixture was stirred for 1 h. Aldehyde (0.30 g, 2.1 mmol) was introduced dropwise through a syringe. The brown suspension turned into white suspension immediately. After stirring for 1.5 h, reaction mixture was sealed and warmed to 50 °C for 20 h. After cooling to rt, white solid was removed by filtration through Celite, and washed with hexanes (2 × 5.0 mL). The combined filtrates were concentrated in vacuo to give crude product. Purification by flash column chromatography (20:1 1 hexanes/EtOAc) yielded pure ester 45 (0.31 g, 70% yield). H NMR (CDCl3, 300 MHz) δ 5.41 (m, 2H), 3.64 (s, 3H), 2.87 (m, 2H), 2.29 (t, J = 7.5 Hz, 2H), 2.09 (m, 4H), 1.62 (q, J = 7.8 Hz, 2H), 1.48 (sextet, J = 7.5 Hz, 2H), 1.38 (q, J = 7.2 Hz, 2H), 0.94 (t, J = 7.2 Hz, 3H); 13C NMR

(CDCl3, 75 MHz) δ 174.47, 130.93, 125.96, 80.36, 78.76, 51.84, 34.33, 29.24, 27.10, 24.93, -1 22.81, 21.17, 17.56, 13.86; IR (neat film) νmax 3006, 2934, 1740, 1436, 1201 cm ; HRMS (EI) + m/z [M ] calcd for C14H22O2 222.1614, found 222.1603.

1 Methyl 5-(5-butylthiophen-2-yl)pentanoate-d0 (47b): H NMR (CDCl3, 300 MHz) δ 6.53 (t, J = 3.6 Hz, 2H), 3.64 (s, 3H), 2.73 (q, J = 7.5 Hz, 4H), 2.32 (t, J = 6.9 Hz, 2H), 1.60 (m, 6H), 1.36 13 (sextet, J = 7.5 Hz, 2H), 0.90 (t, J = 7.5 Hz, 3H); C NMR (CDCl3, 75 MHz) δ 174.41, 143.91, 142.70, 123.98, 123.75, 51.90, 34.21 (2C), 31.49, 30.22, 30.18, 24.78, 22.61, 14.22; IR (neat -1 + film) νmax 3005, 2931, 2860, 1741, 1436, 1170, 798 cm ; HRMS (EI) m/z [M ] calcd for

C14H22O2S 254.1335, found 254.1309.

1 Methyl 5-(5-butylthiophen-2-yl)pentanoate-d1: H NMR (CDCl3, 300 MHz) δ 6.53 (t, J = 3.6 Hz, 2H), 3.65 (s, 3H), 2.73 (q, J = 7.8 Hz, 3H), 2.32 (t, J = 6.9 Hz, 2H), 1.62 (m, 6H), 1.39 13 (sextet, J = 7.0 Hz, 2H), 0.90 (t, J = 7.5 Hz, 3H). C NMR (CDCl3, 75 MHz) δ 174.44, 143.93, 142.72, 124.00, 123.77, 51.93, 34.24, 34.14, 31.51, 30.25, 30.20, 24.80, 22.63, 14.52.

1 Methyl 5-(5-butylthiophen-2-yl)pentanoate-d2: H NMR (CDCl3, 300 MHz) δ 6.53 (t, J = 3.6 Hz, 2H), 3.65 (s, 3H), 2.74 (t, J = 7.2 Hz, 2H), 2.32 (t, J = 6.9 Hz, 2H), 1.65 (m, 6H), 1.37 13 (sextet, J = 6.0 Hz, 2H), 0.90 (t, J = 7.2 Hz, 3H). C NMR (CDCl3, 75 MHz) δ 174.43, 143.92,

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2 142.71, 124.00, 123.77, 51.91, 34.23, 34.00, 31.51, 30.19, 24.80, 22.56, 14.24. H NMR (CDCl3, 300 MHz) δ 6.60 (br), 2.73 (br).

OH OTBDMS TBDMSCl, CH2Cl2 imidazole 63 64 92%

Synthesis of 3-tert-butyldimethylsiloxy-1-octyne (64): To a solution of 1-octyn-3-ol 63 (2.4 g, 19.3 mmol) in methylene chloride (70 mL) was added imidazole (2.6 g, 38.4 mmol) and followed by the addition of TBDMSCl (3.2 g, 21.2 mmol). The pale yellow solution turned to white suspension immediately. After stirring for 2 h, the reaction was quenched with water (30 mL). The layers were separated and aqueous phase was extracted with CH2Cl2 (3 × 15 ml). The combined organic phases were dried over MgSO4 and concentrated under vacuum. The crude product was purified by flash chromatography (30:1 hexanes/ether) to get pure 64 (3.77 g, 81% 1 yield). H NMR (CDCl3, 300 MHz) δ 4.30 (td, J = 6.3, 1.8 Hz, 1H), 2.35 (d, J = 2.1 Hz, 1H), 1.63 (q, J = 6.3 Hz, 2H), 1.30 (m, 6H), 0.87 (m, 12H), 0.10 (s, 3H), 0.08 (s, 3H); 13C NMR

(CDCl3, 75 MHz) δ 86.21, 72.20, 63.18, 38.94, 31.82, 26.17 (3C), 25.17, 22.95, 18.62, 14.38, - -1 4.18, -4.68; IR (neat film) νmax 3313, 2957, 2859, 1472, 1253, 1092, 838, 778 cm .

1. BuLi, Et O, -78 °C OTBDMS 2 2. I2, Et2O I 3. cyclohexene, 4. AcOH, ethanolamine OTBDMS 64 83% 65

Synthesis of (Z)-3-tert-butyldimethylsiloxy-1-iodooctene (65): To a solution of cyclohexene

(0.78 mL, 7.7 mmol) in n-pentane (15 mL) at −78 °C was added BH3 (10 M in Me2S, 0.39 mL, 3.9 mmol) slowly. After stirring for 10 min the reaction mixture was warmed to rt for 1 h, and a white slurry was obtained. Compound 64 (1.19 g, 3.3 mmol) was added as a solution in n- pentane (5.0 mL) at rt and the mixture was kept stirring for 2 h. Then it was cooled to 0 °C, and AcOH (0.5 mL) was added slowly. After 30 min, 2-amino-ethanol (0.5 mL) was added followed by (after 15 min) addition of water (20 mL). The layers were separated. After general work-up

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procedures, the crude product was purified by flash column chromatography (20:1 n- 1 pentane/ether) to get pure 65 (1.1 g, 88% yield). H NMR (CDCl3, 300 MHz) δ 6.15 (m, 2H), 13 4.31 (m, 1H), 1.39 (m, 8H), 0.87 (m, 12H), 0.07 (s, 3H), 0.03 (s, 3H); C NMR (CDCl3, 75 MHz) δ 145.24, 79.90, 75.84, 37.20, 32.13, 26.24 (3C), 25.00, 23.00, 18.50, 14.42, -3.84, -4.30; IR -1 (neat film) νmax 2956, 2857, 1471, 1257, 1085, 837, 776 cm .

1. Pd(PPh ) I 3 4 C5H11 2. 1-pentyne, Et3N, CuI OTBDMS OH 3. TBAF, THF 65 66% 66

Synthesis of (Z)-tridec-6-en-4-yn-8-ol (66): To a solution of compound 65 (0.53 g, 1.5 mmol)

in benzene (5.0 mL) at rt was added Pd(PPh3)4 (0.06 g, 0.05 mmol) and the mixture was stirred for 1 h. Then 1-pentyne (0.22 g, 3.2 mmol) in Et3N (3.5 mL) was added dropwise and followed by addition of CuI (0.10 g, 0.5 mmol). The yellow solution was kept stirring at rt overnight.

After that, ether (20 mL) was added and the mixture was poured into saturated NH4Cl (40 mL). After general work-up procedure, the crude product was purified by flash column (hexanes) to get pure product (0.44 g, 66% yield).

The mixture of previous product (0.44 g, 1.4 mmol), TBAF (1.0 M in THF, 2.5 mL) and THF (5.0 mL) was stirred at rt in a plastic tube for 2 h. Then the solvent was removed and the crude product was purified by column chromatography (10:1 hexanes/EtOAc), to yield pure 66 (0.22 g, 1 99% yield). H NMR (CDCl3, 300 MHz) δ 5.78 (dd, J = 10.8, 8.1 Hz, 1H), 5.52 (dm, J = 12 Hz, 1H), 4.62 (qd, J = 7.2, 1.0 Hz, 1H), 2.29 (td, J = 6.9, 2.1 Hz, 2H), 1.54 (m, 4H), 1.32 (m, 6H), 13 0.98 (t, J = 7.5 Hz, 3H), 0.87 (t, J = 6.9 Hz, 3H); C NMR (CDCl3, 75 MHz) δ 144.02, 110.44,

96.10, 76.58, 70.11, 36.59, 31.74, 24.84, 22.59, 22.13, 21.49, 14.02, 13.51; IR (neat film) νmax 3346, 2960, 2932, 2860, 2213, 1459, 955 cm-1; HRMS (Electrospray) m/z [M + Na+] calcd for: + C13H22ONa 217.1563, found 217.1559.

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C H C5H11 5 11 PPh3, DIAD, AcSH OH 85% S 66 67 O

Synthesis of (Z)-tridec-6-en-4-yn-8-yl thioacetate (67): To a solution of PPh3 (0.97 g, 3.7 mmol) in dry THF (12 mL) at 0 °C was added DIAD (0.75 g, 3.7 mmol) dropwise. After stirring for 30 min, a white slurry was obtained. To this white slurry a mixture of the secondary alcohol 66 (0.36 g, 1.9 mmol) and thiolacetic acid (0.28 g, 3.7 mmol) in THF (0.2 mL) was added. The final mixture was stirred at 0 °C for 1 h to give a greenish-black slurry. The reaction was then allowed to warm to rt for 1 h to give a clear yellow solution. The reaction was quenched by adding aqueous NaHCO3 (15 mL) and ethyl ether (20 mL). The layers were separated and the organic phase was washed with aqueous NaHCO3 (3 × 15 mL). After drying over MgSO4 and filtering, the solvent was removed with a rotary evaporator. The residue was dissolved in petroleum ether (3.0 mL) and run through silica gel pad (petroleum ether). The collected solution was concentrated in vacuo to give a pale yellow oil. The crude product was purified by flash column chromatography (100:1 petroleum ether/ether) to afford pure 67 (0.4 g, 85% yield). 1H

NMR (CDCl3, 300 MHz) δ 5.70 (t, J = 10.5 Hz, 1H), 5.50 (dt, J = 8.4, 2.1 Hz, 1H), 4.56 (m, 1H), 2.29 (td, J = 7.8, 2.1 Hz 2H), 2.28 (s, 3H), 1.57 (m, 4H), 1.28 (br, 6H), 0.99 (t, J = 7.5 Hz, 3H), 13 0.86 (t, J = 6.6 Hz, 3H); C NMR (CDCl3, 75 MHz) δ 194.74, 140.02, 111.58, 97.35, 76.52,

43.68, 35.21, 31.39, 30.62, 26.67, 22.48, 22.14, 21.62, 14.00, 13.50; IR (neat film) νmax 3006, 2931, 2859, 2250, 1693, 1108, 951, 631 cm-1; HRMS (Electrospray) m/z [M + Na+] calcd for + C15H24OSNa 275.1440, found 275.1446.

C H 5 11 1. LAH, THF, 0 °C C5H11 S 2. MeI, LiOH SMe 53% 53 67 O

Synthesis of methyl (Z)-tridec-6-en-4-yn-8-yl thioether (53): To a suspension of LAH (0.045 g, 1.18 mmol) in dry Et2O (5.0 mL) at 0 °C, thioester (0.30 g, 1.19 mmol) was added slowly as a solution in dry Et2O (5.0 mL). After stirring for 30 min, the reaction mixture was allowed to

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warm to rt for 30 min. The mixture was subsequently cooled to 0 °C and HCl (3 M, 5.0 mL) was added slowly to consume the excess LAH. The layers were separated, and the aqueous layer was extracted with ether (3 × 3 mL). The combined organic phases were washed with aqueous

NaHCO3 (10 mL) and brine (10 mL) and dried over MgSO4. After the solvent was removed, the crude product was obtained as pale yellow oil.

To the solution of crude product (obtained above) in dry THF (5.0 mL) at rt was added LiOH (0.11 g, 4.6 mmol, preactivated at 100 °C for at least 1 h) and MeI (0.17 g, 1.2 mmol). The final mixture was stirred for 2.5 h, and distilled water (15 mL) was added followed by addition of ether (10 mL). The layers were separated and the aqueous layer was extracted with ether (3 × 10

mL). The combined organic layers were washed with brine (10 mL), dried over MgSO4, and filtered. The solvent was removed with a rotary evaporator to give crude product. Purification by flash column chromatography (100:1 petroleum ether/ether) yielded pure 53 (0.14 g, 53% yield). 1 H NMR (CDCl3, 300 MHz) δ 5.53 (m, 2H), 3.79 (m, 1H), 2.27 (td, J = 6.9, 1.4 Hz, 2H), 2.03 (s, 3H), 1.52 (m, 4H), 1.26 (br, 6H), 0.97 (t, J = 7.5 Hz, 3H), 0.86 (t, J = 6.9 Hz, 3H); 13C NMR

(CDCl3, 75 MHz) δ 142.54, 110.44, 95.27, 77.59, 46.08, 24.46, 31.93, 27.35, 22.89, 22.65, 21.89, -1 14.43 (2C), 13.94; IR (neat film) νmax 3019, 2930, 2858, 2219, 1464, 954 cm ; HRMS (EI) m/z + 1 1 [M ] calcd for: C14H24S 224.1593, found 224.1601. DEPT135, H- H COSY and HETCOR spectra are available.

OTBDMS OTBDMS 1. cyclohexene, BH3, n-pentane, 1-iodopentyne C5H11

2. Pd(PPh3)4, NaOEt/EtOH 64 50% 68

Synthesis of (E)-8(-tert-butyldimethylsiloxy)tridec-6-en-4-yne (68): To the solution of cyclohexene (0.4 mL, 4.0 mmol) in n-pentane (5.0 mL) at −78 °C was added BH3 (10 M in Me2S, 0.2 mL, 2.0 mmol). After 10 min, the mixture was warmed to rt for 1 h. Compound 64 (0.48 g. 2.0 mmol) was then added by syringe, which caused the white slurry to turn clear. Stirring was continued for 1 h (Solution I).

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In a second flask 1-iodopentyne (0.39 g, 2.0 mmol) was stirred with benzene (5.0 mL) and

Pd(PPh3)4 (53 mg, 0.05 mmol) at 0 °C for 30 min. Solution I was added through a cannula to the second flask followed by the addition of NaOEt (0.27 g in 4.0 mL EtOH). The reaction mixture was allowed to warm to rt for 30 min followed by heating to reflux for 20 h. The reaction was

quenched with NaOH (2 M, 1.0 mL). H2O2 (50%, 1.0 mL) was added after 30 min. After the general work-up procedures, the crude product was purified by flash column chromatography 1 (hexanes) to get pure 68 (0.38 g, 50% yield). H NMR (CDCl3, 300 MHz) δ 6.00 (dd, J = 15.8, 5.7 Hz, 1H), 5.69 (dq, J = 15.8, 1.9 Hz, 1H), 4.10 (q, J = 5.7 Hz, 1H), 2.24 (td, J = 7.1, 2.0 Hz, 2H), 1.52 (m, 4H), 1.24 (br, 6H), 0.97 (t, J = 7.5 Hz, 3H), 0.86 (m, 12 H), 0.02 (s, 3H), 0.01 (s, 13 3H); C NMR (CDCl3, 75 MHz) δ 145.52, 109.40, 90.66, 79.31, 73.12, 38.38, 32.23, 26.27,

25.05, 22.99, 22.61, 21.83, 18.61, 14.42, 13.93, -4.04, -4.45; IR (neat film) νmax 2930, 2858, 2215, 1710, 1464, 1255, 837 cm-1.

OTBDMS OH TBAF, THF C5H11 C5H11 98% 68 69

Synthesis of (E)-tridec-6-en-4-yn-8-ol (69): A mixture of compound 68 (0.25 g, 0.8 mmol), TBAF (1.0 M in THF, 2.0 mL), and THF (4.0 mL) was stirred in a plastic tube at rt for 2 h. After the reaction was complete, the solvent was removed and the crude product was purified by flash column chromatography (10:1 hexanes/EtOAc) to get pure 69 (0.154 g, 98% yield). 1H NMR

(CDCl3, 300 MHz) δ 6.02 (dd, J = 15.9, 6.4 Hz, 1H), 5.66 (dq, J = 15.9, 2.2 Hz, 1H), 4.10 (qd, J = 6.4, 1.0 Hz, 1H), 2.26 (td, J = 7.1, 2.1 Hz, 2H), 1.52 (m, 5H), 1.30 (br, 6H), 0.97 (t, J = 7.5 Hz, 13 3H), 0.86 (t, J = 6.9 Hz, 3H); C NMR (CDCl3, 75 MHz) δ 144.61, 110.93, 91.50, 78.90, 72.91,

37.37, 32.11, 25.35, 22.97, 22.55, 21.78, 14.41, 13.90; IR (neat film) νmax 3346, 2932, 2860, 2215, 1464, 956 cm-1.

OH SAc

PPh3, DIAD, AcSH C5H11 C5H11 85% 69 70

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Synthesis of (E)-tridec-6-en-4-yn-8-yl thioacetate (70): Following the similar procedure for 1 making compound 67, compound 70 was synthesized in 85% yield. H NMR (CDCl3, 300 MHz) δ 5.89 (dd, J = 15.0, 9.0 Hz, 1H), 5.66 (dq, J = 15.0, 1.2 Hz, 1H), 4.02 (q, J = 9.0 Hz, 1H), 2.28 (s, 3H), 2.26 (td, J = 7.2, 2.1 Hz, 2H), 1.53 (m, 4H), 1.25 (br, 6H), 0.96 (t, J = 7.5 Hz, 3H), 0.85 13 (t, J = 7.2 Hz, 3H); C NMR (CDCl3, 75 MHz) δ 195.04, 141.44, 112.19, 91.64, 78.92, 46.42,

34.34, 31.79, 31.10, 27.12, 22.84, 22.52, 21.81, 14.38, 13.92; IR νmax (neat film) 3005, 2931, 2872, 2254, 1694, 1459, 1109, 951, 631 cm-1; HRMS (EI) m/z [M + Na+] calcd for: + C15H24OSNa 275.1400, found 275.1450.

SAc SH LAH, THF, 0 °C C5H11 C5H11 98% 70 71

Synthesis of (E)-tridec-6-en-4-yn-8-thiol (71): To a stirred suspension of LAH (0.045 g, 1.2 mmol) in dry Et2O (5.0 mL) at 0 °C was slowly added thioester (0.30 g, 1.2 mmol) as a solution in dry Et2O (5.0 mL). After 30 min, the reaction mixture was allowed to warm to rt for 30 min. During the workup, the mixture was cooled to 0 °C and HCl (3 M, 5.0 mL) was added slowly to consume the excess LAH. The phases were separated and aqueous layer was extracted with ether

(3 × 3 mL). The combined organic phases were washed with aqueous NaHCO3 (10 mL) and brine (10 mL) and were dried over MgSO4. After solvent was removed, crude product was obtained as pale yellow oil. The crude product was purified through column chromatography 1 (5:1 hexanes/EtOAc) to give pure 71 (0.25 g, 98% yield). H NMR (CDCl3, 300 MHz) δ 5.75 (dd, J = 15.6, 9.6 Hz, 1H), 5.38 (td, J = 15.5, 2.1 Hz, 1H), 2.94 (q, J =8.4 Hz, 1H), 2.24 (td, J = 7.2, 1.8 Hz, 2H), 1.94 (s, 1H), 1.52 (m, 4H), 1.25 (br, 6H), 0.96 (t, J = 7.2 Hz, 3H), 0.84 (t, J = 13 6.9 Hz, 3H); C NMR (CDCl3, 75 MHz) δ 142.97, 110.87, 90.50, 78.94, 50.08, 34.39, 31.95,

27.42, 22.88, 22.57, 21.78, 14.42, 13.94; IR (neat film) νmax 2930, 2858, 2218 (C≡C), 1459, 954 cm-1. DEPT135, 1H-1H COSY and HETCOR are available.

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SH SMe MeI, LiOH C H C5H11 5 11 55%

71 54

Synthesis of methyl (E)-tridec-6-en-4-yn-8-yl thioether (54): Purified 71 (0.25 g, 1.2 mmol) was dissolved in dry THF (5.0 mL) and LiOH (0.11 g, 4.6 mmol, preactivated at 100 °C for at least 1 h) and MeI (0.17 g, 1.2 mmol) were added sequentially. The final mixture was stirred at rt for 2.5 h. Distilled water (15 mL) was added followed with ether (10 mL). The phases were separated and aqueous layer was extracted with ether (3 × 10 mL). The combined organic layers

were washed with brine (10 mL) and dried over MgSO4. The solvent was evaporated with rotary evaporator to give crude product that was purified by flash column chromatography (100:1 1 petroleum ether/ether) to yield pure 54 (0.14 g, 53% yield). H NMR (CDCl3, 300 MHz) δ 5.76 (dd, J = 9.6 Hz, 1H), 5.39 (dt, J = 15.6,1.8 Hz 1H), 3.01 (q, J = 8.3 Hz, 1H), 2.26 (td, J = 6.9, 2.0 Hz, 2H), 1.96 (s, 3H), 1.53 (m, 4H), 1.27 (br, 6H), 0.97 (t, J = 7.2 Hz, 3H), 0.85 (t, J = 6.9 Hz, 13 3H); C NMR (CDCl3, 75 MHz) δ 142.98, 110.87, 90.54, 78.95, 50.09, 34.40, 31.95, 27.42, -1 22.88, 22.57, 21.79, 14.42, 14.34, 13.94; IR (neat film) νmax 2930, 2871, 2215, 1459, 954 cm ; + 1 1 HRMS (EI) m/z [M ] calcd for C14H24S 224.1593, found. 224.1601. DEPT135, H- H COSY and HETCOR are available.

PPh Br 3 O NaH + H THF/HMPA 62 46 63%

Synthesis of (E)-tridec-7-en-4-yne (46): To the mixture of phosphonium salt 62 (1.05 g, 2.4 mmol) and hexanal (0.24 g, 2.4 mmol) in 10:1 THF (15 mL)/HMPA (1.5 mL) at rt was added NaH (95%, 61 mg, 2.4 mmol) in one portion. The obtained pale brown suspension was stirred at rt for 20 h. The reaction mixture was filtered through Celite and washed with n-pentane to give a brown solution. Solvent was removed with a rotary evaporator and the crude product was then purified by flash column chromatography (n-pentane) to afford pure 46 (0.27 g, 63% yield). 1H

NMR (CDCl3, 300 MHz) δ 5.41 (m, 2H), 2.87 (m, 2H), 2.10 (tt, J = 7.1, 2.4 Hz, 2H), 2.02 (q, J =

89

7.2 Hz, 2H), 1.49 (sextet, J = 7.3 Hz, 2H), 1.29 (br, 6H), 0.94 (t, J = 7.3 Hz, 3H), 0.86 (t, J = 6.9 13 Hz, 3H); C NMR (CDCl3, 75 MHz) δ 131.74, 125.40, 80.24, 78.97, 31.85, 29.46, 27.46, 22.93, -1 22.83, 21.19, 17.55, 14.43, 13.86; IR (neat film) νmax 30.19, 2960, 2929, 2858, 1465 cm .

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