Scalable and efficient synthesis of the mycolactone core

Abstract

A highly efficient, scalable, and stereoselective synthesis of the mycolactone core is reported. The synthesis consists of 14 longest linear steps, with 19% overall yield.

1. Introduction

Buruli ulcer is a devastating, but neglected disease of the skin and soft tissue caused by M. ulcerans, the third most common mycobacterial pathogen of humans after M. tuberculosis and M. leprae.¹ The disease is characterized by the formation of necrotic lesions that are usually painless and can extend to over 15% of a patient’s skin surface if left untreated. In 1999, Small and co-workers isolated two equilibrating toxins, mycolactone A and B, from West African strains of M. ulcerans and later demonstrated with animal tests that these toxins are responsible for the observed pathology of the disease.² The gross structures of mycolactone A and B were elucidated primarily through 2-D NMR experiments,³ and their stereochemistry was subsequently predicted via the NMR database approach and confirmed by total synthesis.⁴ In addition, several other groups have described syntheses of the mycolactone core or unsaturated fatty acid side chain.⁵ Because mycolactone A and B exist as an equilibrium mixture of geometric isomers, they are referred to as mycolactone A/B in this paper.

Since the structure of mycolactone A/B was reported, five more structurally distinct mycolactones have been described from different strains of M. ulcerans, and close relatives from fish and frogs (Figure 1). All of the mycolactones described to date are composed of a common and conserved 12-membered macrolactone core and a highly unsaturated fatty acid side chain that differs amongst the members of this class of natural products. Mycolactone C, which lacks a C12’ hydroxyl group, was identified as the major toxin in Australian strains of M. ulcerans in 2003.⁶ Later, in 2005, a toxin bearing methyl substitution at the C2’ position, mycolactone D, was proposed as the major metabolite in Chinese strains of M. ulcerans.⁷ Mycobacteriosis was then discovered in African clawed frogs (Xenopus laevis and Xenopus tropicalis), leading to the isolation of the tetraene-containing mycolactone E from M. liflandii.⁸

Figure 1.

Structures of the mycolactones.

Most recently, mycolactone F has been isolated from mycobacterial pathogens infecting saltwater fish,⁹ and mycolactone dia-F has been isolated from pathogens of freshwater fish.¹⁰

While much has been learned about Buruli ulcer throughout the last decade, many questions still remain unanswered. For example, many researchers have wondered whether it would be feasible to develop a diagnostic method for the early detection of Buruli ulcer that relies on recognition of mycolactone toxins in human skin.^1a Other highly important research areas are centered on the study of the trafficking and cellular actions of mycolactones,¹¹ as well as the identification of the cellular target of these toxins. In order for research into these, and other, fields of study to be practically conducted, we feel it is necessary to have reliable and scalable methods for the production of mycolactones of high purity.^1e This is particularly important considering that the mycolactones isolated from laboratory-scale cultivation of these slow-growing mycobacteria are often scant in quantity (typically microgram) and difficult to isolate cleanly given that mycolactones of varying side chain structures are often present in the crude lipid extracts from the cultured mycobacteria.^1e

2. Results and discussion

Based on the knowledge accumulated through our first- and second-generation total syntheses,⁴ we recognized the need for improvements in two areas. First, we wished to develop shorter, more efficient, and easily scalable syntheses of the fragments used to assemble the mycolactone core. Second, we wished to prepare the mycolactone core with a high level of stereochemical homogeneity, i.e., no contamination of minor stereoisomers.

2.1. Synthesis of the C1–C7 fragment

We began our synthesis of the mycolactone core by turning our attention to the C1–C7 fragment (Scheme 1). Starting from known aldehyde 1,¹² the C5 and C6 stereocenters were installed using Brown crotylboration¹³ in 86% ee as determined by (R)- and (S)-Mosher’s ester analysis. Given that we wished to prepare the mycolactone core in optically pure form, it was important to be mindful of the minor enantiomer present in 2, as downstream products resulting from this contaminant would need to be removed at some later stage in the synthesis. Notably, we mentioned in our previous synthesis that a significant amount of the δ-lactone was often formed during the Brown crotylboration to convert 1 to 2;^4d however, in this work we found that shortened reaction times during the oxidative workup step allowed us to avoid this byproduct altogether.

Scheme 1.

Reagents and conditions: (a) Z-2-butene, t-BuOK, n-BuLi, (+)-(Ipc)₂BOMe, BF₃·OEt₂, THF, −78 °C; then NaOH, H₂O₂, 1h, 78%, 86% ee; (b) PMB-trichloroacetimidate, Sc(OTf)₃, PhMe, 0 °C, 81%; (c) (i) OsO₄, NMO, 3:1 acetone-H₂O; (ii) Pb(OAc)₄, PhH, rt; (iii) NaBH₄, MeOH, 0 °C, 71% for 3 steps; (d) Ph₃P, imidazole, I₂, CH₂CL₂, rt, 92%. PMB=para-methoxybenzyl, THF=tetrahydrofuran, Tf=trifluoromethane-sulfonate, NMO=N-methylmorpholine N-oxide, Ac=acetyl.

To continue with the synthesis, we wished to convert alcohol 2 to the corresponding p-methoxybenzyl (PMB) ether. Under the majority of conditions examined, however, mixtures of the desired product 3 and the δ-lactone were observed. Fortunately, on treatment of 2 with p-methoxybenzyl trichloroacetimidate in the presence of a catalytic amount of scandium(III) triflate, we were able to obtain 3 in 81% yield without observing a significant quantity of the δ-lactone. Using standard conditions (OsO₄/NMO followed by Pb(OAc)₄), alkene 3 was oxidatively cleaved to furnish the aldehyde, which was reduced to alcohol 4 with NaBH₄ in good overall yield (71% for 3 steps). Lastly, 4 was converted to the corresponding alkyl iodide 5, providing the desired C1–C7 fragment as a 93:7 mixture with its corresponding enantiomer, ent-5.

2.2. Synthesis of the C8–C13 fragment

Next we focused on a new synthesis of the C8–C13 segment (Scheme 2). Subjection of readily available allylic alcohol 7, prepared from 6 by the protocol of Jamison,¹⁴ to Sharpless asymmetric epoxidation¹⁵ provided the expected epoxide 8. The optical purity of 8 thus obtained was found to be 80% ee.¹⁶ Fortunately, we were able to crystallize and recrystallize 8 from acetone/hexanes (10:1) or toluene/hexanes (1:1) to obtain the optically pure 8 (>99% ee) in 70% yield.

Scheme 2.

Reagents and conditions: (a) LiAlH₄, Et₂O, rt, 98%; (b) Ti(O-i-Pr)₄, TBHP, (−)-DET, 4Å MS, CH₂CL₂, −25 °C, 70%; then recrystallization from 10:1 hexanes/acetone, −24 °C (c) MeLi, CuCN, THF, −20 °C, 79%; (d) p-TsOH, cyclopentanone, rt, 89%; (e) Cp₂Zr(H)Cl, THF, 50 °C, then I₂, THF, 0 °C, 68%. TBHP=tert-butyl hydroperoxide, DET=diethyl tartrate, MS=molecular sieves, p-TsOH=para-toluenesulfonic acid.

As in examples previously reported,¹⁷ optically pure 8 was treated with the higher-order cuprate prepared from CuCN and MeLi to furnish 1,3-diol 9 exclusively. Upon treatment with p-TsOH and cyclopentanone, 9 was uneventfully converted to cyclopentylidene acetal 10.

The last step required for the synthesis of the C8–C13 fragment was the conversion of alkyne 10 to vinyl iodide 11. We examined several protocols for this transformation, all of which relied on the use of the Schwartz hydrozirconation reagent, [Cp₂Zr(H)Cl].¹⁸ First, we attempted hydrozirconation using Cp₂Zr(H)Cl prepared in situ from DIBAL-H and Cp₂ZrCl₂,¹⁹ but found that this reagent gave poor regioselectivity (1:1.3 11/12 at room temperature and 6:1 11/12 at 50 °C) as well as multiple byproducts. We eventually found that the Schwartz reagent, prepared from Red-Al and Cp₂ZrCl₂ following the reported protocol,^15c gave improved regioselectivity as well as attenuated levels of byproduct formation. After optimization of temperature, reagent equivalents, and reaction duration, we were consistently able to obtain close to 70% yield of 11 as a 22:1 mixture with 12. It is noteworthy that 8 was obtained in optically pure form and therefore 11 was free from any enantiomeric contaminants.

2.3. Synthesis of the C14–C20 fragment

Having completed syntheses of the C1–C7 and C8–C13 fragments, we set out to prepare the remaining C14–C20 segment required for the synthesis of the mycolactone core (Scheme 3). The synthesis commenced with (R)-propylene oxide 13 (Alfa Aesar, >99% optical purity), which was opened with the lithium anion of ethynyltrimethylsilane. The resultant secondary alcohol was protected as a TBS ether to furnish 14. Upon treatment with NIS and AgNO₃, 14 was converted to the alkynyl iodide, which underwent subsequent hydroboration with Cy₂BH to provide 15 after quenching with acetic acid. The overall yield for these four steps was 95%.

Scheme 3.

Reagents and conditions: (a) (i) n-BuLi, ethynyltrimethyl-silane, BF₃·OEt₂, Et₂O, −78 °C; (ii) TBSCl, imidazole, DMF, rt; (b) (i) NIS, AgNO₃, DMF, rt; (ii) Cy₂BH, THF, 0 °C, then AcOH, 95% for 4 steps; (c) propyne, PdCl₂(PPh₃)₂, CuI, Et₂NH, rt, 96%; (d) Ti(O-i-Pr)₄, 17, 4,4'-thiobis(6-t-butyl-m-cresol), H₂O₂, phosphate buffer, CH₂CL₂, 40 °C, 91%, ≥50:1 dr; (e) LiAlMe₄, BF₃·OEt₂, CH₂CL₂, −78 °C, 87%; (f) TBSCl, imidazole, DMF, rt, 99%; (g) Cp₂Zr(H)Cl, THF, 50 °C, then I₂, THF, 0 °C, 68%. TBS=tert-butyldimethylsilyl, DMF=N,N-dimethylformamide, NIS=N-iodosuccinimide, Cy=cyclohexyl.

To continue the synthesis of the C14–C20 fragment, vinyl iodide 15 was coupled with propyne under Sonogashira conditions²⁰ to produce enyne 16. At this stage we wished to oxidize the cis-olefin in 16 in a stereoselective manner. We hoped that an enantioselective epoxidation could override the pre-existing stereocenter at C19, thereby allowing us to introduce the C-O bond at C16 stereoselectively. A literature search revealed ample precedent for enyne epoxidation,²¹ and we felt that ligand 17 recently reported by Katsuki and coworkers could meet our need.^20b Indeed, on treatment with Ti(O-i-Pr)₄ and H₂O₂ in the presence of 17, cis-olefin 16 was smoothly converted to propargylic epoxide 18 in excellent yield with outstanding selectivity (≥50:1 dr, as estimated by ¹H NMR). Satisfied with this result, we proceeded forward with the synthesis of the C14–C20 fragment.

Upon treatment with LiAlMe₄, a highly regio- and stereospecific opening of the propargylic epoxide in 18 was observed to furnish homopropargylic alcohol 19.²² To complete the synthesis, alcohol 19 was protected as the TBS ether 20, and subsequent hydrozirconation-iodination under the same conditions optimized for the conversion of 10 → 11 provided vinyl iodide 21, this time with complete regioselectivity.

2.4. Assembly of the mycolactone core

With scalable and efficient routes established for the requisite fragments, we turned our attention to their assembly to construct the mycolactone core. The first two fragments coupled were 5 and 11, and this was accomplished using a Negishi coupling (Scheme 4).²³ Notably, we chose to prepare the alkylzinc iodide species via zinc insertion using an active Zn-Cu couple²⁴ rather than by transmetallation from Li to Zn due to the electrophilic ester present in 5. In the event, the coupling proceeded smoothly in the presence of eight equivalents of LiCl in N-methylpyrrolidinone (freshly distilled) to furnish 22 in excellent yield (95%). Notably, we were able to detect minor diastereoisomers in the ¹H NMR of 22. These minor products resulted from coupling of alkyl iodide 5, which was obtained as a 93:7 mixture of enantiomers (Scheme 1), with vinyl iodide 11, which was synthesized in enantiomerically pure form but contained a small amount of regioisomer 12 (Scheme 2). We were unable to estimate the exact ratio of the components in the mixture by ¹H NMR, as the signals overlapped significantly. However, if we assume the rates of formation of the various products are similar, statistically 22, 23, 24, and 25 should be present in a ratio of 89:6.7:4:0.3. In accordance with this, it was possible to detect 22, 23 and 24 by ¹H NMR, although we were unable to observe 25 as it was present in such low quantity.

Scheme 4.

Reagents and conditions: (a) Zn, Cu(OAc)₂, Pd(PPh₃)₄, LiCl, NMP, 55 °C, 95% (for mixture of 23, 24, and 25). NMP=N-methyl-2-pyrrolidinone.

At this point, we began to search for a point at which the minor contaminants could be removed (Scheme 5). Fortunately, once the cyclopentylidene acetal had been cleaved under acidic conditions to form 26, we were readily able to chromatographically separate 1,3-diol 28, derived from 24. However, at this stage, it was not practical to separate the minor diastereoisomer 27 resulting from 23. Despite this, we carried on with the synthesis with the hope that the remaining diastereoisomeric contaminant could be removed at a later stage.

Scheme 5.

Regents and conditions: (a) 1:4:16 TFA/H₂O/CH₂CL₂, 91%. (for mixture of 27 and 28). TFA=trifluoroacetic acid.

Accordingly, we proceeded to convert 26 to seco-acid 29 in a two-step sequence consisting of primary alcohol protection and saponification of the methyl ester (Scheme 6). In the previous study, selective protection of the primary alcohol was cleanly achieved under standard conditions (TIPSCl, imidazole, DMF). In this study, however, we observed mixtures of the primary- and secondary-protected alcohols in varying ratios. The reason for the discrepancy in these studies is not clear at this time. Nevertheless, we were eventually able to selectively protect the primary position using triisopropylsilyl triflate and 2,6-lutidine at −78 °C. After hydrolysis of the methyl ester to form 29, the stage was set for macrolactonization. On exposure to standard Yamaguchi conditions,²⁵ seco-acid 29 was converted to macrolactone 31. To our delight, the remaining minor diastereoisomeric lactone, 32, was readily removed chromatographically at this stage to afford 31 in optically pure form (>99% ee).

Scheme 6.

Reagents and conditions: (a) (i) TIPSOTf, 2,6-lutidine, CH₂CL₂, −78 °C; (ii) LiOH, 1:1:4 H₂O/MeOH/THF, 88% for 2 steps (as mixture of 29 and 30); (b) Cl₃C₆H₂COCl, i-Pr₂NEt, benzene, then DMAP, benzene, 74% (for single compound, 31). TIPS=triisopropylsilyl, DMAP=4-(dimethylamino)pyridine.

To complete the synthesis of the mycolactone core, the triisopropyl ether of 31 was cleaved,²⁶ and the resultant primary alcohol, 33, was converted to alkyl iodide 34 (Scheme 7). Under the same Negishi coupling conditions optimized for the case of 5 + 11 → 22, alkyl iodide 34 was coupled with 21 (1.5 equiv) to provide 35 in 88% yield.

Scheme 7.

Reagents and conditions: (a) HF·pyr, pyr, CH₃CN, 0 °C, 92%; (b) Ph₃P, I₂, imidazole, CH₂CL₂, rt, 99%; (c) Zn, Cu(OAc)₂, 20, Pd(PPh₃)₄, LiCl, NMP, 60 °C, 88%. pyr=pyridine.

To summarize, the synthesis of the mycolactone core described herein proceeds in 14 linear steps and 19% overall yield. This synthesis features scalable and efficient methods for the preparation of the C1–C7, C8–C13, and C14–C20 fragments, and has allowed for the preparation of 6.0 grams of protected mycolactone core 35 with relative ease. Furthermore, we anticipate that it should be straightforward to prepare even larger quantities of this material should the need arise. Importantly, by synthesizing the C8–C13 fragment in optically pure form and removing the minor contaminants chromatographically, we can be confident that the material produced through this sequence is >99% optically pure.

3. Conclusion

A third synthesis of the mycolactone core has been described. While our first and second syntheses allowed us to unambiguously confirm the relative and absolute stereochemistry of the mycolactones and optimize protecting group strategy, respectively, the synthetic efforts described herein have resulted in a scalable and efficient route to the mycolactone core which has allowed for the preparation of multi-gram quantities in high purity. We feel that this material will be useful for research efforts aimed at addressing some of the many unsolved questions still surrounding Buruli ulcer.

4. Experimental

4.1. General procedures and methods

NMR spectra were recorded on Varian Inova 500 MHz or 600 MHz spectrometers. Chemical shifts are reported in parts per million (ppm). For ¹H NMR spectra (CDCl₃ or C₆D₆), the residual solvent peak was used as the internal reference (7.26 ppm in CDCl₃; 7.15 ppm in C₆D₆). For ¹³C NMR spectra, the central solvent peak was used as the internal reference (77.0 ppm in CDCl₃; 128.0 ppm in C₆D₆). Mass spectrometry was performed on an Agilent 6210 Time-of-Flight mass spectrometer with an electrospray ionization source. Analytical thin layer chromatography (TLC) was performed with E. Merck pre-coated TLC plates, silica gel 60F-254, layer thickness 0.25 mm. Flash chromatography separations were performed on E. Merck Kieselgel 60 (230–400) mesh silica gel. Reagents and solvents are of commercial grade and were used as supplied. All reactions were conducted under an inert atmosphere. Reaction vessels were flame-dried or oven-dried and allowed to cool to rt under inert atmosphere.

4.2. Synthesis outlined in Scheme 1

4.2.1. Preparation of 2

To a solution of potassium t-butoxide (13.45 g, 120 mmol) in THF (60 mL) at −78 °C was added condensed cis-2-butene (42 mL, 461 mmol) followed by n-BuLi in hexanes (1.6 M in hexanes, 75 mL, 120 mmol) dropwise. The resultant bright yellow suspension was stirred for 10 min at −78 °C, 20 min at −50 °C, and 10 min at −78 °C. A solution of (+)-Ipc₂-BOMe (43.80 g, 151 mmol) in Et₂O (100 mL) was added dropwise via cannula. The colorless solution was stirred at −78 °C for 40 min, and then BF₃·OEt₂ (19.2 mL, 151 mmol) was added dropwise. Aldehyde 1¹¹ (12.0 g, 92.2 mmol) was then added dropwise as a solution in THF (90 mL). The resultant mixture was stirred a further 3 h at −78 °C before being quenched with 3 M aqueous NaOH (73.2 mL) and diluted with EtOAc (500 mL). 30% H₂O₂ (35.4 mL) was added carefully, the cold bath was removed, and the resultant mixture was stirred for 1 h. The layers were separated, and the aqueous extracted with EtOAc (3 × 200 mL). The combined organics were washed with saturated aqueous NaCl, dried (MgSO₄), and concentrated in vacuo. The crude residue was purified by flash column chromatography in a solvent gradient (SiO₂, 5% → 10% → 30% EtOAc/hexanes) to provide 2 (13.38 g, 78% yield, 86% ee as determined by conversion to the corresponding (R)- and (S)-Mosher’s esters) as a colorless oil. [α]²⁷ _D −25.4 (c=1.0, CHCL₃); ¹H NMR (500 MHz, CDCl₃) δ 1.04 (3H, d, J=7.0 Hz), 1.36–1.45 (1H, m), 1.51–1.60 (2H, m), 1.64–1.75 (1H, m), 1.80–1.90 (1H, m), 2.25–2.33 (1H, m), 2.37 (2H, t, J=7.5 Hz), 3.47–3.54 (1H, m), 3.69 (3H, s), 5.07–5.13 (2H, m), 5.75–5.83 (1H, m); ¹³C NMR (125 MHz, CDCl₃) δ 14.16, 21.41, 33.32, 33.83, 43.51, 51.50, 74.23, 115.40, 140.77, 174.17; HR-MS (ESI) exact mass calculated for [M+Na]⁺ (C₁₀H₁₈O₃Na) requires m/z 209.1148, found 209.1141.

4.2.2. Preparation of 3

Alcohol 2 (0.90 g, 4.83 mmol) and PMB-trichloroacetimidate (2.44 g, 9.66 mmol) were dissolved in PhMe (48 mL) and cooled to 0 °C. Scandium(III) trifluoromethanesulfonate (0.12 g, 0.24 mmol) was added portionwise over 2 min. Stirring at 0 °C was continued for a further 15 minutes. The reaction was diluted with hexanes and filtered over celite. The filtrate was concentrated in vacuo, and the crude residue was purified by flash column chromatography (SiO₂, 7.5% EtOAc/hexanes) to provide 3 (1.20 g, 81% yield) as a colorless oil: [α]²⁷ _D −25.4 (c=1.0, CHCL₃); ¹H NMR (500 MHz, CDCl₃) δ 1.05 (3H, d, J=7.0 Hz), 1.43–1.59 (2H, m), 1.60–1.72 (1H, m), 1.75–1.86 (1H, m), 2.24–2.36 (2H, m), 2.51 (1H, apparent sextet, J=6.5 Hz), 3.23–3.29 (1H, m), 3.68 (3H, s), 3.81 (3H, s), 4.48 (2H, AB quartet, J=11.5 Hz, 33.0 Hz), 5.01–5.09 (2H, m), 5.80–5.89 (1H, m), 6.86–6.92 (2H, m), 7.26–7.31 (2H, m); ¹³C NMR (125 MHz, CDCl₃) δ 15.63, 21.00, 30.40, 34.04, 40.60, 51.42, 55.25, 71.36, 82.11, 113.71, 114.45, 129.35, 130.89, 140.75, 159.09, 174.06; HRMS (ESI) exact mass calculated for [M+Na]⁺ (C₁₈H₂₆O₄Na) requires m/z 329.1723, found 329.1730.

4.2.3. Preparation of 4

To a solution of 3 (9.00 g, 29.4 mmol) in 3:1 acetone/H₂O (488 mL) was added 4-methylmorpholine N-oxide·H₂O (7.94 g, 58.8 mmol) and OsO₄ (0.1M in H₂O, 5.9 mL, 0.59 mmol). The resultant solution was stirred at rt for 14 h. Saturated aqueous Na₂S₂O₃ (300 mL) and EtOAc (300 mL) were added, and the mixture was stirred for 1 h. The phases were separated, and the aqueous extracted with EtOAc (3 × 200 mL). The combined organic phase was washed with H₂O and brine, dried (Na₂SO₄), and concentrated in vacuo to yield an inconsequential 3:1 mixture of diol diastereoisomers which was carried on to the following step without further purification.

To a solution of the diol from the above step in benzene (185 mL) was added Pb(OAc)₄ (20.6 g, 46.4 mmol). The resultant mixture was stirred at rt for 20 min. Saturated aqueous NaHCO₃ was added until the aqueous phase reached pH 7.0, and the mixture was extracted with EtOAc (3 × 200 mL). The combined organic phase was washed with brine, dried (MgSO₄), and concentrated in vacuo to provide the crude aldehyde, which was used in the following step without further purification.

The crude aldehyde from the preceding step was dissolved in MeOH (300 mL) and cooled to 0 °C. NaBH₄ (2.22 g, 58.8 mmol) was added portionwise over 5 min, and the solution was stirred at 0 °C for 1 h. Saturated aqueous NH₄Cl was added to quench the reaction, and the mixture was extracted with EtOAc (3 × 200 mL). The combined organic phase was washed with brine, dried (Na₂SO₄), and concentrated in vacuo. The crude residue was purified by flash column chromatography (SiO₂, 25% EtOAc/hexanes) to provide alcohol 4 (6.51 g, 71% over three steps) as a colorless oil: [α]²³_D +0.8 (c=1.77, CHCL₃); ¹H NMR (500 MHz, CDCl₃) δ 0.87 (3H, d, J=7.0 Hz), 1.45–1.56 (1H, m), 1.56–1.67 (2H, m), 1.71–1.82 (1H, m), 2.05–2.14 (1H, m), 2.25–2.37 (2H, m), 3.47–3.52 (1H, m), 3.54 (1H, dd, J=5.0, 11.0 Hz), 3.665 (1H, apparent dd, J=8.0, 11.0 Hz), 3.67 (3H, s), 3.80 (3H, s), 4.49 (2H, AB quartet, J=11.0, 26.5 Hz), 6.85–6.90 (2H, m), 7.24–7.28 (2H, m). ¹³C NMR (100 MHz, CDCl₃) δ 173.9, 159.2, 130.3, 129.5 (2C), 113.8 (2C), 81.4, 71.4, 65.8, 55.2, 51.5, 36.7, 33.9, 29.3, 21.6, 11.9 LRMS (ES) calculated for [M+H]⁺ (C₁₇H₂₇O₅) requires m/z 311, found 311.

4.2.4. Preparation of 5

To a solution of alcohol 4 (0.217 g, 0.699 mmol) in CH₂CL₂ (8 mL) was added imidazole (0.143 g, 2.10 mmol), PPh₃ (0.385 g, 1.47 mmol), and iodine (0.373 g, 1.47 mmol). The resultant brown reaction mixture was stirred at rt for 1 h. The reaction was diluted with EtOAc and poured into saturated aqueous Na₂S₂O₃. The aqueous layer was separated and extracted with EtOAc (3 × 20 mL). The combined organics were washed with saturated aqueous NaHCO₃ and brine, dried (MgSO₄), and concentrated in vacuo. The crude material was purified by flash column chromatography (SiO₂, 10% EtOAc/hexanes) to provide alkyl iodide 5 (0.269 mg, 92% yield) as a colorless oil: [α]²⁶_D +18.2 (c=1.0, CHCL₃); ¹H NMR (500 MHz, CDCl₃) δ 1.04 (3H, d, J=6.5 Hz), 1.45–1.54 (1H, m), 1.54–1.78 (3H, m), 1.89–1.98 (1H, m), 2.27–2.39 (2H, m), 3.09 (1H, dd, J=7.6, 9.6 Hz), 3.37 (1H, dd, J=6.0, 9.6 Hz), 3.42–3.48 (1H, m), 3.68 (3H, s), 3.82 (3H, s), 4.48 (2H, AB quartet, J=11.5, 21.0 Hz), 6.87–6.92 (2H, m), 7.25–7.29 (2H, m); ¹³C NMR (125 MHz, CDCl₃) δ 12.17, 15.40, 20.90, 29.96, 33.74, 38.88, 51.34, 55.09, 71.70, 80.57, 113.62, 129.15, 130.53, 159.02, 173.60; HRMS (ESI) exact mass calculated for [M+Na]⁺ (C₁₇H₂₅IO₄Na) requires m/z 443.0690, found 443.0659.

4.3. Synthesis outlined in Scheme 2

4.3.1. Preparation of 7

To a 0 °C solution of LiAlH₄ (1M in Et₂O from Aldrich, 179 mL, 179 mmol) was added diynol 6¹³ (19.34 g, 179 mmol) in Et₂O (200 mL) dropwise. The resultant mixture was heated to a gentle reflux and stirred for 9 hr. The reaction was diluted with Et₂O (310 mL) and treated, in sequence, with H₂O (6.5 mL), 2N NaOH (6.5 mL) and H₂O (6.5 mL). After stirring for a further 15 min, the mixture was filtered through celite, dried (MgSO₄), and concentrated in vacuo to provide pure 7 (19.37 g, 98% yield) as a colorless oil whose ¹H NMR was identical to that published previously.¹⁴

4.3.2. Preparation of 8

To a suspension of activated 4Å molecular sieves (30.0 g, powdered) in anhydrous CH₂CL₂ (430 mL) at −10 °C were added (−)-diethyl D-tartrate (3.21 mL, 18.8 mmol) and titanium tetraisopropoxide (4.40 mL, 15.0 mmol). The resultant mixture was cooled to −25 °C and treated with tert-butyl hydroperoxide (78.6 mL, 5.5 M solution in decane, 432 mmol). After 20 min, a solution of alcohol 7¹⁴ (20.7 g, 188 mmol) in anhydrous CH₂CL₂ (110 mL) was added dropwise via cannula over 30 min. The resultant mixture was stirred 4 h at −25 °C, then warmed to rt over 2 h. After 30 minutes at rt, H₂O (52 mL) and 4 M aqueous NaOH (26 mL) were added. The resultant mixture was stirred for 30 min, and the cloudy suspension was filtered through celite. After diluting with CH₂CL₂ (300 mL), the organic phase was washed with 10% Na₂SO₃. The layers were separated, and the aqueous phase extracted with CH₂CL₂ (2 × 300 mL). The combined organic phase was washed with brine, dried (Na₂SO₄), and concentrated in vacuo. The crude residue was purified by flash column chromatography (SiO₂, 15% acetone/hexanes) to afford 8 (19.8 g, 83%) as white solid. It was determined by conversion of 9 (for preparation of 9, see the following step) to the corresponding (R)- and (S)-Mosher esters that the Sharpless asymmetric epoxidation had proceeded in 80% ee. Optically pure 8 (16.5 g, 70%, >99% ee) was obtained by recrystallization from hexanes/acetone (10/1) or toluene/hexanes (1/1) in a −20 °C freezer: R_f 0.35 (hexanes/acetone, 4/1); [α]²⁰_D +18.7 (c=0.4, CHCL₃); ¹H NMR (600 MHz, CDCl₃) δ 1.75 (1H, dd, J = 5.4, 7.8 Hz), 1.83 (3H, t, J = 1.8 Hz), 2.48–2.65 (2H, m), 3.14–3.20 (2H, m), 3.67–3.75 (1H, m), 3.95–4.05 (1H, m); ¹³C NMR (125 MHz, CDCl₃) δ 3.44, 21.63, 53.50, 57.80, 61.16, 73.08, 78.15; HRMS (ESI) exact mass calculated for [M+Na]⁺ (C₇H₁₀O₂Na) requires m/z 149.0573, found 149.0576.

4.3.3. Preparation of 9

To a suspension of CuCN (34.6 g, 387 mmol) in anhydrous THF (560 mL) was added MeLi (1.6 M in ether, 524 mL, 838 mmol) dropwise at −20 °C under argon. The mixture was stirred until it became clear, then epoxide 8 (16.2 g, 129 mmol) was added as a solution in anhydrous Et₂O (250 mL) via cannula over 30 min. The resultant mixture was stirred at −20 °C overnight, then warmed to 0 °C. The reaction was diluted with Et₂O (500 mL) and saturated aqueous NH₄Cl (115 mL) and stirred vigorously for 10 min. The precipitate was removed by filtration through celite, then the organic layer was separated, washed with brine, dried (Na₂SO₄), and concentrated in vacuo. The residue was dissolved in acetone (40 mL) and H₂O (50 mL), and was treated with NaIO₄ (6.10 g, 28.5 mmol). After 2 h, the mixture was quenched by the addition of Na₂SO₃ (7.2 g, 57.0 mmol), diluted with Et₂O, and filtered through celite. The organic phase was separated, washed with brine, dried (Na₂SO₄), and concentrated in vacuo. The residue was purified by flash column chromatography in a solvent gradient (SiO₂, 33% EtOAc/hexanes → 50% EtOAc/hexanes) to give pure diol 9 (19.4 g, 79%): R_f 0.25 (50% EtOAc/hexanes); [α]²⁰_D = +17.6 (c=0.4, CHCL₃); ¹H NMR (600 MHz, CDCl₃) δ 0.92 (3H, d, J = 7.2 Hz), 1.85 (3H, t, J = 3.0 Hz), 1.85–1.93 (1H, m), 2.36–2.40 (1H, m), 2.50–2.54 (1H, m), 2.77–3.83 (2H, m), 3.63–3.67 (1H, m), 3.68–3.72 (1H, m), 3.73–3.78 (1H, m); ¹³C NMR (125 MHz, CDCl₃) δ 3.39, 13.54, 25.81, 38.94, 67.00, 74.84, 75.06, 78.31; HRMS (ESI) exact mass calculated for [M+Na]⁺ (C₈H₁₄O₂Na) requires m/z 165.0886, found 165.0884.

4.3.4. Preparation of 10

To a solution of diol 9 (18.7 g, 131 mmol) in cyclopentanone (200 mL) was added p-TsOH (monohydrate, 2.50 g, 13.1 mmol). The mixture was stirred 12 h at rt, then treated with Et₃N (3.64 mL, 26.3 mmol). The mixture was concentrated in vacuo, and the residue purified by flash column chromatography (SiO₂, 10% EtOAc/hexanes → 50% EtOAc/hexanes) to provide 10 (22.3 g, 89% based on consumed diol) along with recovered diol 9 (1.63 g, 5%): R_f 0.40 (10% EtOAc/hexanes); [α]²⁰_D −4.1 (c=0.3, CHCL₃); ¹H NMR (500 MHz, C₆D₆) δ 0.42 (3H, d, J = 6.5 Hz), 1.54 (3H, t, J = 2.5 Hz), 1.50–1.63 (4H, m), 1.69–1.82 (2H, m), 1.85–1.95 (1H, m), 2.04–2.13 (2H, m), 2.28–2.43 (2H, m), 3.17 (1H, t, J = 11 Hz), 3.24–3.30 (1H, m), 3.58 (1H, dd, J = 11.5, 5.0 Hz); ¹³C NMR (125 MHz, C₆D₆) δ 3.51, 12.30, 22.84, 24.23, 24.66, 30.85, 33.69, 40.43, 67.21, 75.89, 76.95, 110.41, 127.69; HRMS (ESI) exact mass calculated for [M+Na]⁺ (C₁₃H₂₀O₂Na) requires m/z 231.1356, found 231.1357.

4.3.5. Preparation of 11

Schwartz reagent (~70% pure homemade reagent,^18c 13.5 g, 36.6 mmol) was transferred to a round bottom flask under inert atmosphere, then treated with a solution of alkyne 10 (5.08 g, 24.4 mmol) in THF (150 mL). The resultant suspension was heated to 50 °C and stirred for 30 minutes. The reaction was cooled to rt, then to 0 °C. A solution of iodine (9.28 g, 36.6 mmol) in THF (122 mL) was added dropwise to the 0 °C suspension until a brown color just persisted. At this point the addition of iodine/THF was ceased, and the remaining iodine/THF solution was discarded. The reaction was quenched after 5 min by dilution with 1:1 saturated aqueous NaHCO₃/saturated aqueous Na₂S₂O₃ (200 mL) and EtOAc (100 mL). The biphasic mixture was stirred vigorously for 10 minutes, then the layers were separated and the aqueous extracted with EtOAc (3 × 100 mL). The combined organics were washed with brine, dried (MgSO₄), and concentrated in vacuo. The crude residue was purified by flash column chromatography (SiO₂, 1% EtOAc/hexanes) to provide vinyl iodide 11 as an inseparable 22:1 mixture with the undesired regioisomer 12 as a clear oil (5.57 g, 68% yield). Spectroscopic data for the major regioisomer: [α]²⁶_D +10.9 (c=1.0, CHCL₃); ¹H NMR (500 MHz, C₆D₆) δ 0.28 (3H, d, J=6.7 Hz), 1.42–1.56 (5H, m), 1.56–1.64 (1H, m), 1.66–1.73 (1H, m), 1.85–1.96 (3H, m), 1.96–2.03 (1H, m), 2.12 (3H, d, J=1.5 Hz), 3.05 (1H, ddd, J=3.5, 8.0, 11.0 Hz), 3.09 (1H, apparent t, J=11.5 Hz), 3.51 (1H, dd, J=5.0, 11.5 Hz), 6.33 (1H, dt, J=1.7, 7.2 Hz); ¹³C NMR (125 MHz, C₆D₆) δ 137.51, 110.21, 94.84, 76.00, 67.21, 40.33, 34.14, 33.70, 30.72, 27.72, 24.55, 22.73, 12.28; HRMS (ESI) exact mass calculated for [M+Na]⁺ (C₁₃H₂₁IO₂Na) requires m/z 359.0478, found 359.0470.

4.4. Synthesis outlined in Scheme 3

4.4.1. Preparation of 14

A solution of n-BuLi (91.5 mL of a 1.87 M solution in hexanes, 171 mmol) was added dropwise to a solution of ethynyltrimethylsilane (32.0 mL, 214 mmol) in Et₂O (950 mL) at −78 °C. Once addition was complete, the reaction mixture was warmed to 0 °C for 40 min, then recooled to −78 °C. (R)-2-methyloxirane 13 (Alfa Aesar, >99% optical purity, 10.0 mL, 143 mmol) was added via syringe, and the resultant solution was stirred for 10 min. A solution of BF₃·OEt₂ (freshly distilled, 21.5 mL, 171 mmol) in Et₂O (21.5 mL) was added dropwise over 3 min. The mixture was stirred at −78 °C for 1 h, then quenched with saturated aqueous NaHCO₃ (100 mL). The layers were separated, and aqueous was extracted with Et₂O (3 × 100 mL). The combined organic phase was dried (MgSO₄), and the solvent was removed by distillation through a Vigreux column (10 cm height, 55 °C oil bath) until 200 mL of solution remained. The crude residue, whose ¹H and ¹³C NMR spectral data were consistent with those reported,²⁷ was used for the next step without purification.

To a solution of crude alcohol (143 mmol) and imidazole (19.4 g, 285 mmol) in DMF (150 mL) was added TBSCl (30.1 g, 200 mmol). The resultant solution was stirred at rt for 48 h. The mixture was diluted with Et₂O (1.5 L) and washed with brine (2 × 250 mL), H₂O (2 × 250 mL), and brine (250 mL). The organic phase was dried (MgSO₄), and gently concentrated in vacuo until 100 mL of solution remained. The crude residue of 14, whose ¹H and ¹³C NMR spectral data were consistent with those reported,²⁸ was used for the next step without purification.

4.4.2. Preparation of 15

To a solution of crude alkyne 14 (143 mmol) and NIS (43.3 g, 192 mmol) in DMF (475 mL) was added AgNO₃ (3.63 g, 21.4 mmol). The resultant mixture was stirred for 5 h at rt. The mixture was quenched with brine (150 mL), filtered over celite, and diluted with hexanes/Et₂O (1:1, 2 L). The separated organic phase was washed with brine (2 × 1 L) and H₂O (3 × 1 L), dried (MgSO₄), and gently concentrated in vacuo until 100 mL of solution remained. The crude residue was unstable and used for the next step immediately without purification.

THF (715 mL) was bubbled with Ar for 15 min at 0 °C, then cyclohexene (37.5 mL, 371 mmol) was added. To the resultant solution was cautiously added BH₃·S(CH₃)₂ (17.6 mL, 185 mmol) dropwise over 20 min. The reaction was stirred for 2 h at 0 °C and 1h at rt with constant bubbling of Ar through the solution. The resultant Cy₂BH solution was cooled to 0 °C, and the crude alkynyl iodide from the preceding step was added dropwise over 10 min. The reaction was stirred 3 h at rt, then quenched by addition of AcOH (32.6 mL, 570 mmol) at 0 °C over 5 min. After stirring at rt for 30 min, the reaction was allowed to stand at 5 °C for 12 h. The mixture was gently concentrated in vacuo to 200 mL, then K₂CO₃ (95 g in 300 mL water) was added to neutralize the mixture. Following dilution with hexanes (1.5 L), the organics were washed with saturated aqueous NaHCO₃ (3 × 400 mL) and brine (400 mL), dried (MgSO₄), and concentrated in vacuo. The crude residue was purified by flash chromatography (SiO₂, 100% hexanes → 5% CH₂CL₂/hexanes) to provide vinyl iodide 15 (44.1 g, 95% over 4 steps) as a colorless oil, whose ¹H and ¹³C NMR spectral data were consistent with those reported.²⁹

4.4.3. Preparation of 16

Propyne was bubbled (via a balloon) through a solution of vinyl iodide 15 (44.1 mL, 135 mmol) in Et₂NH (0.9 L) for 10 min at 0 °C. PdCl₂(PPh₃)₂ (4.12 g, 5.4 mmol) and CuI (2.06 g, 10.8 mmol) were added. Propyne was continually bubbled into the resultant solution as the reaction stirred for 3 h at rt. The mixture was diluted with pentane (4 L) and washed with saturated aqueous NH₄Cl (3 × 2 L), H₂O (2 × 2 L) and brine (2 L). The organic phase was dried (MgSO₄) and concentrated in vacuo. The residue was purified by flash chromatography (SiO₂, 100% pentane → 5% Et₂O/pentane) to afford enyne 16 (30.5 g, 96%) as a colorless oil; [α]²⁵_D +4.9 (c=1.51, CHCL₃); ¹H NMR (600 MHz, CDCl₃) δ 0.04 (3H, s), 0.04 (3H, s), 0.87 (9H, s), 1.13 (3H, d, J = 6.0 Hz), 1.95 (3H, d, J = 2.4 Hz), 2.46-2.35 (2H, m), 3.87 (1H, dq, J = 6.0, 6.0 Hz), 5.49-5.44 (1H, m), 5.85 (1H, dt, J = 10.2, 7.5 Hz); ¹³C NMR (125 MHz) δ − 4.78, −4.61, 4.36, 18.15, 23.49, 25.86, 40.11, 68.22, 76.61, 89.83, 110.89, 138.74; HRMS (ESI) exact mass calculated for [M+H]⁺ (C₁₄H₂₇OSi) requires m/z 239.1831, found 239.1826.

4.4.4. Preparation of 18

To a solution of ligand 17^21b (5.02 g, 9.38 mmol) in CH₂CL₂ (200 mL) was added Ti(O-i-Pr)₄ (2.54 mL, 8.53 mmol). The reaction was stirred for 1 h at rt. Enyne 16 (20.0 g, 85.3 mmol), 4,4'-thiobis(6-t-butyl-m-cresol) (Sumitomo Chemical, 2.0 g, 5.58 mmol), H₂O₂ (30% solution in H₂O, 20 mL, 176 mmol), and phosphate buffer (pH 7.4, 67 mM, 10 mL) were added. The resultant reaction mixture was stirred at 40 °C for 4 h, then H₂O₂ (30% solution in H₂O, 20 mL, 196 mmol) was added (repeated twice more, stirring 4 h between each H₂O₂ addition). The reaction was stirred at 40 °C for another 18 h until all of the enyne was converted to the epoxide. Solid NH₄Cl (30 g) was added, and the resultant mixture was diluted with hexanes (2 L) and filtered over celite. The filtrate was washed with saturated NH₄Cl solution (3 × 400 mL), dried (MgSO₄), and concentrated in vacuo. The residue was purified by flash chromatography in a solvent gradient (SiO₂, 3% CH₂CL₂/hexanes → 10% CH₂CL₂/hexanes → 10% Et₂O/hexanes) to afford epoxide 18 (19.8 g, 91%, ≥ 50:1 dr as determined by ¹H-NMR) as a colorless oil; [α]²⁵_D −11.9 (c=1.80, CHCL₃); ¹H NMR (600 MHz, CDCl₃) δ 0.04 (3H, s), 0.05 (3H, s), 0.87 (9H, s), 1.22 (3H, d, J = 6.0 Hz), 1.75 (1H, dt, J = 14.1, 6.0 Hz), 1.84 (3H, d, J = 1.8 Hz), 1.86 (1H, dt, J = 14.1, 6.0 Hz), 3.12 (1H, dt, J = 6.0, 1.8 Hz), 3.38-3.35 (1H, m), 4.05 (1H, dq, J = 6.0, 6.0 Hz); ¹³C NMR (125 MHz, CDCl₃) δ −4.89, −4.53, 3.65, 18.04, 23.85, 25.79, 38.92, 44.85, 55.26, 66.37, 74.25, 82.13; HRMS (ESI) exact mass calculated for [M+Na]⁺ (C₁₄H₂₆O₂SiNa) requires m/z 277.1600, found 277.1592.

4.4.5. Preparation of 19

To a solution of epoxide 18 (19.8 mL, 78.0 mmol) in CH₂CL₂ (780 mL) at −78 °C was added AlMe₃ (2 M solution in PhMe, Aldrich, 78.0 mL, 156 mmol) dropwise. After stirring for 10 min, MeLi (1.6 M solution in Et₂O, Aldrich, 98 mL, 156 mmol) was added dropwise. The reaction was stirred a further 20 min, then treated with a solution of BF₃·OEt₂ (freshly distilled, 19.8 mL, 156 mmol) in Et₂O (19.8 mL). After 1 h at −78 °C, the reaction mixture was carefully quenched by dropwise addition of MeOH (100 mL) over 30 min while still stirring at −78 °C. The resultant mixture was warmed to rt, saturated aqueous NH₄Cl (100 mL) was added, and stirring was continued for 30 min. The mixture was then treated with saturated aqueous sodium potassium tartrate (150 mL) and stirred for 2 h. The phases were separated, and aqueous extracted with Et₂O (3 × 250 mL). The combined organic phase was dried (MgSO₄), concentrated in vacuo, and purified by flash chromatography (SiO₂, 33% CH₂CL₂/hexanes → 50% CH₂CL₂/hexanes → 10% Et₂O/hexanes) to afford alcohol 19 (18.3 g, 87%) as a colorless oil; [α]²⁵_D −8.5 (c=1.13, CHCL₃); ¹H NMR (600 MHz, CDCl₃) δ 0.09 (3H, s), 0.10 (3H, s), 0.88 (9H, s), 1.14 (3H, d, J = 4.8 Hz), 1.19 (3H, d, J = 6.0 Hz), 1.55 (1H, dt, J = 14.4, 9.6 Hz), 1.78 (3H, d, J = 1.8 Hz), 1.83 (1H, ddd, J = 14.4, 3.6, 1.8 Hz), 2.47-2.40 (1H, m), 3.57-3.53 (1H, m), 3.58 (1H, d, J = 1.8 Hz), 4.11-4.06 (1H, m); ¹³C NMR (125 MHz, CDCl₃) δ −4.79, −3.93, 3.57, 17.07, 17.88, 24.53, 25.82, 33.05, 42.75, 70.13, 74.68, 77.36, 81.00; HRMS (ESI) exact mass calculated for [M+Na]⁺ (C₁₅H₃₀O₂SiNa) requires m/z 293.1913, found 293.1907.

4.4.6. Preparation of 20

TBSCl (14.3 g, 94.9 mmol) was added to a solution of alcohol 19 (18.3 g, 67.8 mmol) and imidazole (13.8 g, 203 mmol) in DMF (225 mL), and the reaction was stirred at rt for 48 h. The mixture was diluted with Et₂O/hexanes (1:1, 1.4 L). The organic layer was washed with brine (2 × 500 mL), H₂O (2 × 500 mL), and brine (500 mL), dried (MgSO₄), and concentrated. The residue was purified by flash chromatography (SiO₂, 100% hexanes → 9% CH₂CL₂/hexanes) to afford alkyne 20 (25.9 g, 99%) as a colorless oil: [α]²³_D +10.8 (c=1.28, CHCL₃); ¹H NMR (500 MHz, CDCl₃) δ 0.057 (3H, s), 0.061 (3H, s), 0.07 (3H, s), 0.08 (3H, s), 0.90 (18H, overlapped two singlets), 1.07 (3H, J=6.0 Hz), 1.14 (3H, d, J=6.0 Hz), 1.54 (1H, ddd, J=6.0, 6.0, 13.5 Hz), 1.77 (3H, d, J=2.5 Hz), 1.82 (1H, ddd, J=6.0, 6.0, 13.5 Hz), 2.46–2.54 (1H, m), 3.68 (1H, apparent q, J=6.0 Hz), 3.98 (1H, apparent sextet, J=6.0 Hz); HRMS (ESI) exact mass calculated for [M+H]⁺ (C₂₁H₄₄O₂Si₂) requires m/z 385.2958, found 385.2954.

4.4.7. Preparation of 21

Schwartz reagent (~70% pure homemade reagent,^18c 1.44 g, 3.90 mmol) was transferred to a round bottom flask under inert atmosphere, then treated with a solution of alkyne 20 (1.00 g, 2.60 mmol) in THF (4.3 mL). The resultant suspension was heated to 50 °C and stirred for 1 h. The reaction was cooled to rt, then to 0 °C. A solution of iodine (0.99 g, 3.90 mmol) in THF (4.3 mL) was added dropwise to the 0 °C suspension until a brown color just persisted. At this point the addition of iodine/THF was ceased, and the remaining iodine/THF solution was discarded. The reaction was quenched by dilution with 1:1 saturated aqueous NaHCO₃/saturated aqueous Na₂S₂O₃ (10 mL) and EtOAc (10 mL). The biphasic mixture was stirred vigorously for 10 minutes, then the layers were separated. The aqueous phase was extracted with EtOAc (3 × 10 mL). The combined organics were washed with brine, dried (MgSO₄), filtered, and concentrated. The crude residue was purified by flash column chromatography (SiO₂, 1% EtOAc/hexanes) to provide vinyl iodide 21 as a clear oil (0.88 g, 60% yield): [α]²⁷_D +10.9 (c=1.0, CHCL₃); ¹H NMR (500 MHz) δ 0.05–0.09 (12H, m), 0.90 (9H, s), 0.91 (9H, s), 0.93 (3H, d, J=6.5 Hz), 1.15 (3H, d, J=6.3 Hz), 1.54–1.67 (2H, m), 2.40 (3H, d, J=1.5 Hz), 2.49–2.58 (1H, m), 3.65–3.70 (1H, m), 3.89 (1H, apparent sextet, J=6.0 Hz), 6.15 (1H, dd, J=1.5, 10.3 Hz); ¹³C NMR (125 MHz) δ −4.66, −4.55, −4.18, 14.10, 18.06, 24.06, 25.86, 25.91, 25.96, 27.89, 40.32. 44.90, 65.85, 71.87, 93.09, 145.04; HRMS (ESI) exact mass calculated for [M+Na]⁺ (C₂₁H₄₅IO₂Si₂Na) requires m/z 535.1895, found 535.1889.

4.5. Synthesis outlined in Scheme 4

4.5.1. Preparation of 22

To prepare active Zn-Cu couple, Cu(OAc)₂ (87.3 mg, 0.437 mmol) was suspended in glacial AcOH (20 mL). To this was added Zn dust (1.43 g, 21.9 mmol), and the resultant suspension was heated to reflux for one minute or until all of the copper had deposited onto the zinc (disappearance of blue color in the supernatant). Stirring was stopped, and the reddish-gray silt was allowed to settle. The AcOH was removed by pipette, then another portion of AcOH (20 mL) was added and the suspension was heated to reflux for one minute. The AcOH was removed again by pipette, and this process was repeated once more. Finally, the Zn-Cu couple was rinsed with Et₂O (3 × 20 mL), with each rinse being removed by pipette. The resultant active couple was dried under vacuum for 30 min.

Alkyl iodide 5 (1.84 g, 4.37 mmol) dissolved in anhydrous 15:1 PhH/DMF (17.5 mL) was cannulated into to the active Zn-Cu couple, and the resultant suspension was heated at 55 °C for 1 h. Meanwhile, LiCl (1.11 g, 26.2 mmol, dried over flame/vacuum) and Pd(PPh₃)₄ (361 mg, 0.312 mmol) were combined and purged with argon. Freshly distilled anhydrous NMP (12.5 mL) was added, followed by the vinyl iodide 11 (1.05 g, 3.12 mmol) in NMP (4.1 mL, plus a little more to aid in transfer). Then the colorless alkylzinc iodide solution was added via cannula (the excess settled Zn was left behind in the original flask as much as possible). The reaction mixture was degassed with one freeze-pump-thaw cycle, and the mixture was heated to 55 °C for 35 min. The reaction was cooled to rt, diluted with EtOAc, and poured into saturated aqueous NaHCO₃. The layers were separated, and the aqueous extracted with EtOAc (3 × 50 mL). The combined organics were washed with H₂O (3 × 100 mL) and brine (100 mL), dried (Na₂SO₄) and concentrated in vacuo. The crude residue was purified by flash column chromatography in a solvent gradient (SiO₂, 5% → 10% EtOAc/hexanes) to provide 22 (1.50 g, 95%) as a colorless oil, which contained small amounts of the diastereomeric products 23 and 24. Spectral data is reported only for the major product, 22: [α]²⁶_D +5.2 (c=1.0, CHCL₃); ¹H NMR (500 MHz, CDCl₃) δ 0.75 (3H, d, J=6.8 Hz), 0.83 (3H, d, J=6.8 Hz), 1.44–1.55 (2H, m), 1.55–1.70 (5H, m), 1.70–1.87 (6H, m), 1.87–1.96 (2H, m), 2.08–2.18 (1H, m), 2.24 (1H, dd, J=4.6, 12.9 Hz), 2.28–2.39 (3H, m), 3.26 (1H, ddd, J=3.5, 3.5, 7.0 Hz), 3.33–3.39 (1H, m), 3.40 (1H, t, J=11.0 Hz), 3.68 (3H, s), 3.73 (1H, dd, J=4.5, 11.0 Hz), 3.81 (3H, s), 4.46 (2H, AB quartet, J=11.0, 25.5 Hz), 5.27 (1H, dd, J=6.0, 7.0 Hz), 6.86–6.91 (2H, m), 7.25–7.31 (2H, m); ¹³C NMR (125 MHz) δ 12.60, 14.46, 15.99, 21.47, 22.47, 24.24, 30.01, 30.53, 31.52, 33.29, 33.83, 34.02, 40.13, 42.67, 51.34, 55.13, 67.41, 71.34, 77.16, 81.89, 110.04, 113.60, 122.14, 129.09, 131.13, 134.89, 158.93, 173.93; HRMS (ESI) m/z exact mass calculated for [M+H]⁺ (C₃₀H₄₇O₆) requires m/z 503.3372, found 503.3372.

4.6. Synthesis outlined in Scheme 5

4.6.1. Preparation of 26

A mixture of CH₂CL₂ (663 mL), H₂O (166 mL) and TFA (41 mL) was shaken vigorously in a separatory funnel. The lower organic layer was drained into a round bottom flask containing 22 (6.29 g, 12.5 mmol). The resultant solution was stirred at rt for 3.5 h. The reaction solution was cautiously and slowly poured into ice-cold saturated aqueous NaHCO₃. The layers were separated, and the aqueous extracted with EtOAc (3 × 250 mL). The combined organics were washed with brine, dried (Na₂SO₄), and concentrated in vacuo. The crude residue was purified by flash column chromatography in a solvent gradient (SiO₂, 25% → 50% EtOAc/hexanes) to provide 1,3-diol 26 (4.97 g, 91%, contaminated with ~6% of 27) as a white waxy solid. During this chromatographic separation, it was possible to remove minor contaminant 28. Data for 26: [α]²⁷_D +1.9 (c=1.0, CHCL₃); ¹H NMR (400 MHz, CDCl₃) δ 0.82 (3H, d, J=6.8 Hz), 0.89 (3H, d, J=7.2 Hz), 1.40–1.80 (8H, m), 1.63 (3H, s), 1.84 (1H, dd, J=9.0, 13.2 Hz), 2.16–2.34 (5H, m), 3.19–3.26 (1H, m), 3.50 (1H, dt, J=4.0, 8.0 Hz), 3.63 (1H, dd, J=7.6, 10.8 Hz), 3.67 (3H, s), 3.70 (1H, dd, J=3.2, 10.8 Hz), 3.80 (3H, s), 4.43 (2H, AB quartet, J=11.4, 23.4 Hz), 5.13 (1H, apparent t, J=7.2 Hz), 6.84–6.90 (2H, m), 7.22–7.29 (2H, m); ¹³C NMR (125 MHz) δ 13.82, 15.39, 16.08, 21.51, 29.41, 32.86, 33.91, 34.20, 39.62, 42.89, 51.39, 55.17, 67.66, 71.35, 77.05, 82.26, 113.63, 121.35, 129.36, 130.77, 138.05, 159.03, 174.00; HRMS (ESI) exact mass calculated for [M+H]⁺ (C₂₅H₄₁O₆) requires m/z 437.2903, found 437.2879.

4.7. Synthesis outlined in Scheme 6

4.7.1. Preparation of 29

To a stirred −78 °C solution of diol 26 (9.70 g, 22.2 mmol) in CH₂CL₂ (222 mL) was added 2,6-lutidine (6.20 mL, 53.3 mmol), followed by TIPSOTf (6.57 mL, 24.4 mL) dropwise. The resultant solution was stirred at −78 °C for 20 min, then poured into saturated aqueous NH₄Cl. The layers were separated, and the aqueous phase extracted with EtOAc (3 × 200 mL). The combined organics were washed with 1M HCl, saturated aqueous NaHCO₃, and brine, then dried (MgSO₄) and concentrated in vacuo. The crude residue was carried on to the following step without further purification.

To the crude ester from the preceding step dissolved in 4:1:1 THF/MeOH/H₂O (1.11 L) was added 1M LiOH (193 mL). The resultant solution was stirred at rt for 1.5 h. The reaction was cautiously and slowly poured into 1M HCl, then the mixture was extracted with EtOAc (4 × 500 mL). The combined organics were washed with H₂O (2 × 500 mL) and brine, dried (Na₂SO₄), and concentrated in vacuo. The crude residue was purified by flash column chromatography (SiO₂, 25% → 50% methyl t-butyl ether/hexanes) to provide seco-acid 29 (14.85 g, 88% over 2 steps, contaminated with ~6% of 30) as a colorless oil: [α]²⁶_D +1.1 (c=1.0, CHCL₃); ¹H NMR (500 MHz, CDCl₃) δ 0.85 (3H, d, J=6.5 Hz), 0.91 (3H, d, J=7.0 Hz), 1.05–1.18 (21H, m), 1.50–1.62 (3H, m), 1.62 (3H, s), 1.62–1.72 (1H, m), 1.72–1.82 (2H, m), 1.82 (1H, dd, J=8.5, 13.0 Hz), 1.88–1.98 (1H, m), 2.22–2.30 (3H, m), 2.35 (2H, t, J=7.3 Hz), 3.26–3.32 (1H, m), 3.63 (1H, apparent dt, J=5.0, 7.0 Hz), 3.71 (1H, dd, J=7.3, 9.8 Hz), 3.81 (3H, s), 3.91 (1H, dd, J=3.9, 9.8 Hz), 4.47 (2H, AB quartet, J=11.5, 18.0 Hz), 5.25 (1H, apparent t, J=6.5 Hz), 6.86–6.90 (2H, m), 7.26–7.31 (2H, m); ¹³C NMR (125 MHz) δ 11.73, 13.73, 14.83, 16.17, 17.94, 21.32, 29.68, 29.97, 33.33, 33.75, 33.99, 39.27, 42.97, 55.25, 68.42, 71.42, 76.53, 81.80, 113.71, 122.37, 129.28, 131.15, 135.70, 159.03, 178.22; HRMS (ESI) exact mass calculated for [M+H]⁺ (C₃₃H₅₉O₆Si) requires m/z 579.4075, found 579.4043.

4.7.2. Preparation of 31

To a solution of seco-acid 29 (14.9 g, 25.7 mmol) in PhMe (260 mL) was added i-Pr₂NEt (26.8 mL, 153.9 mmol) and 2,4,6-trichlorobenzoyl chloride (12.1 mL, 77.0 mmol). The resultant solution was stirred for 1.5 h at rt. In a separate flask, DMAP (9.41 g, 77.0 mmol) was dissolved in PhMe (1.3 L). To this solution was added the anhydride solution dropwise via cannula over 4 h, and the resultant cloudy suspension was stirred a further 12 h at rt. The reaction mixture was poured into 1M HCl, and the layers were separated. The aqueous phase was extracted with EtOAc (3 × 150 mL), and the combined organics were washed with saturated aqueous NaHCO₃, H₂O, and brine. After drying (MgSO₄), the organic phase was concentrated in vacuo. The crude residue was purified by flash column chromatography in a solvent gradient (SiO₂, 100% hexanes → 2% EtOAc/hexanes) to provide macrolactone 31 (10.61 g, 74%) in pure form as a white waxy solid. During this chromatography, 32 (0.86 g, 6%) was removed. Data for the 31: [α]²⁷_D −17.9 (c=.84, CHCL₃); ¹H NMR (500 MHz, CDCl₃) δ 0.99 (3H, d, J=6.8 Hz), 1.04 (3H, d, J=7.0 Hz). 1.04–1.14 (21H, m), 1.40–1.52 (1H, m), 1.57–1.67 (1H, m), 1.66 (3H, s), 1.67–1.84 (3H, m), 1.85–1.92 (2H, m), 1.92–2.00 (1H, apparent septet, J=6.5 Hz), 2.06–2.13 (1H, m), 2.07 (1H, dt, J=3.5, 12.0 Hz), 2.42–2.53 (2H, m), 3.10–3.16 (1H, m), 3.54 (1H, dd, J=7.0, 10.0 Hz), 3.68 (1H, dd, J=5.5, 10.0 Hz), 3.81 (3H, s), 4.42 (2H, AB quartet, J=11.5, 97.0 Hz), 4.98–5.04 (1H, br d, J=10.0 Hz), 5.08 (1H, ddd, J=3.4, 6.1, 12.0 Hz), 6.87–6.90 (2H, m), 7.25–7.30 (2H, m); ¹³C NMR (125 MHz) δ 11.90, 12.80, 15.64, 17.66, 17.97, 19.28, 20.51, 28.99, 30.59, 32.63, 35.84, 40.42, 45.68, 55.17, 65.17, 70.82, 73.62, 83.10, 113.63, 121.91, 129.34, 131.07, 137.10, 159.01, 173.39; HRMS (ESI) exact mass calculated for [M+H]⁺ (C₃₃H₅₇O₅Si) requires m/z 561.3975, found 561.3985.

4.8. Synthesis outlined in Scheme 7

4.8.1. Preparation of 33

To a stirred 0 °C solution of 31 (4.00 g, 7.13 mmol) in CH₃CN (300 mL) in a Teflon bottle was added pyridine (20.1 mL, 249.6 mmol). After 10 minutes, HF·pyridine (70%, 20.1 mL) was added. Stirring was continued at 0 °C for 72 h. Using extreme caution, the reaction was quenched slowly by repeatedly pipetting ~20 mL aliquots of the reaction mixture onto ice-cold saturated aqueous NaHCO₃. After all of the reaction mixture was transferred, the resultant biphasic mixture was stirred for 30 minutes, then the layers were separated. The aqueous phase was extracted with EtOAc (3 × 200 mL), and the combined organics were washed with 1M HCl and brine, dried (MgSO₄), and concentrated in vacuo. The crude residue was purified by flash column chromatography (25% EtOAc/hexanes) to provide alcohol 33 (2.66 g, 92%) as a white solid: [α]²⁵_D −42.6 (c=1.0, CHCL₃); ¹H NMR (500 MHz, CDCl₃) δ 1.04 (3H, d, J=6.5 Hz), 1.06 (3H, d, J=7.0 Hz), 1.43–1.54 (1H, m), 1.55–1.65 (2H, m), 1.67 (3H, s), 1.67–1.77 (3H, m), 1.77–1.85 (1H, m), 1.85–1.96 (2H, m), 2.11 (1H, td, J=3.4, 12.5 Hz), 2.19 (1H, br d, J=12.5 Hz), 2.41 (1H, dt, J=11.0, 14.0 Hz), 2.54 (1H, dt, J=4.4, 12.7 Hz), 3.09–3.14 (1H, m), 3.45 (1H, dd, J=3.4, 11.7), 3.58 (1H, dd, J=3.9, 11.7), 3.81 (3H, s), 4.40 (2H, AB quartet, J=11.0, 98.5 Hz), 4.85 (1H, ddd, J=2.9, 8.8, 11.7 Hz), 4.99 (1H, br d, J=11.0 Hz), 6.85–6.90 (2H, m), 7.24–7.29 (2H, m); ¹³C NMR (125 MHz) δ 13.70, 15.69, 19.00, 20.65, 28.84, 31.58, 32.44, 35.88, 40.20, 45.67, 55.23, 63.97, 70.76, 74.31, 83.16, 113.68, 121.71, 129.36, 130.95, 137.45, 159.06, 175.07; HRMS (ESI) exact mass calculated for [M+H]⁺ (C₂₄H₃₇O₅) requires m/z 405.2641, found 405.2640.

4.8.2. Preparation of 34

To a solution of alcohol 33 (1.67 g, 4.12 mmol) in CH₂CL₂ (82 mL) was added imidazole (841 mg, 12.4 mmol), triphenylphosphine (2.27 g, 8.65 mmol), and iodine (2.20 g, 8.65 mmol). The resultant mixture was stirred at rt for 12 h. The reaction was quenched by dilution with 1:1 saturated aqueous NaHCO₃/saturated aqueous Na₂S₂O₃ and EtOAc. The layers were separated, and the aqueous phase was extracted with EtOAc (3 × 100 mL). The combined organics were washed with brine, dried (MgSO₄), and concentrated in vacuo. The crude residue was purified by flash column chromatography (SiO₂, 5% EtOAc/hexanes) to provide 34 (2.10 g, 99%) as a clear oil: [α]²⁵_D −8.2 (c=1.0, CHCL₃); ¹H NMR (500 MHz, CDCl₃) δ 1.04 (3H, d, J=7.0 Hz), 1.11 (3H, d, J=7.0 Hz), 1.37–1.49 (1H, m), 1.52–1.68 (1H, m), 1.65 (3H, s), 1.68–1.84 (3H, m), 1.84–1.96 (3H, m), 2.04–2.18 (2H, m), 2.37–2.47 (1H, m), 2.47–2.53 (1H, m), 3.01 (1H, td, J=1.0, 9.8 Hz), 3.10–3.14 (1H, m), 3.31 (1H, dd, J=3.9, 9.8 Hz), 3.82 (3H, s), 4.32 (1H, d, J=11.0 Hz), 4.52 (1H, d, J=11.0 Hz), 4.91–5.02 (2H, m), 6.86–6.91 (2H, m), 7.25–7.30 (2H, m); ¹³C NMR (125 MHz) δ 10.55, 15.69, 17.16, 19.19, 20.67, 28.81, 31.21, 32.56, 35.79, 40.34, 45.58, 55.26, 70.81, 74.45, 83.12, 113.68, 120.89, 129.39, 131.00, 137.85, 159.05, 173.38; HRMS (ESI) exact mass calculated for [M+Na]⁺ (C₂₄H₃₅IO₄Na) requires m/z 537.1472, found 537.1434.

4.8.3. Preparation of 35

To prepare active Zn-Cu couple, Cu(OAc)₂ (39 mg, 0.194 mmol) was suspended in glacial AcOH (10 mL). To this was added Zn dust (636 mg, 9.72 mmol), and the resultant suspension was heated to reflux for one minute or until all of the copper had deposited onto the zinc (disappearance of blue color in the supernatant). Stirring was stopped, and the reddish-gray silt was allowed to settle. The AcOH was removed by pipette, then another portion of AcOH (10 mL) was added, and the suspension was heated to reflux for one minute. The AcOH was removed again by pipette, and this process was repeated once more. Finally, the Zn-Cu couple was rinsed with Et₂O (3 × 10 mL), with each rinse being removed by pipette. The resultant active couple was dried under vacuum for 30 min.

Alkyl iodide 34 (1.00 g, 1.95 mmol) dissolved in anhydrous 15:1 PhH/DMF (7.0 mL) was cannulated into to the active Zn-Cu couple, and the resultant suspension was heated at 50 °C for 1 h. Meanwhile, LiCl (494 mg, 11.7 mmol, dried over flame/vacuum) and Pd(PPh₃)₄ (337 mg, 0.292 mmol) were combined and purged with argon. Freshly distilled anhydrous NMP (8.0 mL) was added, followed by vinyl iodide 21 (1.5 g, 2.92 mmol) in NMP (3.7 mL, plus a little more to aid in transfer). Then the colorless alkylzinc iodide solution was added via cannula (the excess settled Zn was left behind in the original flask as much as possible). The reaction mixture was degassed with one freeze-pump-thaw cycle, and the mixture was heated to 60 °C for 17.5 h. The reaction was cooled to rt, diluted with EtOAc, and poured into saturated aqueous NaHCO₃. The layers were separated, and the aqueous extracted with EtOAc (3 × 50 mL). The combined organics were washed with H₂O (3 × 100 mL) and brine (100 mL), dried (Na₂SO₄) and concentrated in vacuo. The crude residue was purified by flash column chromatography in a solvent gradient (SiO₂, 1% → 2% → 3% EtOAc/hexanes) to provide 35 (1.318 g, 88%) as a colorless oil: [α]²⁵_D −8.2 (c=1.0, CHCL₃); ¹H NMR (500 MHz, CDCl₃) δ 0.02–0.07 (12H, m), 0.84 (3H, d, J=7.0 Hz), 0.88 (3H, d, J=6.5 Hz), 0.88 (9H, s), 0.90 (9H, s), 1.03 (3H, d, J=6.5 Hz), 1.14 (3H, d, J=6.5 Hz), 1.40–1.52 (1H, m), 1.52–1.83 (9H, m), 1.59 (3H, d, J=1.0 Hz), 1.66 (3H, s), 1.83–1.95 (2H, m), 2.02–2.08 (1H, m), 2.09 (1H, dd, J=3.5, 12.5 Hz), 2.14 (1H, dd, J=3.5, 13.5 Hz), 2.37–2.52 (3H, m), 3.10–3.15 (1H, m), 3.81 (3H, s), 3.90 (1H, apparent sextet, J=6.5 Hz), 4.31 (1H, d, J=11.0 Hz), 4.51 (1H, d, J=11.0 Hz), 4.84–4.91 (1H, m), 5.00 (1H, m), 5.12 (1H, d, J=9.5 Hz), 6.85–6.90 (2H, m), 7.25–7.30 (2H, m); ¹³C NMR (125 MHz) δ −4.70, −4.39, −4.34, −4.05, 14.47, 15.66, 15.74, 16.01, 18.07, 18.10, 19.26, 20.61, 23.96, 25.89, 25.96, 28.97, 30.25, 32.61, 35.19, 35.67, 37.61, 43.03, 45.03, 45.74, 55.24, 66.00, 70.82, 73.14, 76.04, 83.21, 113.68, 121.89, 129.39, 130.46, 131.09, 131.60, 137.12, 159.05, 173.59; HRMS (ESI) exact mass calculated for [M+H]⁺ (C₄₅H₈₁O₆Si₂) requires m/z 773.5572, found 773.5577.