Total synthesis and absolute configuration of avenolide, extracellular factor in Streptomyces avermitilis

Abstract

The first total synthesis of extracellular factor, “Avenolide”, in Streptomyces avermitilis has been achieved using a convergent approach. The stereogenic centers in two key segments were installed using Sharpless epoxidation and dihydroxylation. This synthetic study allowed the determination of the absolute configuration of avenolide as 4S,10R, and yielded important information on its structure–activity relationship.

Introduction

In several streptomycete microorganisms, γ-butyrolactones have important, sometimes crucial, roles as extracellular factors in determining the onset of secondary metabolite production and morphological differentiation. Up to date, the structures of all extracellular factors are consisted of γ-butyrolactones.^{1, 2} A-factor is required for streptomycin biosynthesis and sporulation in Streptomyces griseus^{1, 2} and virginia butanolide appears to control virginiamycin biosynthesis in Streptomyces virginiae.^{3, 4, 5} Other studies γ-butyrolactones include IM-2 elicits production of showdomycin and minimycin in Streptomyces lavendulae FRI-5^{3, 4, 5} and SCB1 also elicits the precocious production of actinorhodin in Streptomyces coelicolor A3(2).^{3, 4, 5}

Genome-sequenced Streptomyces avermitilis is an important industrial microorganism for the production of anthelmintic and insecticidal macrocyclic lactone, avermectin, which is used as antiparasitic agents in the medical, veterinary and agricultural fields. To identify the extracellular factor(s) controlling avermectin production in Streptomyces avermitilis, about 1 mg of the extracellular factor from 1000 l of culture filtrate was isolated and purified by chromatographic separation steps.⁶ Since spectroscopic analyses of the extracellular factor purified have elucidated that the factor is a new butenolide structure including two stereogenic centers (Scheme 1) but not γ-butyrolactone structure, the extracellular factor is named as “avenolide.” The absolute configuration could not be elucidated, however, due to the trace quantities isolated and the presence of contaminants. Only the configuration of C4 (S, avenolide numbering) was proposed, by comparison of the CD spectra of avenolide and similar butenolide compounds.^{7, 8}

To undertake further biochemical properties of avenolide, the determination of the absolute structure, access to an adequate supply of avenolide and the elucidation of the structure–activity relationship are required. We describe here the first total synthesis of avenolide, which represents significant progress towards these objectives.

Results and discussion

We first embarked on the synthesis of (4S,10R)-avenolide (1). As outlined in Scheme 1, our retrosynthetic strategy is convergent. We speculated that 1 could be derived from epoxide 2 via ring closing metathesis, which itself could be constructed by the coupling of aldehyde 3 and iodide 4. This route should provide a concise route amenable to large-scale synthesis of avenolide as well as various avenolide analogs, including stereoisomers.

The synthesis of 1 commenced with esterification of a commercially available β-methallyl alcohol with p-anisic acid (Scheme 2). Subsequent Sharpless dihydroxylation^{9, 10} using (DHQ)₂PHAL as a chiral ligand afforded 5 (93% ee).^{11, 12, 13} (The enantiomeric excess was determined by chiral HPLC analysis (condition: DAICEL CHIRALPAK AS-3 (0.46 cm φ × 25 cm), 3:1 hexanes/2-propanol mobile phase (flow rate at 1.0 ml per min) and detection at 254 nm (room temperature, rt)). Tosylation of the primary alcohol followed by a substitution reaction with Me₂CuCNLi₂ and methanolysis gave diol 6. After acetal formation, DIBAL reduction and Parikh–Doering oxidation¹⁴ furnished the desired aldehyde 3.

Another key intermediate 4 was derived from a commercially available 1,5-pentanediol. Mono-TBS protection, TEMPO oxidation, Wittig olefination with Ph₃P=CHCO₂Et and DIBAL reduction afforded E-allyl alcohol 7.^{15, 16} This was then converted to 8 by a two-step sequence of protecting group manipulations. Mesylation of the primary alcohol of 8 followed by treatment with sodium iodide gave rise to 4.

The aldehyde 3 was coupled with an alkyl lithium species derived from halogen–lithium exchange of iodide 4 with t-BuLi to furnish alcohol 9 as a diastereomeric mixture. TPAP oxidation and THP deprotection under acidic conditions afforded ketoalcohol 10. Subsequent Sharpless asymmetric epoxidation¹⁷ using (+)-DET as a chiral ligand gave the desired epoxyalcohol 2. Transformation of the primary alcohol to iodide followed by treatment with zinc yielded the corresponding allyl alcohol, which was acylated with acryloyl chloride to provide 11. Finally, deprotection of the PMB group by treatment with DDQ and ring closing metathesis^{18, 19, 20, 21, 22} using Grubbs second-generation catalyst afforded 1.

To determine the absolute configuration of 1, we also synthesized (4S,10S)-avenolide (19), according to Scheme 2, with (DHQD)₂PHAL used instead of (DHQ)₂PHAL as the chiral ligand in Sharpless asymmetric dihydroxylation (Scheme 3).

We next analyzed the synthetic 1 and 19 with a chiral HPLC column (conditions: DAICEL CHIRALPAK IA-3 (0.46 cm φ × 25 cm), EtOH mobile phase (flow rate of 0.3 ml per min) and detection at 200 nm (0 °C)). When a 1:1 mixture of 1 and 19 was injected onto the chiral HPLC system, the two diastereomers were completely resolved, as shown in Figure 1a. Figures 1b and c show the HPLC chromatogram of 1 and 19, respectively. An authentic sample of the natural avenolide was also analyzed by HPLC, and its retention time was identical to that of 1 as shown in Figure 1d. This result establishes the absolute configuration of avenolide as 4S,10R.

Figure 1

HPLC chromatograms of natural and synthetic samples of avenolide (1 and 19). The abscissa axis indicates retention time (min). (a) 1:1 mixture of 1 and 19; (b) 1; (c) 19; (d) natural avenolide.

We also synthesized (4R,10R)-avenolide (22) and 10-deoxy avenolide (28) for structure–activity relationship studies as shown in Scheme 4. (The spectrum data of compounds in Scheme 3 and 4 are provided as Supplementary information.) The synthesis of 22 was achieved according to our synthetic procedure for 1 as shown in Scheme 2, but using (−)-DET as the chiral ligand during Sharpless epoxidation. In contrast, the synthesis of 28 required a slightly altered synthetic route. A commercially available (S)-2-methyl-1-butanol was subjected to TEMPO oxidation and dithioacetalization to afford 23,²³ which was coupled with iodide 4 to give 24. THP deprotection followed by treatment with [bis(trifluoroacetoxy)iodo]benzene furnished ketone 25. The conversion of 25 into 10-deoxy avenolide (28) was carried out following the same synthetic procedure as that developed for the synthesis of 1 shown in Scheme 3.

Synthetic 1 demonstrated identical activity to that of natural 1. In contrast, the rAvaR1-ligand activities of the C10-epimer (19), the C4-epimer (22) and the 10-deoxy avenolide (28) demonstrated only one-half, one twenty-fifth and one hundredth of the activity of the natural product, respectively.⁶ It can therefore be concluded that the stereochemistry at C4 and C10, and the presence of the hydroxyl group at C10, are important to the activity of avenolide as a rAvaR1 ligand.

In conclusion, we have achieved the total synthesis of avenolide and determined its absolute configuration. By extending the synthetic methodology, we have generated a number of structural analogs, which provide information on the structure–activity relationship of avenolide. Further biological studies on the synthetic avenolide are currently underway and will be reported in due course.

Experimental Procedure

General

IR spectra were obtained using a Horiba FT-710 spectrophotometer (Horiba, Kyoto, Japan). ¹H and ¹³C-NMR spectra were obtained on Mercury-300 and UNITY-400 spectrometers (Agilent Technologies, Santa Clara, CA, USA), and chemical shifts are reported on the δ scale, using TMS as an internal reference. MS spectra were measured on JEOL JMS-700, JEOL JMS-T-100LP and JEOL JMS-AX505HA spectrometers. Optical rotations were recorded on a JASCO DIP-1000 polarimeter (Jasco, Hachioji, Japan). Commercial reagents were used without further purification unless otherwise indicated. Organic solvents were distilled and dried over molecular sieves (3 or 4 Å). Reactions were performed in flame-dried glassware under positive Ar pressure while stirring with a magnetic stirrer bar unless otherwise indicated. Flash chromatography was performed on silica gel 60N (spherical, neutral, particle size 40–50 mm). TLC was performed on 0.25 mm Merck silica gel 60 F254 plates (Merck, Darmstadt, Germany) and visualized by UV (254 nm), and using phosphomolybdic acid and p-anisaldehyde as TLC stains.

(R)-2,3-Dihydroxy-2-methylpropyl-4-methoxybenzoate (5)

To a solution of β-methallyl alcohol (3.50 ml, 41.3 mmol) in CH₂Cl₂ (83 ml) were added DMAP (504 mg, 4.13 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (WSC, 8.68 g, 45.4 mmol) and p-anisic acid (6.91 g, 45.5 mmol) at rt under N₂. The reaction mixture was stirred for 2.5 h at rt. The reaction was quenched with H₂O and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (30:1 hexanes/EtOAc) to furnish the corresponding p-methoxybenzoate (8.38 g, 99%) as a colorless oil.

IR (KBr) 3082, 2939, 2842, 1715, 1607, 1511, 1457 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 8.03 (d, J=9.0 Hz, 2H), 6.92 (d, J=9.0 Hz, 2H), 5.06 (brs, 1H), 4.97 (brs, 1H), 4.72 (brs, 2H), 3.85 (s, 3H), 1.83 (brs, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ 166.0, 163.4, 140.2, 131.7, 122.6, 113.7, 112.7, 67.8, 55.4, 19.6; HRMS (ESI⁺, TFA-Na) calcd for C₁₂H₁₄NaO₃ 229.0841 [M+Na]⁺, found m/z 229.0841.

To a solution of the p-methoxybenzoate (4.00 g, 19.4 mmol) in t-BuOH:H₂O (1:1, 194 ml) were added K₃Fe(CN)₆ (19.2 g, 58.2 mmol), K₂CO₃ (8.05 g, 58.2 mmol), K₂OsO₄(OH)₄ (71.5 mg, 0.19 mmol) and (DHQ)₂PHAL (151 mg, 0.19 mmol) at 0 °C under N₂. The reaction mixture was stirred for 4.5 h at 0 °C. The reaction was quenched with a saturated aqueous Na₂S₂O₃ and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (5:1 hexanes/EtOAc) to afford 5 (4.66 g, quant.) as a colorless oil. The enantiomeric excess (93% ee) of 5 was determined by chiral HPLC analysis (conditions: DAICEL CHIRALPAK AS-3 (0.46 cm φ × 25 cm), mobile phase of 3:1 hexanes/2-propanol (flow rate of 1.0 ml per min) and detection at 254 nm (rt.)).

[α]²⁶_D −2.51 (c 1.0 MeOH); IR (KBr) 3478, 2976, 2842, 1706, 1606, 1512, 1462 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.98 (d, J=9.1 Hz, 2H), 6.90 (d, J=9.1 Hz, 2H), 4.36 (d, J=11.3 Hz, 1H), 4.19 (d, J=11.3 Hz, 1H), 3.85 (s, 3H), 3.57 (d, J=11.6 Hz, 1H), 3.46 (d, J=11.4 Hz, 1H), 2.95 (brs, 1H), 1.26 (s, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 166.9, 163.7, 131.8, 121.8, 113.8, 72.2, 68.0, 66.9, 55.5, 21.3; HRMS (ESI⁺, TFA-Na) calcd for C₁₂H₁₆NaO₅ 263.0895 [M+Na]⁺, found m/z 263.0902.

(R)-2-Methylbutane-1,2-diol (6)

To a solution of 5 (1.58 g, 6.59 mmol) in CH₂Cl₂ (66 ml) were added Et₃N (1.80 ml, 13.2 mmol), Me₃N·HCl (63.0 mg, 0.66 mmol) and TsCl (1.90 g, 9.89 mmol) at rt under N₂. The reaction mixture was stirred for 3 h at rt. The reaction was quenched with H₂O and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (1:1 hexanes/EtOAc) to afford the corresponding tosylate (2.57 g, 99%) as a colorless oil.

[α]²⁶_D +1.94 (c 1.0 CHCl₃); IR (KBr) 3519, 2982, 2842, 1712, 1604, 1511, 1462 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 7.87 (d, J=9.0 Hz, 2H), 7.76 (d, J=9.0 Hz, 2H), 7.26 (d, J=7.8 Hz, 2H), 6.90 (d, J=8.8 Hz, 2H), 4.36 (d, J=11.3 Hz, 2H), 4.00 (d, J=8.0 Hz, 2H), 3.88 (s, 3H), 2.35 (s, 3H), 1.28 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ 166.1, 163.7, 145.2, 132.3, 131.8, 130.0, 128.0, 121.7, 113.7, 72.8, 70.8, 67.7, 55.5, 21.6, 21.6; HRMS (ESI⁺, TFA-Na) calcd for C₁₉H₂₂NaO₇S 417.0984 [M+Na]⁺, found m/z 417.0971.

To a solution of CuCN (1.79 g, 19.9 mmol) in THF (20 ml) was added MeLi (1.07 M in diethyl ether, 37.3 ml, 39.9 mmol) at −78 °C under Ar. After stirring for 20 min at 0 °C, a solution of the tosylate (1.57 g, 3.99 mmol) in THF (20 ml) was added to the reaction mixture at −78 °C. The reaction was stirred for 30 min at −78 °C, then allowed to warm to 0 °C and stirred for 20 min. The reaction was quenched with a saturated aqueous NH₄Cl and the aqueous phase was extracted with EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The resulting residue was dissolved in MeOH (40 ml) and K₂CO₃ (276 mg, 1.99 mmol) was added. After stirring for 14 h at rt, the reaction mixture was concentrated in vacuo. The residue was purified by flash column chromatography (5:1 hexanes/EtOAc) to give 6 (373 mg, 90% over 2 steps) as a colorless oil.

[α]²⁵_D +4.90 (c 1.0 CHCl₃); IR (KBr) 3424, 2926, 2856, 1654, 1462 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 3.34 (d, J=11.1 Hz, 1H), 3.37 (d, J=11.1 Hz, 1H), 3.10 (brs, 1H), 2.71 (brs, 1H), 1.50 (q, J=7.3 Hz, 2H), 1.11 (s, 3H), 0.89 (t, J=7.6 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 73.3, 69.3, 31.0, 22.4, 8.03.

(R)-2-((4-Methoxybenzyl)oxy)-2-methylbutanal (3)

To a solution of 6 (776 mg, 7.46 mmol) in CH₂Cl₂ (30 ml) were added PPTS (93.7 mg, 0.37 mmol) and p-anisaldehyde dimethylacetal (1.27 ml, 7.46 mmol) at rt under N₂. The reaction mixture was stirred for 6 h at rt. The reaction was quenched with H₂O and the aqueous phase was extracted with EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (50:1 hexanes/EtOAc) to afford the corresponding p-methoxybenzylidene acetal (1.66 g, quant.) as a colorless oil.

[α]²⁶_D +1.40 (c 1.0 CHCl₃); IR (KBr) 2969, 2929, 2874, 1614, 1515, 1461 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.42 (d, J=8.8 Hz, 2H), 6.90 (d, J=8.8 Hz, 2H), 5.82 (s, 1H), 3.83 (s, 2H), 3.81 (s, 3H), 1.77–1.68 (m, 2H), 1.38 (s, 3H), 1.00 (t, J=7.5 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 160.4, 130.5, 128.1, 113.7, 103.6, 81.7, 75.4, 55.3, 31.2, 24.3, 8.54; HRMS (ESI⁺, TFA-Na) calcd for C₁₃H₁₉O_3: 223.1334 [M+H]⁺, found m/z 223.1342.

To a solution of the p-methoxybenzylidene acetal (697 mg, 3.14 mmol) in CH₂Cl₂ (31 ml) was added DIBAL (1.02 M in hexanes, 9.00 ml, 9.41 mmol) at 0 °C under N₂. After stirring for 1 h at 0 °C, the reaction was cautiously quenched with MeOH, diluted with CH₂Cl₂ and treated with celite (2.80 g) and Na₂SO₄·10H₂O (2.80 g). The mixture was allowed to warm to rt and stirred for 2 h. It was then filtered through a pad of celite and the filtrate was concentrated. The residue was purified by flash column chromatography (10:1 hexanes/EtOAc) to give the corresponding alcohol (615 mg, 87%) as a colorless oil.

[α]²⁶_D +1.40 (c 1.0 MeOH); IR (KBr) 3444, 2970, 2936, 2879, 2839, 1613, 1514 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.27 (d, J=8.7 Hz, 2H), 6.89 (d, J=8.7 Hz, 2H), 4.37 (s, 2H), 3.81 (s, 3H), 3.59 (d, J=4.8 Hz, 1H), 3.55 (d, J=5.0 Hz, 1H), 2.06 (brs, 1H), 1.66 (dq, J=7.6, 2.3 Hz, 1H), 1.23 (s, 3H), 0.94 (t, J=7.5 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 159.0, 131.2, 129.0, 129.0, 113.9, 113.9, 77.8, 66.9, 63.3, 55.3, 27.8, 19.7, 8.09; HRMS (FAB, m-NBA) calcd for C₁₃H₁₉O₃ 223.1334 [M+H]⁺, found m/z 223.1338.

To a solution of the alcohol (239 mg, 0.94 mmol) in CH₂Cl₂ (9.4 ml) were added DMSO (666 μl, 9.37 mmol), Et₃N (1.31 ml, 9.37 mmol) and SO₃·pyr (895 mg, 5.62 mmol) at rt under N₂. The reaction mixture was stirred for 3.5 h at rt. The reaction was quenched with H₂O and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (50:1 hexanes/EtOAc) to furnish 3 (188 mg, 89%) as a colorless oil.

[α]²²_D +6.30 (c 1.0 CHCl₃); IR (KBr) 2974, 2937, 2838, 1734, 1613, 1514 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 9.62 (s, 1H), 7.27 (d, J=8.8 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 4.39 (d, J=17.6 Hz, 1H), 4.35 (d, J=17.6 Hz, 1H), 3.78 (s, 3H), 1.81–1.63 (m, 2H) 1.29 (s, 3H), 0.91 (t, J=7.6 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 205.3, 159.3, 130.4, 129.1, 113.9, 82.8, 65.9, 55.3, 27.7, 17.7, 7.30; HRMS (FAB, m-NBA) calcd for C₁₃H₁₈NaO₃ 245.1154 [M+Na]⁺, found m/z 245.1162.

(E)-7-((tert-Butyldimethylsilyl)oxy)hept-2-en-1-ol (7)

To a solution of NaH (1.94 g, 47.5 mmol) in THF (158 ml) were added 1,5-pentanediol (5.00 ml, 47.5 mmol) and TBSCl (7.31 g, 47.5 mmol) at 0 °C under N₂. The reaction mixture was stirred for 5 min at 0 °C, then allowed to warm to rt and stirred for 2.5 h. The reaction was quenched with H₂O at 0 °C and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (5:1 hexanes/EtOAc) to afford the corresponding TBS ether (8.30 g, 80%) as a colorless oil.

IR (KBr) 3359, 2933, 2860, 1468, 1254, 1100 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 3.64–3.59 (m, 4H), 1.58–1.52 (m, 4H), 1.40–1.39 (m, 2H), 0.88 (s, 9H), 0.04 (s, 6H); ¹³C-NMR (100 MHz, CDCl₃) δ 63.2, 62.9, 32.5, 32.5, 26.0, 22.0, 18.4, –2.28; HRMS (FAB, m-NBA) calcd for C₁₁H₂₇O₂Si 219.1780 [M+H]⁺, found m/z 219.1776.

To a solution of the TBS ether (4.00 g, 18.3 mmol) in CH₂Cl₂ (183 ml) were added iodobenzene diacetate (8.86 g, 27.5 mmol) and TEMPO (573 mg, 3.67 mmol) at rt under N₂. The reaction mixture was stirred for 5 h at rt. The reaction was quenched with an aqueous Na₂S₂O₃ solution, and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (50:1 hexanes/EtOAc) to afford the corresponding aldehyde (3.96 g, quant.) as a colorless oil.

IR (KBr) 2952, 2933, 2859, 1727, 1468, 1254, 1102 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 9.75 (s, 1H), 3.61 (t, J=6.2 Hz, 2H), 2.46–2.42 (m, 2H), 1.70–1.65 (m, 2H), 1.57–1.51 (m, 2H), 0.87 (s, 9H), 0.03 (s, 6H); ¹³C-NMR (100 MHz, CDCl₃) δ 203.7, 62.6, 43.6, 32.1, 25.9, 18.6, 18.3, –5.34; HRMS (FAB, m-NBA) calcd for C₁₁H₂₅O₂Si 217.1624 [M+H]⁺, found m/z 217.1618.

To a solution of the aldehyde (3.19 g, 14.8 mmol) in benzene (148 ml) was added ethyl(triphenylphosphoranylidene)acetate (7.71 g, 22.1 mmol) under N₂. The reaction mixture was stirred for 3 h at 80 °C. The reaction was concentrated in vacuo. The residue was purified by flash column chromatography (80:1 hexanes/EtOAc) to give the corresponding α,β-unsaturated ester (4.19 g, quant.) as a colorless oil.

IR (KBr) 2933, 2859, 1724, 1655, 1468, 1259, 1101 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 6.94 (dt, J=15.7, 7.0 Hz, 1H), 5.79 (dt, J=15.7, 1.6 Hz, 1H), 4.16 (q, J=7.2 Hz, 2H), 3.59 (t, J=6.1 Hz, 2H), 2.21–2.19 (m, 2H), 1.52–1.49 (m, 4H), 1.26 (t, J=7.1 Hz, 3H), 0.87 (s, 9H), 0.02 (s, 6H); ¹³C-NMR (100 MHz, CDCl₃) δ 166.7, 149.1, 121.4, 62.7, 60.1, 32.2, 31.9, 25.9, 24.4, 18.3, 14.3, –5.32; HRMS (ESI⁺, TFA-Na) calcd for C₁₅H₃₀NaO₃Si 309.1862 [M+Na]⁺, found m/z 309.1877.

To a solution of the α,β-unsaturated ester (1.82 g, 8.02 mmol) in CH₂Cl₂ (80 ml) was added DIBAL (1.02 M in hexanes, 15.7 ml, 16.0 mmol) at 0 °C under N₂. After stirring for 1 h, the reaction was cautiously quenched with MeOH, diluted with CH₂Cl₂ and treated with celite (5.90 g) and Na₂SO₄·10H₂O (5.90 g). The mixture was allowed to warm to rt and stirred for 2 h, then filtered through a pad of celite and the filtrate was concentrated. The residue was purified by flash column chromatography (20:1 hexanes/EtOAc) to furnish 7 (1.96 g, quant.) as a colorless oil.

IR (KBr) 3339, 2932, 2859, 1467, 1254, 1101 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 5.68–5.63 (m, 2H), 4.07 (d, J=4.7 Hz, 2H), 3.60 (t, J=6.3 Hz, 2H), 2.09–2.02 (m, 2H), 1.55–1.39 (m, 4H), 0.88 (s, 9H), 0.04 (s, 6H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 133.2, 129.1, 63.8, 63.0, 32.3, 31.9, 26.0, 26.0, 25.4, 18.4, −5.29; HRMS (FAB, m-NBA) calcd for C₁₃H₂₉O₂Si 245.1937 [M+H]⁺, found m/z 245.1934.

(E)-7-((Tetrahydro-2H-pyran-2-yl)oxy)hept-5-en-1-ol (8)

To a solution of 7 (1.96 g, 8.02 mmol) in CH₂Cl₂ (40 ml) were added PPTS (202 mg, 0.80 mmol) and DHP (7.25 ml, 80.2 mmol) at 0 °C under N₂. The reaction mixture was stirred for 6 h at 0 °C. The reaction was quenched with H₂O and the aqueous phase was extracted with EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (50:1 hexanes/EtOAc) to afford the corresponding THP ether (2.63 g, quant.) as a colorless oil.

IR (KBr) 2935, 2859, 1467, 1254, 1102 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 5.72–5.67 (m, 1H), 5.60–5.54 (m, 1H), 4.64–4.62 (m, 1H), 4.17 (ddd, J=11.9, 5.7, 1.2 Hz, 1H), 3.92 (ddd, J=11.9, 5.7, 1.2 Hz, 1H), 3.90–3.84 (m, 1H), 3.59 (t, J=6.5 Hz, 2H), 3.51–3.48 (m, 1H), 2.08–2.03 (m, 2H), 1.82–1.56 (m, 6H) 1.55–1.39 (m, 4H), 0.88 (s, 9H), 0.03 (s, 6H); ¹³C-NMR (100 MHz, CDCl₃) δ 134.5, 126.3, 97.8, 67.9, 63.1, 62.3, 32.4, 32.1, 30.7, 26.0, 25.5, 25.3, 19.6, 18.4, −5.26; HRMS (ESI⁺, TFA-Na) calcd for C₁₈H₃₆NaO₃Si 351.2325 [M+Na]⁺, found m/z 351.2331.

To a solution of the THP ether (2.11 g, 6.42 mmol) in THF (64 ml) was added TBAF (7.71 ml, 7.71 mmol) at rt under N₂. The reaction mixture was stirred for 4.5 h at rt. The reaction was quenched with H₂O, and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (1:1 hexanes/EtOAc) to give 8 (1.34 g, quant.) as a colorless oil.

IR (KBr) 3393, 2939, 2866, 1499, 1351, 1120 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 5.72–5.65 (m, 1H), 5.61–5.54 (m, 1H), 4.63–4.60 (m, 1H), 4.16 (ddd, J=11.9, 5.5, 1.1 Hz, 1H), 3.90 (ddd, J=11.9, 5.5, 1.1 Hz, 1H), 3.89–3.82 (m, 1H), 3.61 (t, J=6.5 Hz, 2H), 3.50–3.46 (m, 1H), 2.10–2.03 (m, 2H), 1.83–1.41 (m, 10H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 133.0, 127.0, 97.8, 67.8, 62.7, 62.2, 32.2, 32.0, 30.6, 25.4, 25.2, 19.5; HRMS (FAB, m-NBA) calcd for C₁₂H₂₂NaO₃ 273.1467 [M+Na]⁺, found m/z 273.1467.

(E)-2-((7-Iodohept-2-en-1-yl)oxy)tetrahydro-2H-pyran (4)

To a solution of 8 (657 mg, 3.07 mmol) in CH₂Cl₂ (31 ml) were added Et₃N (855 μl, 6.14 mmol), Me₃N·HCl (29.3 mg, 0.31 mmol) and MsCl (356 μl, 4.61 mmol) at 0 °C under N₂. The reaction mixture was allowed to warm to rt and stirred for 3 h. The reaction was quenched with H₂O and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (10:1 hexanes/EtOAc) to afford the corresponding mesylate (837 mg, 93%) as a colorless oil.

IR (KBr) 2940, 2866, 1354, 1174, 1121 cm⁻¹, ¹H-NMR (400 MHz, CDCl₃) δ 5.69–5.64 (m, 1H), 5.62–5.55 (m, 1H), 4.61–4.60 (m, 1H), 4.21 (t, J=6.5 Hz, 2H), 4.17 (ddd, J=12.0, 5.3, 1.2 Hz, 1H), 3.91 (ddd, J=12.0, 5.3, 1.2 Hz, 1H), 3.88–3.47 (m, 1H), 3.51–3.47 (m, 1H), 2.99 (s, 3H), 2.12–2.06 (m, 2H), 1.84–1.46 (m, 10H); ¹³C-NMR (100 MHz, CDCl₃) δ 133.0, 127.0, 97.8, 69.8, 67.6, 62.2, 37.2, 31.5, 30.5, 28.5, 25.3, 24.7, 19.4; HRMS (ESI⁺, TFA-Na) calcd for C₁₃H₂₄NaO₅S 315.1242 [M+Na]⁺, found m/z 315.1206.

To a solution of NaI (2.42 g, 16.2 mmol) in acetone (216 ml) was added the mesylate (3.15 g, 10.8 mmol) under N₂. The reaction mixture was stirred for 8.5 h at reflux. After cooling to rt, the reaction was quenched with H₂O and the aqueous phase was extracted with EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (80:1 hexanes/EtOAc) to give 4 (3.49 g, quant.) as a colorless oil.

IR (KBr) 2941, 2869, 1451, 1350, 1121 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 5.71–5.54 (m, 2H), 4.61 (dd, J=4.3, 2.9 Hz, 1H), 4.17 (ddd, J=12.0, 5.4, 1.0 Hz, 1H), 3.91 (ddd, J=12.0, 5.3, 1.2 Hz, 1H), 3.88–3.82 (m, 1H), 3.53–3.47 (m, 1H), 3.17 (t, J=7.0 Hz, 2H), 2.10–2.03 (m, 2H), 1.87–1.44 (m, 10H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 133.5, 126.8, 97.8, 67.7, 62.3, 33.0, 31.2, 30.6, 29.8, 25.5, 19.6, 6.80; HRMS (FAB, m-NBA): calcd for C₁₂H₂₁NaO₂I 347.0484 [M+Na]⁺, found m/z 347.0484.

(3R,E)-3-((4-Methoxybenzyl)oxy)-3-methyl-11-((tetrahydro-2H-pyran-2-yl)oxy)undec-9-en-4-ol (9)

To a solution of iodide 4 (844 mg, 3.8 mmol) in pentane (22 ml) were added Et₂O (8.2 ml) and t-BuLi (1.55 M in pentane, 5.64 ml, 8.74 mmol) at −78 °C under Ar. After stirring for 15 min at −78 °C, a solution of aldehyde 3 (1.35 g, 4.18 mmol) in Et₂O (7.0 ml) was added. The reaction mixture was allowed to warm to 0 °C and stirred for 3 h. The mixture was quenched with an aqueous NH₄Cl solution and the aqueous phase was extracted with EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (15:1 hexanes/EtOAc) to afford 9 (1.54 g, 96%) as a colorless oil.

[α]²²_D +3.88 (c 1.0 CHCl₃); IR (KBr) 3561, 2837, 1612, 1512, 1458 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.26 (d, J=8.8 Hz, 2H), 6.88 (d, J=8.8 Hz, 2H), 5.75–5.68 (m, 1H), 5.62–5.55 (m, 1H), 4.64 (dd, J=3.5, 3.0 Hz, 1H), 4.34 (s, 2H), 4.19 (ddd, J=11.9, 5.6, 1.1 Hz, 1H), 3.92 (ddd, J=11.9, 6.7, 0.8 Hz, 1H), 3.88–3.82 (m, 1H), 3.80 (s, 3H), 3.68–3.64 (m, 1H), 3.54–3.47 (m, 1H), 2.17–2.05 (m, 2H), 1.86–1.15 (m, 14H), 1.14 (s, 3H), 0.92 (t, J=7.5 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 159.0, 134.9, 131.0, 128.9, 126.1, 113.8, 97.7, 80.1, 73.9, 67.8, 62.8, 62.2, 55.2, 32.3, 30.8, 30.6, 29.6, 29.1, 26.5, 26.4, 25.4, 19.5, 17.6, 14.1, 7.34; HRMS (ESI⁺, TFA-Na) calcd for C₂₅H₄₀NaO₅ 443.2773 [M+Na]⁺, found m/z 443.2753.

(R,E)-11-Hydroxy-3-((4-methoxybenzyl)oxy)-3-methylundec-9-en-4-one (10)

To a solution of 9 (674 mg, 1.60 mmol) in CH₂Cl₂ (16 ml) were added NMO (380 mg, 3.21 mmol), TPAP (28.2 mg, 0.08 mmol) and 4 Å molecular sieves (674 mg) at rt under N₂. The reaction mixture was stirred for 1.5 h at rt. The mixture was filtered through a pad of celite. The filtrate was diluted with H₂O and extracted with CH₂Cl₂ followed by EtOAc. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (40:1 hexanes/EtOAc) to afford the corresponding ketone (652 mg, 97%) as a colorless oil.

[α]²⁷_D +7.80 (c 1.0 CHCl₃); IR (KBr) 2940, 2868, 1712, 1613, 1514, 1459 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.27 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 5.71–5.51 (m, 1H), 5.60–5.51(m, 1H), 4.63–4.25 (m, 1H), 4.32 (d, J=10.7 Hz, 1H), 4.28 (d, J=10.7 Hz, 1H), 4.17 (ddd, J=12.0, 5.5, 1.1 Hz, 1H), 3.91 (ddd, J=12.0, 5.5, 1.1 Hz, 1H), 3.88–3.83 (m, 1H) 3.80 (s, 3H), 3.80–3.46 (m, 1H), 2.64 (dt, J=7.3, 3.5 Hz, 2H), 2.09–2.01 (m, 2H), 1.87–1.35 (m, 10H), 1.42–1.35 (m, 2H), 1.32 (s, 3H), 0.84 (t, J=7.5 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 214.9, 159.0, 134.2, 130.7, 128.6, 126.4, 113.8, 97.8, 84.9, 67.8, 65.1, 62.6, 55.3, 36.8, 32.2, 30.7, 29.3, 28.8, 25.5, 23.0, 19.9, 19.6, 7.89; HRMS (ESI⁺, TFA-Na) calcd for C₂₅H₃₈NaO₅ 441.2617 [M+Na]⁺, found m/z 441.2605.

To a solution of the ketone (597 mg, 1.43 mmol) in MeOH (14 ml) was added PPTS (359 mg, 1.43 mmol) at rt under N₂. The reaction mixture was stirred for 5 h at rt. The reaction was quenched with H₂O and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (10:1 hexanes/EtOAc) to afford 10 (475 mg, 99%) as a colorless oil.

[α]²²_D +4.35 (c 1.0 CHCl₃); IR (KBr) 3561, 3298, 2866, 1712, 1612, 1513, 1458 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.27 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 5.69–5.58 (m, 2H), 4.32 (d, J=10.7 Hz, 1H), 4.29 (d, J=10.7 Hz, 1H), 4.07–4.05 (m, 2H), 3.80 (s, 3H), 2.64 (dt, J=7.3, 3.8 Hz, 2H), 2.08–2.01 (m, 2H), 1.87–1.68 (m, 2H), 1.62–1.52 (m, 2H), 1.41–1.34 (m, 2H), 1.32 (s, 3H), 0.84 (t, J=7.5 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 215.0, 159.0, 132.8, 130.7, 129.3, 128.6, 113.8, 113.8, 65.1, 63.7, 55.3, 36.8, 32.1, 29.2, 28.8, 23.0, 19.9, 7.90; HRMS (ESI⁺, TFA-Na) calcd for C₂₀H₃₀NaO₄ 357.2042 [M+Na]⁺, found m/z 357.2031.

(R)-8-((2S,3S)-3-(Hydroxymethyl)oxiran-2-yl)-3-((4-methoxybenzyl)oxy)-3-methyloctan-4-one (2)

To a suspension of 4 Å molecular sieves (134 mg) and (+)-DET (177 μl, 1.03 mmol) in CH₂Cl₂ (5.0 ml) was added Ti(OiPr)₄ (305 μl, 1.03 mmol) at −20 °C under Ar. After stirring for 0.5 h, t-BuOOH (413 μl, 2.06 mmol) was slowly added to the suspension at −20 °C and the resulting mixture was stirred for 0.5 h. A solution of 10 (335 mg, 1.03 mmol) in CH₂Cl₂ (5.3 ml) was then added dropwise to the reaction mixture and the mixture was stirred for 18 h at −20 °C. The reaction was quenched with Me₂S (105 μl, 1.43 mmol), diluted with CH₂Cl₂ and treated with celite (1.10 g) and Na₂SO₄·10H₂O (1.10 g). The suspension was allowed to warm to rt and then stirred for 2 h. The resulting mixture was filtered through a pad of celite and the filtrate was concentrated in vacuo. The residue was purified by flash column chromatography (5:1 hexanes/EtOAc) to afford 2 (311 mg, 86%) as a colorless oil.

[α]²⁸_D −2.89 (c 1.0 CHCl₃); IR (KBr) 3460, 2935, 1711, 1613, 1514, 1461 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 7.27 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 4.33 (d, J=10.7 Hz, 1H), 4.29 (d, J=10.7 Hz, 1H), 3.90–3.82 (m, 1H), 3.80 (s, 3H), 3.64–3.62 (m, 1H), 2.96–2.88 (m, 2H), 2.66 (dt, J=7.2, 2.6 Hz, 2H), 1.88–1.75 (m, 2H), 1.73–1.54 (m, 4H), 1.48–1.39 (m, 2H), 1.33 (s, 3H), 0.84 (t, J=7.5 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 214.9, 159.0, 130.7, 128.6, 113.8, 84.9, 65.1, 61.7, 58.4, 55.8, 55.3, 36.8, 31.4, 29.2, 25.7, 23.1, 20.0, 7.91; HRMS (ESI⁺, TFA-Na) calcd for C₂₀H₃₀NaO₅ 373.1991 [M+Na]⁺, found m/z 373.1975.

(3S,9R)-9-((4-Methoxybenzyl)oxy)-9-methyl-8-oxoundec-1-en-3-yl acrylate (11)

To a solution of 2 (195 mg, 0.56 mmol) in THF:MeCN (4:1, 5.6 ml) were added imidazole (227 mg, 3.33 mmol), PPh₃ (437 mg, 1.67 mmol) and I₂ (423 mg, 1.67 mmol) at 0 °C under N₂. After warming to rt, the reaction mixture was stirred for 1 h. The reaction was quenched with H₂O and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (30:1 hexanes/EtOAc) to give the corresponding iodide (204 mg, 80%) as a colorless oil.

[α]²⁵_D +6.68 (c 1.0 CHCl₃); IR (KBr) 2936, 2864, 1712, 1613, 1514, 1460 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.28 (d, J=8.8 Hz, 2H), 6.90 (d, J=8.8 Hz, 2H), 4.33 (d, J=10.7 Hz, 1H), 4.30 (d, J=10.7 Hz, 1H), 3.80 (s, 3H), 3.26–3.21 (m, 1H), 3.05–3.00 (m, 1H), 2.97 (ddd, J=4.7, 2.3 Hz, 1H), 2.79 (ddd, J=5.6, 5.5, 1.9 Hz, 1H), 2.66 (dt, J=7.0, 3.8 Hz, 2H), 1.88–1.66 (m, 2H), 1.64–1.52 (m, 4H), 1.48–1.40 (m, 2H), 1.33 (s, 3H), 0.84 (t, J=7.5 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 214.7, 159.1, 130.7, 128.6, 113.9, 84.9, 65.1, 62.4, 58.3, 36.8, 31.6, 29.2, 25.6, 23.2, 20.0, 7.93, 5.02; HRMS (ESI⁺, TFA-Na) calcd for C₂₀H₂₉INaO₄ 483.1008 [M+Na]⁺, found m/z 483.1012.

To a solution of the iodide (163 mg, 0.35 mmol) in MeOH (707 μl) were added NaI (133 mg, 0.88 mmol) and Zn (69.4 mg, 1.06 mmol) under N₂. After stirring for 2.5 h at 90 °C, the reaction was quenched with H₂O and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (10:1 hexanes/EtOAc) to afford the corresponding allyl alcohol (118 mg, quant.) as a colorless oil.

[α]²⁶_D +8.93 (c 1.0 CHCl₃); IR (KBr) 3518, 3075, 2941, 1710, 1613, 1513 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.28 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 5.90–5.79 (m, 1H), 5.20 (ddd, J=17.2, 1.4 Hz, 1H), 5.09 (ddd, J=10.4, 1.4 Hz, 1H), 4.32 (d, J=10.8 Hz, 1H), 4.29 (d, J=10.8 Hz, 1H), 4.12–4.05 (m, 1H) 3.80 (s, 3H), 2.65 (dt, J=7.3, 4.3 Hz, 2H), 1.87–1.68 (m, 2H), 1.61–1.34 (m, 6H), 1.32 (s, 3H), 0.84 (t, J=7.5 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 215.0, 159.0, 141.1, 130.7, 128.6, 128.6, 114.5, 113.8, 113.8, 84.8, 72.9, 65.1, 55.2, 36.8, 36.8, 29.2, 25.0, 23.2, 20.0, 7.84; HRMS (ESI⁺, TFA-Na) calcd for C₂₀H₃₀NaO₄ 357.2042 [M+Na]⁺, found m/z 357.2025.

To a solution of the allyl alcohol (179 mg, 0.53 mmol) in CH₂Cl₂ (5.3 ml) were added acryloyl chloride (65 μl, 0.80 mmol), Et₃N (223 μl, 1.60 mmol) and DMAP (cat.) at 0 °C under N₂. After stirring for 1 h at rt, the reaction was quenched with H₂O and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (30:1 hexanes/EtOAc) to afford 11 (187 mg, 90%) as a colorless oil.

[α]²⁴_D +6.32 (c 1.0 CHCl₃); IR (KBr) 2940, 1718, 1615, 1514, 1460 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.27 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 6.40 (dd, J=17.3, 1.5 Hz, 1H), 6.12 (dd, J=17.3, 10.4 Hz, 1H), 5.84–5.73 (m, 2H), 5.31–5.27 (m, 1H), 5.24 (ddd, J=17.3, 9.3, 1.3 Hz, 1H), 5.16 (ddd, J=10.5, 5.9, 1.2 Hz, 1H), 4.32 (d, J=10.7 Hz, 1H), 4.29 (d, J=10.7 Hz, 1H), 3.81 (s, 3H), 2.64 (dt, J=7.3, 3.9 Hz, 2H), 1.87–1.53 (m, 8H), 1.32 (s, 3H), 0.84 (t, J=7.5 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 214.8, 165.5, 159.0, 136.3, 130.7, 130.6, 128.7, 128.6, 116.8, 113.8, 113.8, 84.5, 74.8, 65.1, 55.3, 36.8, 34.1, 29.2, 24.8, 23.3, 20.0, 7.90; HRMS (ESI⁺, TFA-Na) calcd for C₂₃H₃₂NaO₅ 411.2147 [M+Na]⁺, found m/z 411.2136.

(4S,10R)-avenolide (1)

To a solution of 11 (187 mg, 0.48 mmol) in CH₂Cl₂:H₂O (20:1, 4.6 ml) was added DDQ (146 mg, 0.53 mmol) at rt under N₂. After stirring for 1 h at rt, the reaction was quenched with H₂O and the aqueous phase was extracted with CH₂Cl₂. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (40:1 hexanes/EtOAc) to give the corresponding alcohol (159 mg, quant.) as a colorless oil.

[α]²⁵_D −8.51 (c 1.0 CHCl₃); IR (KBr) 3486, 2936, 2863, 1721, 1637, 1459 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 6.41 (dd, J=17.3, 1.5 Hz, 1H), 6.12 (dd, J=17.3, 10.4 Hz, 1H), 5.85–5.73 (m, 2H), 5.34–5.16 (m, 2H), 3.82 (brs, 1H), 2.54–2.45 (m, 2H), 1.75–1.59 (m, 8H), 1.33 (s, 3H), 0.79 (t, J=7.4 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 214.3, 165.5, 136.2, 130.8, 128.6, 116.9, 78.9, 74.6, 35.5, 34.0, 32.3, 25.1, 24.7, 23.3, 7.65; HRMS (ESI⁺, TFA-Na) calcd for C₁₅H₂₄NaO₄ 291.1572 [M+Na]⁺, found m/z 291.1576.

To a solution of the alcohol (38.1 mg, 0.14 mmol) in CH₂Cl₂ (5.0 ml) was added a solution of Grubbs second-generation catalyst (6.0 mg, 0.01 mmol) in CH₂Cl₂ (9.2 ml) at rt under N₂. The reaction mixture was stirred for 2 h at 40 °C. After the reaction was complete, Quadrasil AP (500 mg) was added to the reaction mixture. The suspension was stirred for 5 min at rt and then allowed to stand for 10 min. The mixture was filtered through a pad of celite and the filtrate was washed with H₂O. The organic layers were dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash column chromatography (5:1 hexanes/EtOAc) to afford 1 (43.2 mg, quant.) as a colorless oil.

[α]²⁶_D +2.27 (c 1.0 CHCl₃); IR (KBr) 3480, 2929, 2858, 1746, 1710, 1459 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.44 (dd, J=5.8, 1.5 Hz, 1H), 6.11 (dd, J=5.7, 2.0 Hz, 1H), 5.05–5.02 (m, 1H), 3.76 (brs, 1H), 2.56–2.47 (m, 2H), 1.83–1.61 (m, 6H), 1.61–1.42 (m, 2H), 1.33 (s, 3H), 0.79 (t, J=7.4 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 214.2, 173.0, 156.1, 121.7, 83.0, 78.9, 35.4, 33.0, 32.4, 25.2, 24.6, 23.2, 7.66; HRMS (FAB, m-NBA) calcd for C₁₃H₂₁O₄ 241.1440 [M+H]⁺, found m/z 241.1447.

(4S,10S)-avenolide (19)

[α]²⁴_D +48.91 (c 1.0, CHCl₃); IR (KBr) 3483, 2940, 2874, 1748, 1711, 1459 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.45 (dd, J=5.7, 1.5 Hz, 1H), 6.12 (dd, J=5.7, 2.1 Hz, 1H), 5.08–5.03 (m, 1H), 3.77 (brs, 1H), 2.60–2.45 (m, 2H), 1.87–1.41 (m, 8H), 1.34 (s, 3H), 0.81 (t, J=7.5 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 214.1, 173.0, 156.0, 121.7, 83.0, 78.9, 35.4, 33.0, 32.4, 25.2, 24.6, 23.1, 7.66; HRMS (FAB, m-NBA) calcd for C₁₃H₂₁O₄ 241.1440 [M+H]⁺, found m/z 241.1438.

(4R,10R)-avenolide (22)

[α]²⁶_D −51.07 (c 1.0. CHCl₃); IR (KBr) 3482, 3093, 2930, 1748, 1458 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.45 (dd, J=5.7, 1.5 Hz, 1H), 6.12 (dd, J=5.7, 2.1 Hz, 1H), 5.07–5.02 (m, 1H), 3.76 (brs, 1H), 2.59–2.45 (m, 2H), 1.86–1.34 (m, 8H), 1.34 (s, 3H), 0.80 (t, J=7.4 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 214.4, 173.2, 156.2, 121.9, 83.2, 79.1, 35.6, 33.2, 32.6, 35.4, 24.9, 23.3, 7.86; HRMS (ESI⁺, TFA-Na) calcd for C₁₃H₂₀NaO₄ 263.1259 [M+Na]⁺, found m/z 263.1271.

10-Deoxy avenolide (28)

[α]²⁵_D +47.75 (c 1.0. CHCl₃); IR (KBr) 3503, 3091, 2930, 1753, 1708 cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 7.45 (dd, J=5.7, 1.5 Hz, 1H), 6.11 (dd, J=5.7, 2.1 Hz, 1H), 5.07–5.02 (m, 1H), 2.47–2.40 (m, 3H), 1.81–1.28 (m, 8H), 1.05 (d, J=7.0 Hz, 3H), 0.87 (t, J=7.5 Hz, 3H); ¹³C-NMR (75.0 MHz, CDCl₃) δ 214.4, 173.1, 156.2, 121.6, 83.1, 47.9, 40.6, 33.1, 25.9, 24.7, 23.1, 15.9, 11.7; HRMS (ESI⁺, TFA-Na) calcd for C₁₃H₂₀NaO₃ 247.1310 [M+Na]⁺, found m/z 247.1322.

Evaluation of biological activity

Biological activity of the compounds were measured either as the DNA dissociation activity from the rAvaR1–DNA complex in gel-shift experiment or by avermectin-inducing activity of S. avermitilis aco mutant.⁶

Retrosynthetic analysis of (4S,10R)-avenolide (1).

Reagents and conditions: (a) p-anisic acid, WSC, DMAP, CH₂Cl₂, rt, 99%; (b) K₃Fe(CN)₆, K₂CO₃, (DHQ)₂PHAL, K₂OsO₄(OH)₄, t-BuOH:H₂O=1:1, 0 °C, quant., 93% ee; (c) p-TsCl, Et₃N, Me₃N·HCl, CH₂Cl₂, rt, 99%; (d) CuCN, MeLi, THF, 0 °C; (e) K₂CO₃, MeOH, rt, 90% (two steps); (f) p-methoxybenzaldehyde dimethylacetal, PPTS, rt, CH₂Cl₂, quant.; (g) DIBAL, CH₂Cl₂, 0 °C, 87%; (h) SO₃·Py, DMSO, Et₃N, CH₂Cl₂, rt, 89%; (i) NaH, TBSCl, THF, rt, 80%; (j) PhI(OAc)₂, TEMPO, CH₂Cl₂, rt, quant.; (k) Ph₃P=CHCO₂Et, benzene, 80 °C, quant.; (l) DIBAL, CH₂Cl₂, 0 °C, quant.; (m) PPTS, DHP, CH₂Cl₂, 0 °C, quant.; (n) TBAF, THF, rt, quant.; (o) MsCl, Et₃N, Me₃N·HCl, CH₂Cl₂, rt, 93%; (p) NaI, acetone, reflux, quant.; (q) t-BuLi, pentane, Et₂O, −78 °C, then 3, −78 °C to 0 °C, 96%; (r) TPAP, NMO, CH₂Cl₂, rt, 97%; (s) PPTS, MeOH, rt, 99%; (t) (+)-DET, Ti(Oi-Pr)₄, 4 Å molecular sieves, t-BuOOH, CH₂Cl₂, −20 °C, 86%; (u) PPh₃, I₂, imidazole, THF:MeCN=4:1, rt, 80%; (v) NaI, Zn, MeOH, 90 °C, quant.; (w) acryloyl chloride, DMAP, Et₃N, CH₂Cl₂, rt, 90%; (x) DDQ, CH₂Cl₂:H₂O=2:1, rt, quant.; (y) Grubbs second-generation catalyst, CH₂Cl₂, 40 °C, quant.

Reagents and conditions: the same reaction conditions as those described in Scheme 2 were used, unless otherwise noted. (b) K₃Fe(CN)₆, K₂CO₃, (DHQD)₂PHAL, K₂OsO₄(OH)₄, t-BuOH:H₂O=1:1, 0 °C, quant., 93% ee^{11, 12, 13}; (q) t-BuLi, pentane, Et₂O, −78 °C, then 14, −78 °C to 0 °C, 88%.

Reagents and conditions: the same reaction conditions as those described in Scheme 2 were used unless otherwise noted. (z) (−)-DET, Ti(Oi-Pr)₄, 4 Å molecular sieves, t-BuOOH, CH₂Cl₂, −20 °C, 70%; (aa) PhI(OAc)₂, TEMPO, CH₂Cl₂, rt; (bb) 1,3-pentanedithiol, BF₃·Et₂O, −5 °C, 70% (two steps); (cc) t-BuLi, THF:HMPA=10:1, −78 °C, then 4, 82%; (dd) [bis(trifluoroacetoxy)iodo]benzene, CH₃CN:H₂O=9:1, quant.