Total Synthesis and Stereochemical Assignment of Alternapyrone

Abstract

Alternapyrone, a bioactive polyketide produced by the fungal host Aspergillus oryzae, is biosynthesized by a polyketide synthase encoded by the alt1-5 gene cluster. Despite its known bioactivity, the stereochemical configuration of the three stereogenic centers in its polyketide backbone has remained unresolved. In this study, we determined the complete stereostructure of alternapyrone using an integrative approach that combines predictive, rule-based stereochemical analysis with experimental validation through total synthesis. The efficient total synthesis enabled the precise assignment of the hypothesized stereochemistry by matching the synthetic product to the natural compound. This comprehensive study conclusively established the absolute configuration of alternapyrone.

1. Introduction

Fungi have been the source of many groundbreaking drugs, playing a critical role in the treatment of chronic conditions. These fungal-derived compounds have proven invaluable in managing chronic infections, autoimmune diseases, and high cholesterol. Recently, metabolites derived from fungi have entered clinical trials, showing promise in treating chronic diseases such as cancer and drug-resistant depression. Fungi-derived natural products often include polyketides, a large and diverse group of secondary metabolites known for their structural variety [1]. Among these, polyketides featuring an α-pyrone structure with a 6-alkenyl chain display diverse methylation patterns, leading to various metabolites such as citreoviridin [2], verrucosidin [3], and aurovertin B [4] (Figure 1). The carbon backbone of these polyketides is biosynthesized by polyketide synthases (PKSs), a class of enzymes with catalytic domains resembling those of mammalian fatty acid synthases. Fungal PKSs are classified as iterative type I PKSs [5]. During the assembly of polyketide-derived backbones, specific auxiliary domains within fungal PKSs participate selectively in particular condensation cycles, introducing functional diversity into the polyketide chain. In collaboration with our research partners, we have developed a unified stereochemical model that elucidates the configuration of complex polyhydroxylated polyketides. This work has led to the formulation of a biochemistry-based rule, enabling the prediction of the absolute configuration of fungal-derived reduced polyketides [6].

In 2005, Isao Fujii and colleagues introduced the alt1-5 genes, originally cloned from the fungal pathogen Alternaria solani, into the fungal host Aspergillus oryzae. These genes encode a polyketide synthase responsible for producing a polyketide compound called alternapyrone [7]. Since then, researchers have continued to explore methods for synthesizing alternapyrone analogs [8,9]. Using high-resolution electron ionization mass spectrometry (EI-MS), ¹³C NMR, ¹H NMR, and two-dimensional NMR techniques, the two-dimensional structure of alternapyrone was elucidated, and a biosynthetic pathway for its catalytic production was proposed. In 2018, Yit-Heng Chooi’s team tested the biological activity of alternapyrone. Their findings showed that alternapyrone exhibited cytotoxicity against mouse myeloma cells, with a minimum inhibitory concentration (MIC) of 3.1 μg/mL. Notably, its activity against neonatal foreskin fibroblast non-tumor cells was eight times lower (MIC = 25 μg/mL), suggesting a degree of specificity for tumor cells [8]. In 2022, Wattana-Amorn and colleagues investigated the role of C-methylation in alternapyrone biosynthesis. Their work provided insights into the methylation programming of highly reducing polyketide synthases (HR-PKSs) [10]. We have long been interested in the stereochemistry of polyketides, particularly in developing reliable methods to resolve the stereochemical complexity of natural products. To this end, we integrated established stereochemical principles with insights into the biosynthetic pathway to propose the absolute stereochemistry of alternapyrone. This study outlines our efforts to achieve the total synthesis of alternapyrone and confirm its proposed stereochemistry.

The biochemistry-based rule offers a promising approach to simplifying the stereochemical determination of fungal-derived polyketide compounds [6]. We propose that this rule could be applied to predict the absolute configuration of fungal-reduced alternapyrone, potentially optimizing synthetic processes and minimizing redundant efforts. Fungal HR-PKSs (highly reducing polyketide synthases) execute a series of complex reactions, including initiation, elongation, methylation, reduction, and dehydration. According to the “Biochemistry-guided Rule” [6], the keto reductase (KR) domain reduces keto groups to hydroxyl groups with a °R-configuration, the enoyl reductase (ER) domain reduces enoyl groups to methyl groups with a °S-configuration, and the methyltransferase (MT) domain incorporates methyl groups with a °R-configuration (Figure 2a). It is worth noting that the °R/°S configurations described by the biochemistry-based rule differ slightly from the conventional R/S stereochemical notation. The °R-configuration is determined using the following steps: identify and label the group on the polyketide chain elongation side as RC; label the group on the chain initiation side as RM; assign priority to the groups in this order: OH > RC > RM > Me > H; view the stereogenic center from the side opposite to the hydrogen atom (the group with the lowest priority) and arrange the remaining three groups in descending order of priority. If the sequence follows a clockwise pattern, it is designated as a °R configuration. If the sequence is counterclockwise, it is classified as a °S configuration. This systematic approach enhances clarity and provides a robust framework for stereochemical prediction of the absolute configuration of fungal-derived polyketides.

The absolute configuration of alternapyrone was predicted based on the rule mentioned above and is illustrated in Figure 2b. Specifically, the enoyl reductase (ER) domain sequentially reduces the intermediate, resulting in an °S-configuration for the three methyl groups. To validate this stereochemical prediction, it is crucial to synthesize the compound. Experimental verification will confirm the proposed structure and offer valuable insights into the reliability of our biochemistry-based prediction model, supporting its application to other fungal polyketide systems.

The retrosynthetic analysis of alternapyrone (1) is outlined in Scheme 1. To address the potential instability of the conjugated diene group, we propose synthesizing alternapyrone via a Stille coupling in the final steps. This strategy allows alternapyrone to be retrosynthetically traced back to alkenyl iodide (2) and alkenyl stannane (3). The α-pyrone core can be constructed through a retro Diels–Alder reaction, which simplifies its derivation from compound (4). This intermediate contains a methylene-protected group and can be converted into allyl iodide (2). Intermediate (4) can be synthesized via an aldol reaction between chiral aldehyde (6) and compound (5). In parallel, the long-chain stannane (3) can be readily obtained using a samarium diiodide-mediated Kagan–Molander coupling reaction between aldehyde (7) and allyl bromide (8). Following this retrosynthetic pathway, the complex structure of alternapyrone can be systematically constructed from simpler, commercially available compounds, ensuring precise stereochemical control at each step.

2. Results and Discussion

Our synthesis, as shown in Scheme 2, is commenced with the preparation of vinyl iodide (2) starts from the known acid 9 [11]. Thus, the condensation of acid (9) with (S)-4-benzyl-2-azolidinone (10) produced the acyloxazolidinone 11 in 86% yield. Subsequent alkylation of the resulting acyloxazolidinone (11) using methyl iodide in the presence of NaHMDS proceeded with excellent diastereocontrol, providing the methylated adduct (12) as a single isomer in 65% yield [12]. The Evans auxiliary was then removed using sodium borohydride, and the resulting primary alcohol was oxidized under Swern oxidation conditions [13] to generate the corresponding aldehyde (6) in 82% yield over two steps. This aldehyde was then added to a lithio derivative of the cyclic compound (5), generated in situ from (5) and LDA at −78 °C to give the corresponding secondary alcohol intermediate [14] which was directly oxidized with the Dess−Martin periodinane reagent [15] to furnish ketone (4) in 70% yield over two steps. A Retro-Diels–Alder reaction of ketone (4) proceeded smoothly in refluxing toluene, providing the corresponding α-pyrone derivative in 91% yield [16], which was subsequently converted into fragment (2) using MOMCl (chloromethyl methyl ether) in the presence of triethylamine.

The synthesis of allyl bromide (8) commenced with the enantiomerically pure alcohol (14), which was prepared via the Mukaiyama aldol reaction (Scheme 3) [17]. The auxiliary was reductively removed using sodium borohydride, providing diol (15) in 91% yield. The primary alcohol in diol (15) was selectively protected as a TBDPS-ether, giving compound (16). Subsequent tosylation of the secondary alcohol in (16), followed by deoxygenation using lithium triethylborohydride [18], afforded the deoxygenated product (17) in 73% yield over two steps. Removal of the TBDPS group with tetrabutylammonium fluoride (TBAF) in THF afforded the primary alcohol (18) in 82% yield. This primary alcohol (18) was then subjected to the Appel reaction with triphenylphosphine and carbon tetrabromide, furnishing allylic bromide (8) in 85% yield [19].

With allyl bromide (8) in hand, we turned our attention to the synthesis of fragment (3, Scheme 4). Deprotection of the benzyl group [20] from the known compound (19) [21] was achieved using boron trichloride. The exposed primary hydroxyl group was subsequently oxidized with Dess–Martin periodinane (DMP) to afford the corresponding aldehyde (7) [15]. A mixture of allyl bromide (8) and aldehyde (7) was then treated with samarium diiodide in THF, yielding the homoallylic alcohol (21) [22] as a mixture of two diastereomers (d.r. = 1.4:1). These diastereomers were separable by flash chromatography, giving a combined overall yield of 42% from aldehyde (7). Since the newly formed stereogenic center would ultimately be destroyed in a later stage of the synthesis, both isomers of (21) were deemed suitable for use in the total synthesis. To simplify the acquisition of clean spectra for the synthetic product, we chose to proceed with the major isomer in subsequent synthetic studies. Palladium-catalyzed hydrostannation of the alkynyl group in the major isomer of (21) afforded the alkenylstannane compound (3) with >95:5 E/Z selectivity [23].

With both key intermediates (2) and (3) in hand, our focus shifted to the critical Stille cross-coupling reaction to join these two units, which is outlined in Scheme 5. The treatment of alkenylstannane (3) and alkenyl iodide (2) in the presence of tetrakis (triphenylphosphine)palladium, CuTC (copper thiophene-2-carboxylate), and cesium fluoride proceeded smoothly, yielding the desired dienyl adduct (22) [24]. The exposed hydroxyl group in (22) then underwent a deoxygenation process. First, reaction of (22) with iodomethane and carbon disulfide in the presence of NaHMDS formed a xanthate intermediate. This intermediate subsequently underwent a Barton–McCombie deoxygenation reaction using tributyltin hydride and triethylborane as the radical initiator, producing the corresponding hydrocarbon (23) [25]. Finally, the MOM protecting group in (23) was removed under mild acidic conditions [26], resulting in the formation of alternapyrone (1) with a yield of 67%. The structure of the synthetic alternapyrone (1) was confirmed through comprehensive spectroscopic analysis. The HRMS, ¹H NMR, and ¹³C NMR data matched closely with reported research values for natural alternapyrone (see Supplementary Materials for details). The agreement in chemical shifts and coupling constants provided strong evidence for the successful synthesis. Additionally, further measurement of the CD spectrum of synthetic alternapyrone, along with a comparison to the calculated spectra [27,28] of the two possible enantiomers, confirmed the configuration of the synthetic sample. Consequently, the structure of alternapyrone (1) was unambiguously assigned as depicted in Figure 2b.

3. Materials and Methods

3.1. General

All reactions were conducted under an atmosphere of dry nitrogen or argon. Oxygen and/or moisture-sensitive reagents were transferred appropriately. All reaction solvents were purified and dried before use. Flash column chromatography was performed using the indicated solvents on silica gel 60 (230–400 mesh ASTM E. Qingdao, Tsingtao, China). The reactions were monitored using thin-layer chromatography. ¹H- and ¹³C-NMR were taken in CDCl₃ at 400 MHz and 75.0 MHz (Bruker, Karlsruhe, Germany). Chemical shifts were reported in parts per million (ppm) using the solvent resonance internal standard (deuterochloroform, 7.26 and 77.16 ppm). Data are reported as follows: chemical shift, multiplicity (ovrlp = overlapping, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constant, and integration. The ratios of diastereomers (dr) were determined by ¹H-NMR (400 MHz) operating at a signal/noise ratio of >200:1. High-resolution mass spectra were measured on an ABI Q-star Elite (Beijing, China). Optical rotations were recorded on a Rudolph AutoPol-I polarimeter (Shanghai, China) at 589 nm with a 50 mm cell. Data are reported as follows: specific rotation (c (g/100 mL), solvent).

3.2. General Experimental Procedures

(S,E)-4-benzyl-3-(5-iodo-4-methylpent-4-enoyl)oxazolidin-2-one (11)

To a cooled (−10 °C) solution of compound 9 (3.68 g, 15.34 mmol, 1.0 equiv.) in anhydrous THF (80 mL, 0.19 M) under argon, TEA (5.3 mL, 38.35 mmol, 2.5 equiv.) and PivCl (1.9 mL, 15.34 mmol, 1.0 equiv.) were added. After being stirred at −10 °C for 1 h, LiCl (976 mg, 23.01 mmol, 1.5 equiv.) and (S)-4-benzyl-2-azolidinone 10 (2.66 g, 15.03 mmol, 0.98 equiv.) were added. The reaction mixture was warm to ambient temperature, at which point the starting material was consumed, as verified by TLC analysis, and the aqueous saturated NH₄Cl (20 mL) was slowly added at 0 °C. The organic layer was separated and the aqueous phase was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with brine (20 mL) and dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (20% EtOAc/hexanes) to give compound 11 as a colorless oil (5.26 g, 86%).

• R_f = 0.40 (20% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +25.5 (c 0.50, CHCl₃);
• HRMS (ESI, m/z) for C₁₆H₁₈INO₃Na⁺ [M + Na]⁺: calcd. 422.0224; found: 422.0225.
• ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.24 (m, 3H), 7.23–7.17 (m, 2H), 6.03 (q, J = 1.1 Hz, 1H), 4.67 (ddt, J = 12.8, 7.1, 3.3 Hz, 1H), 4.26–4.14 (m, 2H), 3.28 (dd, J = 13.4, 3.4 Hz, 1H), 3.22–2.98 (m, 2H), 2.77 (dd, J = 13.4, 9.6 Hz, 1H), 2.68–2.52 (m, 2H), 1.90 (d, J = 1.0 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 172.1, 153.5, 146.2, 135.3, 129.5, 129.1, 127.5, 76.4, 66.4, 55.3, 38.0, 33.9, 33.8, 24.1.

(S)-4-benzyl-3-((S,E)-5-iodo-2,4-dimethylpent-4-enoyl)oxazolidin-2-one (12)

To a stirred solution of 11 (3.30 g, 8.27 mmol, 1.0 equiv.) in anhydrous THF (50 mL), NaHMDS (5.4 mL, 10.75 mmol, 2.0 M in THF. 1.3 equiv.) was added dropwise at −78 °C under argon. After being stirred for 30 min, MeI (0.77 mL, 12.41 mmol, 1.5 equiv.) was added. This mixture was cooled to –78 °C, and stirred for 1 h, then slowly warmed to −40 °C and stirred at this temperature for 3 h, at which point 11 had been consumed,, as verified by TLC analysis; then, aqueous saturated NH₄Cl (20 mL) was slowly added. The aqueous phase was extracted with MTBE (3 × 50 mL). The combined organic phases were washed with H₂O (10 mL) and brine (10 mL) in sequence, and then dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (12% EtOAc/hexanes) to give compound 11 as a colorless oil (2.22 g, 65%).

• R_f = 0.40 (20% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +35.3 (c 0.50, CHCl₃);
• HRMS (ESI, m/z) for C₁₇H₂₀INO₃Na⁺ [M + Na]⁺: calcd. 436.0380; found: 436.0380.
• ¹H NMR (500 MHz, CDCl₃) δ 7.36–7.25 (m, 3H), 7.23–7.18 (m, 2H), 5.97 (s, 1H), 4.64 (tt, J = 10.5, 3.1 Hz, 1H), 4.26–4.14 (m, 2H), 4.05–3.95 (m, 1H), 3.25 (dd, J = 13.4, 3.3 Hz, 1H), 2.77 (dd, J = 13.4, 9.6 Hz, 1H), 2.66 (dd, J = 13.5, 7.2 Hz, 1H), 2.29 (dd, J = 13.6, 7.4 Hz, 1H), 1.86 (s, 3H), 1.19 (d, J = 6.8 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 176.2, 153.2, 145.3, 135.3, 129.6, 129.1, 127.5, 77.3, 66.3, 55.5, 43.2, 38.0, 36.0, 23.9, 17.1.

(S,E)-5-iodo-2,4-dimethylpent-4-en-1-ol (13)

To a stirred solution of compound 12 (1.11 g, 2.69 mmol, 1.0 equiv.) in THF (20 mL)/H₂O (10 mL) at 0 °C, NaBH₄ (508 mg, 13.44 mmol, 5.0 equiv.) was added. The reaction mixture was allowed to warm to ambient temperature and stirred continuously for 3 h, at which point 12 had been consumed, as verified by TLC analysis; the reaction was quenched by adding aqueous saturated NH₄Cl (5 mL) slowly at 0 °C. The organic layer was separated and the aqueous phase was extracted with MTBE (3 × 20 mL). The combined organic phases were washed with brine (10 mL) and dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (25% EtOAc/hexanes) to give compound 13 as a colorless oil (607 mg, 94%). Spectroscopic data were in agreement with those reported previously [29].

• R_f = 0.40 (25% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{28}}$ = −9.6 (c 1.25, CHCl₃);
• ¹H NMR (500 MHz, CDCl₃) δ 5.92–5.86 (m, 1H), 3.52–3.38 (m, 2H), 2.35 (ddd, J = 13.5, 6.1, 1.2 Hz, 1H), 2.01 (ddd, J = 13.5, 8.4, 0.9 Hz, 1H), 1.92–1.81 (m, 1H), 1.83 (d, J = 1.1 Hz, 3H), 0.87 (d, J = 6.7 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 146.7, 75.8, 67.8, 43.7, 33.9, 23.9, 16.4.

(S,E)-5-iodo-2,4-dimethylpent-4-enal (6)

To a solution of (COCl)₂ (0.18 mL, 2.13 mmol, 5.0 equiv.) in anhydrous DCM (12 mL) at −78 °C, DMSO (0.30 mL, 4.25 mmol, 10.0 equiv.) was added dropwise. After being stirred for 10 min, a solution of compound 13 (102 mg, 0.43 mmol, 1.0 equiv.) in DCM (1 mL) was added, and the mixture was stirred at −78 °C for 20 min. The mixture was warmed to −40 °C and kept for 20 min, before it was re-cooled to −78 °C, and then TEA (1.1 mL, 7.65 mmol, 18.0 equiv.) was added dropwise. The reaction mixture was allowed to warm to ambient temperature and stirred continuously for 2 h, at which point 13 had been consumed, as verified by TLC analysis; then, the reaction was quenched by slowly adding aqueous saturated NH₄Cl (3 mL) at 0 °C. The organic layer was separated and the aqueous phase was extracted with EA (3 × 10 mL). The combined organic phases were washed with brine (5 mL) and dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (20% EtOAc/hexanes) to give compound 8 as a colorless oil (88 mg, 87%). Spectroscopic data were in agreement with those reported previously [30].

• R_f = 0.50 (20% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{21}}$ = −22.7 (c 0.72, CHCl₃);
• ¹H NMR (500 MHz, CDCl₃) δ 9.62 (d, J = 1.5 Hz, 1H), 5.99–5.95 (m, 1H), 2.64 (ddd, J = 13.9, 6.0, 1.3 Hz, 1H), 2.59–2.48 (m, 1H), 2.19 (ddd, J = 13.9, 8.4, 0.8 Hz, 1H), 1.82 (d, J = 1.1 Hz, 3H), 1.06 (d, J = 7.0 Hz, 3H).
• ¹³C NMR (126 MHz, CDCl₃) δ 203.8, 144.6, 77.2, 44.3, 40.2, 23.8, 13.2.

6-((4S,E)-7-iodo-4,6-dimethyl-3-oxohept-6-en-2-yl)-2,2,5-trimethyl-4H-1,3-dioxin-4-one (4)

To a cooled (−78 °C) stirred solution of DIPA (0.33 mL, 2.35 mmol, 7.0 equiv.) in anhydrous THF (7 mL), nBuLi (1.5 mL, 2.35 mmol, 1.6 M in hexane, 7.0 equiv.) was added dropwise. After being stirred at −78 °C for 15 min, a solution of compound 5 (400 mg, 2.35 mmol, 7.0 equiv.) in THF (2 mL) was added dropwise and the mixture was kept at −78 °C for 30 min before a solution of compound 6 (80 mg, 0.336 mmol, 1.0 equiv.) in THF (1 mL) was added dropwise. The mixture was stirred at −78 °C overnight, at which point all the starting materials had been consumed, as verified by TLC analysis; then, the reaction was quenched by slowly adding aqueous saturated NH₄Cl (3 mL). After acidified to pH 1~2 with 1 M HCl, the organic layer was separated and the aqueous phase was extracted with EA (3 × 50 mL). The combined organic phases were washed with brine (10 mL) and dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (33% EtOAc/hexanes), which was directly used in the next step.

To a cooled (0 °C) solution of the above intermediate compound (113 mg, 0.277 mmol, 1.0 equiv.) in DCM (6 mL, 0.05 M.), DMP (590 mg, 1.39 mmol, 5.0 equiv.) was added. The mixture was allowed to slowly warm to ambient temperature for 1 h, at which point the starting material had been consumed, as verified by TLC analysis; then, the reaction was quenched by slowly adding aqueous saturated Na₂S₂O₃ (10 mL) and NaHCO₃ (10 mL) at 0 °C. The organic layer was separated and the aqueous phase was extracted with DCM (3 ×50 mL). The combined organic phases were washed with brine (10 mL) and dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (20% EtOAc/hexanes) to give compound 4 as a colorless oil (95 mg, dr =1:1.5, 70% for two steps).

• R_f = 0.40 (20% EtOAc/hexanes), UV and PMA stain.
• HRMS (ESI, m/z) for C₁₆H₂₃IO₄Na⁺ [M + Na]⁺: calcd. 429.0533; found: 429.0521.
• ¹H NMR (400 MHz, CDCl₃) δ 5.95–5.90 (m, 2.5H), 3.76 (q, J = 6.9 Hz, 1H), 3.54 (q, J = 6.9 Hz, 1.5H), 2.94–2.78 (m, 2.5H), 2.56 (ddd, J = 14.0, 6.4, 1.1 Hz, 1H), 2.47 (ddd, J = 13.4, 7.8, 1.0 Hz, 1.5H), 2.23–2.13 (m, 2.5H), 1.91 (s, 3H), 1.90 (s, 4.5H), 1.82 (d, J = 1.1 Hz, 4.5H), 1.76 (d, J = 1.1 Hz, 3H), 1.64 (s, 3H), 1.63 (s, 4.5H), 1.63–1.59 (m, 7.5H), 1.26 (d, J = 6.9 Hz, 3H), 1.23 (d, J = 6.9 Hz, 4.5H), 1.05 (d, J = 7.1 Hz, 3H), 1.02 (d, J = 6.7 Hz, 4.5H).
• ¹³C NMR (101 MHz, CDCl₃) δ 209.1, 208.2, 162.7, 162.6, 162.4, 144.7, 144.7, 105.6, 105.5, 102.5, 102.3, 77.9, 77.6, 48.8, 46.8, 44.2, 42.8, 42.3, 41.9, 26.3, 26.0, 24.0, 24.0, 23.9, 23.8, 17.4, 16.7, 12.4, 12.3, 10.4, 10.4.

(S,E)-6-(5-iodo-4-methylpent-4-en-2-yl)-4-(methoxymethoxy)-3,5-dimethyl-2H-pyran-2-one (2)

To a flask equipped with a reflux condenser, toluene (54 mL) was added, and heated to 110 °C, to which a solution of compound 4 (23 mg, 0.0566 mmol, 1.0 equiv.) in toluene (2 mL) was added dropwise. The mixture was then refluxed for 65 h and then cooled to ambient temperature. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (100% EtOAc/hexanes) to give the intermediate compound S1 as a white solid (18 mg, 91%).

• R_f = 0.50 (100% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +88.1 (c 0.50, CHCl₃);
• HRMS (ESI, m/z) for C₁₃H₁₇IO₃Na⁺ [M + Na]⁺: calcd. 371.0115; found: 371.0109.
• ¹H NMR (400 MHz, CDCl₃) δ 5.90 (q, J = 1.1 Hz, 1H), 3.16–3.02 (m, 1H), 2.57 (ddd, J = 13.7, 8.4, 1.0 Hz, 1H), 2.37 (ddd, J = 13.7, 6.7, 1.0 Hz, 1H), 2.01 (s, 3H), 1.96 (s, 3H), 1.79 (d, J = 1.1 Hz, 3H), 1.17 (d, J = 6.9 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 166.3, 165.0, 160.5, 145.1, 106.9, 98.5, 77.5, 43.9, 33.2, 24.1, 18.1, 9.7, 8.8.

To a cooled (0 °C) solution of intermediate compound S1 (128 mg, 0.368 mmol, 1.0 equiv.) in anhydrous DCM (30 mL, 0.012 M), TEA (0.26 mL, 1.84 mmol, 5.0 equiv.) was added dropwise. After being stirred at 0 °C for 10 min, MOMCl (0.14 mL, 1.84 mmol, 5.0 equiv.) was added dropwise. The reaction mixture was allowed to warm to ambient temperature and vigorous stirring was continued for 12 h, at which point TLC analysis indicated that only a trace of aldehyde S1 remained. The reaction was quenched by slowly adding H₂O at 0 °C. The organic layer was separated and the aqueous phase was extracted with DCM (3 × 30 mL). The combined organic phases were washed with brine (10 mL) and dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (33% EtOAc/hexanes) to give compound 2 as a white solid (110 mg, 76%).

• R_f = 0.40 (33% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +109.1 (c 0.50, CHCl₃);
• HRMS (ESI, m/z) for C₁₅H₂₁IO₄Na⁺ [M + Na]⁺: calcd. 415.0377; found: 415.0370.
• ¹H NMR (500 MHz, CDCl₃) δ 5.88 (q, J = 1.1 Hz, 1H), 5.04 (s, 2H), 3.56 (s, 3H), 3.11–3.00 (m, 1H), 2.58 (ddd, J = 13.7, 8.3, 1.0 Hz, 1H), 2.39 (ddd, J = 13.7, 6.7, 1.0 Hz, 1H), 2.01 (s, 3H), 1.92 (s, 3H), 1.80 (d, J = 1.1 Hz, 3H), 1.18 (d, J = 6.8 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 166.3, 165.7, 160.2, 145.2, 110.1, 109.1, 99.2, 77.4, 58.0, 44.0, 33.5, 24.1, 17.9, 11.0, 10.5.

(4R,5S,E)-2,4-dimethylhex-2-ene-1,5-diol (15)

To a cooled (0 °C) stirred solution of compound 14 (4.30 g, 15.98 mmol, 1.0 equiv.) in THF (80 mL)/H₂O (40 mL), NaBH₄ (2.42 g, 63.92 mmol, 4.0 equiv.) was added. The reaction mixture was allowed to warm to ambient temperature and stirred continuously for 2 h, at which point the starting material had been consumed, as verified by TLC analysis; then, the reaction was quenched by adding aqueous saturated NH₄Cl (20 mL) slowly at 0 °C. The organic layer was separated and the aqueous phase was extracted with MTBE (3 × 80 mL). The combined organic phases were washed with brine (20 mL) and dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (150% EtOAc/hexanes) to give compound 15 as a colorless oil (2.10 g, 91%).

• R_f = 0.50 (150% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +20.0 (c 0.50, CHCl₃);
• HRMS (ESI, m/z) for C₈H₁₆O₂Na⁺ [M + Na]⁺: calcd. 167.1043; found: 167.1043.
• ¹H NMR (400 MHz, CDCl₃) δ 5.32–5.23 (m, 1H), 4.02 (d, J = 1.5 Hz, 2H), 3.59–3.48 (m, 1H), 2.45–2.31 (m, 1H), 2.03–1.82 (m, 2H), 1.69 (d, J = 1.5 Hz, 3H), 1.18 (d, J = 6.2 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 137.1, 127.9, 72.0, 68.6, 40.3, 20.5, 17.1, 14.3.

(2S,3R,E)-6-((tert-butyldiphenylsilyl)oxy)-3,5-dimethylhex-4-en-2-ol (16)

To a cooled (0 °C) stirred solution of compound 15 (2.14 g, 14.87 mmol, 1.0 equiv.) in DCM (100 mL), 4-DMAP (363 mg, 2.97 mmol, 0.2 equiv.), TEA (3.1 mL, 22.31 mmol, 1.5 equiv.) and TBDPSCl (4.6 mL, 17.84 mmol, 1.2 equiv.).were sequentially added. The reaction mixture was allowed to warm to ambient temperature and stirred continuously for 2 h, at which point the starting material had been consumed, as verified by TLC analysis; then, the reaction was quenched by adding H₂O (20 mL) slowly at 0 °C. The organic layer was separated and the aqueous phase was extracted with EA (3 × 40 mL). The combined organic phases were washed with brine (20 mL) and dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (12.5% EtOAc/hexanes) to give compound 16 as a colorless oil (5.46 g, 96%). Spectroscopic data were in agreement with those reported previously [31].

• R_f = 0.50 (12.5% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{22}}$ = +26.0 (c 1.00, CHCl₃);
• ¹H NMR (400 MHz, CDCl₃) δ 7.71–7.63 (m, 4H), 7.46–7.33 (m, 6H), 5.27 (dq, J = 10.0, 1.5 Hz, 1H), 4.08 (d, J = 1.5 Hz, 2H), 3.53–3.42 (m, 1H), 2.45–2.31 (m, 1H), 1.64 (d, J = 1.4 Hz, 3H), 1.17 (d, J = 6.2 Hz, 3H), 1.06 (s, 9H), 0.94 (d, J = 6.8 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 137.0, 135.7, 133.9, 129.8, 127.8, 126.5, 71.9, 68.9, 40.3, 27.0, 20.1, 19.4, 17.0, 14.2.

(S,E)-tert-butyl((2,4-dimethylhex-2-en-1-yl)oxy)diphenylsilane (17)

To a cooled (0 °C) stirred solution of compound 16 (5.34 g, 13.97 mmol, 1.0 equiv.) in pyridine (140 mL, 0.1 M), 4-DMAP (171 mg, 1.4 mmol, 0.10 equiv.) and TsCl (13.32 g, 69.85 mmol, 5.0 equiv.) were added. The reaction mixture was allowed to warm to ambient temperature and stirred for 6 h, at which point the starting material had been consumed, as verified by TLC analysis; then, the reaction was quenched by adding aqueous saturated NH₄Cl (40 mL) slowly at 0 °C. The organic layer was separated and the aqueous phase was extracted with DCM (3 × 80 mL). The combined organic phases were washed with aqueous saturated NaHCO₃ (2 × 40 mL) and brine (40 mL), and dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was directly used in the next step without further purification.

To a cooled (0 °C) stirred solution of the above intermediate compound in anhydrous THF (140 mL, 0.1M), LiBHEt₃ (84 mL, 83.82 mmol, 6.0 equiv.) was added dropwise. The reaction mixture was allowed to warm to ambient temperature and stirred continuously for 10 h, at which point the starting material had been consumed, as verified by TLC analysis; then, the reaction was quenched by adding aqueous saturated NH₄Cl (20 mL) slowly at 0 °C. The organic layer was separated and the aqueous phase was extracted with EA (3 × 40 mL). The combined organic phases were washed with brine (10 mL), and then dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (100% hexanes) to give compound 17 as a colorless oil (3.73 g, 73% for two steps).

• R_f = 0.80 (10% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +15.0 (c 0.10, CHCl₃);
• HRMS (ESI, m/z) for C₂₄H₃₄OSiNa⁺ [M + Na]⁺: calcd. 389.2271; found: 389.2273
• ¹H NMR (500 MHz, CDCl₃) δ 7.74–7.66 (m, 4H), 7.45–7.33 (m, 6H), 5.22–5.15 (m, 1H), 4.06 (d, J = 1.5 Hz, 2H), 2.36–2.24 (m, 1H), 1.62 (d, J = 1.4 Hz, 3H), 1.39–1.17 (m, 2H), 1.06 (s, 9H), 0.93 (d, J = 6.6 Hz, 3H), 0.85 (t, J = 7.4 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 135.7, 134.2, 132.8, 131.3, 129.6, 127.7, 69.4, 33.7, 30.5, 27.0, 20.9, 19.5, 13.9, 12.1.

(S,E)-2,4-dimethylhex-2-en-1-ol (18)

To a cooled (0 °C) stirred solution of compound 17 (304 mg, 0.83 mmol, 1.0 equiv.) in THF (3 mL), TBAF (1.2 mL, 1.23 mmol, 1 M in THF, 1.5 equiv.) was added. The reaction mixture was allowed to warm to ambient temperature and stirred continuously for 2 h, at which point the starting material had been consumed, as verified by TLC analysis; then, the reaction was quenched by adding H₂O (1 mL) slowly at 0 °C. The organic layer was separated and the aqueous phase was extracted with Et₂O (3 × 5 mL). The combined organic phases were washed with brine (2 mL), and then dried over anhydrous Na₂SO₄. The solvent was removed in vacuo at 0 °C and the crude product was purified by flash chromatography on silica gel (20% Et₂O/pentane) to give compound 18 as a colorless oil (87 mg, 82%). Spectroscopic data were in agreement with those reported previously [14].

• R_f = 0.40 (20% Et₂O/pentane), PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}$ = +34.3 (c 1.05, CHCl₃);
• ¹H NMR (500 MHz, CDCl₃) δ 5.17 (dq, J = 9.5, 1.3 Hz, 1H), 4.00 (d, J = 1.2 Hz, 2H), 2.36–2.21 (m, 1H), 1.67 (d, J = 1.4 Hz, 3H), 1.41—1.15 (m, 2H), 0.93 (d, J = 6.7 Hz, 3H), 0.83 (t, J = 7.4 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 133.5, 132.9, 69.3, 33.9, 30.4, 20.8, 14.0, 12.1.

(S,E)-1-bromo-2,4-dimethylhex-2-ene (8)

To a cooled (0 °C) stirred solution of compound 18 (434 mg, 3.39 mmol, 1.0 equiv.) in anhydrous DCM (26 mL, 0.13 M), CBr₄ (1.46 g, 4.41 mmol, 1.3 equiv.) and PPh₃ (1.33 g, 5.09 mmol, 1.5 equiv.) were added. The reaction mixture was allowed to warm to ambient temperature and stirred for 0.5 h. The solvent was removed in vacuo at 0 °C and the crude product was purified by flash chromatography on silica gel (pentane) to give compound 8 as a colorless oil (548 mg, 85%).

• R_f = 0.90 (100% pentane), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +9.7 (c 0.50, CHCl₃);
• ¹H NMR (400 MHz, CDCl₃) δ 5.39–5.32 (m, 1H), 3.99–3.95 (m, 2H), 2.32–2.17 (m, 1H), 1.76 (d, J = 1.4 Hz, 3H), 1.43–1.15 (m, 2H), 0.93 (d, J = 6.6 Hz, 3H), 0.83 (t, J = 7.4 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 137.9, 130.9, 42.3, 34.6, 30.2, 20.3, 15.0, 12.0.

(S)-3-methylhex-4-yn-1-ol (20)

To a cooled (−78 °C) stirred solution of compound 19 (1.53 g, 7.57 mmol, 1.0 equiv.) in anhydrous DCM (76 mL, 0.1 M), BCl₃ (23 mL, 22.71 mmol, 1.0 M in DCM. 3.0 equiv.) was added dropwise. The reaction mixture was continuously stirred at −78 °C for 1 h before it was quenched by adding MeOH (20 mL) and aqueous saturated NaHCO₃ (20 mL) slowly. The organic layer was separated and the aqueous phase was extracted with Et₂O (3 × 50 mL). The combined organic phases were washed with brine (10 mL), and then dried over anhydrous Na₂SO₄. The solvent was removed in vacuo at 0 °C and the crude product was purified by flash chromatography on silica gel (33.3% Et₂O/pentane) to give compound 20 as a colorless oil (798 mg, 94%).

• R_f = 0.30 (33.3% Et₂O/pentane), PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +21.7 (c 0.50, CHCl₃);
• HRMS (ESI, m/z) for C₇H₁₂ONa⁺ [M + Na]⁺: calcd. 135.0780; found: 135.0783.
• ¹H NMR (500 MHz, CDCl₃) δ 3.83–3.71 (m, 2H), 2.61–2.48 (m, 1H), 1.85 (s, 1H), 1.77 (d, J = 2.4 Hz, 3H), 1.72–1.54 (m, 2H), 1.15 (dd, J = 6.9, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 83.3, 76.7, 61.5, 39.8, 23.1, 21.7, 3.6.

(S)-3-methylhex-4-ynal (7)

To a cooled (0 °C) stirred solution of compound 20 (300 mg, 2.68 mmol, 1.0 equiv.) dissolved in anhydrous DCM (27 mL, 0.1 M.), DMP (2.27 g, 5.36 mmol, 2.0 equiv.) was added. The reaction mixture was allowed to warm to ambient temperature and stirred continuously for 3 h, at which point the starting material had been consumed, as verified by TLC analysis; then, pentane (27 mL) was added and the reaction mixture was re-cooled to −50 °C. The mixture was rapidly filtered through a short pad of silica gel (25% Et₂O/pentane, −50 °C). The combined organic phases were washed with aqueous saturated Na₂S₂O₃ (2×30 mL), aqueous saturated NaHCO₃ (2 × 30 mL) and brine (10 mL) in sequence, and then dried over anhydrous Na₂SO₄. The solvent was removed in vacuo at 0 °C and the crude product was purified by flash chromatography on silica gel (25% Et₂O/pentane) to give compound 7 as a colorless oil (251 mg, 85%).

• R_f = 0.50 (25% Et₂O/pentane), PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +12.0 (c 0.50, CHCl₃);
• HRMS (ESI, m/z) for C₇H₁₀ONa⁺ [M + Na]⁺: calcd. 133.0624; found: 133.0624.
• ¹H NMR (500 MHz, CDCl₃) δ 9.79 (t, J = 2.1 Hz, 1H), 2.98—2.86 (m, 1H), 2.53 (ddd, J = 16.5, 7.6, 2.2 Hz, 1H), 2.45 (ddd, J = 16.5, 6.3, 2.0 Hz, 1H), 1.77 (d, J = 2.4 Hz, 3H), 1.20 (d, J = 6.9 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 201.8, 81.7, 77.2, 50.4, 21.4, 20.9, 3.5.

(4S,10S,E)-4,8,10-trimethyldodec-8-en-2-yn-6-ol (21)

To a cooled (0 °C) stirred solution of compound 7 (560 mg, 5.08 mmol, 1.0 equiv.) and compound 8 (966 mg, 5.08 mmol, 1.0 equiv.) in THF (50 mL, 0.1 M) under argon, SmI₂ (152 mL, 15.24 mmol, 0.1 M in THF, 3.0 equiv.) was added dropwise. After being stirred at 0 °C for 0.5 h, additional SmI₂ (102 mL, 10.16 mmol, 0.1 M in THF, 2.0 equiv.) was added into the reaction system dropwise, and the resulting mixture was stirred at 0 °C for 12 h. The reaction was quenched by adding aqueous saturated NH₄Cl (60 mL) slowly. The organic layer was separated and the aqueous phase was extracted with EA (3 × 100 mL). The combined organic phases were washed with aqueous saturated Na₂S₂O₃ (80 mL) and brine (40 mL) in sequence, and then dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (8.3% EtOAc/hexanes) to give compounds 21 and 21′ as colorless oils (474 mg, dr = 1.4:1, 42%).

• 21: R_f = 0.40 (10% EtOAc/hexanes), PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +45.3 (c 0.10, CHCl₃);
• HRMS (ESI, m/z) for C₁₅H₂₆ONa⁺ [M+Na]⁺: calcd. 245.1876; found: 245.1876.
• ¹H NMR (500 MHz, CDCl₃) δ 5.01 (dq, J = 9.5, 1.3 Hz, 1H), 3.94 (tt, J = 8.6, 4.1 Hz, 1H), 2.76–2.65 (m, 1H), 2.33–2.23 (m, 1H), 2.18 (ddd, J = 13.2, 4.4, 1.1 Hz, 1H), 2.06 (ddd, J = 13.2, 9.0, 0.9 Hz, 1H), 1.91 (s, 1H), 1.80 (d, J = 2.4 Hz, 3H), 1.66 (d, J = 1.4 Hz, 3H), 1.51–1.39 (m, 2H), 1.40–1.28 (m, 1H), 1.27–1.18 (m, 1H), 1.16 (d, J = 7.0 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H), 0.84 (t, J = 7.4 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 135.7, 130.6, 83.4, 76.4, 66.8, 48.5, 44.4, 34.3, 30.5, 23.0, 22.1, 21.3, 16.5, 12.1, 3.7.
• 21′: R_f = 0.50 (10% EtOAc/hexanes), PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +42.7 (c 0.10, CHCl₃);
• HRMS (ESI, m/z) for C₁₅H₂₆ONa⁺ [M+Na]⁺: calcd. 245.1876; found: 245.1876.
• ¹H NMR (500 MHz, CDCl₃) δ5.00 (dq, J = 9.5, 1.3 Hz, 1H), 3.84 (ddt, J = 8.9, 8.0, 4.4 Hz, 1H), 2.60–2.49 (m, 1H), 2.35–2.22 (m, 1H), 2.19 (ddd, J = 13.4, 4.1, 1.2 Hz, 1H), 2.02 (ddd, J = 13.3, 8.8, 0.9 Hz, 1H), 1.79 (d, J = 2.4 Hz, 3H), 1.65 (d, J = 1.4 Hz, 3H), 1.68–1.58 (m, 1H), 1.49 (ddd, J = 13.6, 6.5, 4.7 Hz, 1H), 1.39–1.28 (m, 1H), 1.26–1.15 (m, 1H), 1.17 (d, J = 6.9 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H), 0.84 (t, J = 7.4 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 135.5, 130.6, 83.8, 76.6, 67.5, 48.0, 44.0, 34.4, 30.5, 23.3, 21.6, 20.9, 16.5, 12.3, 3.6.

(2E,4S,8E,10S)-4,8,10-trimethyl-2-(tributylstannyl)dodeca-2,8-dien-6-ol (3)

A 25 mL round-bottom flask was charged with Pd(OAc)₂ (17.3 mg, 0.077 mmol, 0.1 equiv.) and PCy₃ (53.9 mg, 0.19 mmol, 0.25 equiv.), and dissolved in degassed anhydrous hexane (9 mL). The reaction mixture was purged with argon for 5 min, and stirred for 1 h before a solution of compound 21 (171 mg, 0.77 mmol, 1.0 equiv.) in degassed anhydrous hexane (1 mL). After being stirred for 1 h, Bu₃SnH (2.1 mL, 7.7 mmol, 10.0 equiv.) was added, and the resulting mixture was stirred for 72 h. The solvent was removed in vacuo and the residue was by flash chromatography on silica gel (4% EtOAc/hexanes, neutral Al₂O₃) to give compound 3 as a colorless oil (150 mg, 38% brsm).

• R_f = 0.70 (10% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +25.8 (c 0.10, CHCl₃);
• HRMS (ESI, m/z) for C₂₇H₅₄OSnNa⁺ [M+Na]⁺: calcd. 537.3089; found: 537.3093.
• ¹H NMR (400 MHz, CDCl₃) δ 5.21 (dq, J = 9.1, 1.9 Hz, 1H), 5.03–4.96 (m, 1H), 3.67–3.56 (m, 1H), 2.99–2.85 (m, 1H), 2.34–2.19 (m, 1H), 2.13 (dd, J = 13.1, 4.4 Hz, 1H), 2.02 (ddd, J = 13.2, 8.8, 0.9 Hz, 1H), 1.87 (d, J = 1.8 Hz, 3H), 1.66–1.55 (m, 2H),1.60 (d, J = 1.4 Hz, 3H), 1.54–1.38 (m, 7H), 1.37–1.16 (m, 10H), 0.98–0.75 (m, 21H).
• ¹³C NMR (101 MHz, CDCl₃) δ 147.2, 136.6, 135.6, 130.7, 67.0, 48.9, 44.9, 34.3, 30.5, 29.3, 29.1, 27.5, 21.9, 21.3, 19.4, 16.4, 13.9, 12.1, 9.2.

6-((2S,4E,6E,8S,12E,14S)-10-hydroxy-4,6,8,12,14-pentamethylhexadeca-4,6,12-trien-2-yl)-4-(methoxymethoxy)-3,5-dimethyl-2H-pyran-2-one (22)

A 25 mL round-bottom flask was charged with compound 2 (66 mg, 0.168 mmol, 1.0 equiv.) and compound 3 (108 mg, 0.210 mmol, 1.25 equiv.) and dissolved in degassed anhydrous DMF (3.4 mL, 0.05 M). The reaction mixture was purged with argon for 5 min, before Pd(PPh₃)₄ (19.4 mg, 0.0168 mmol, 0.1 equiv.), CuTC (64 mg, 0.330 mmol, 1.97 equiv.) and CsF (52 mg, 0.342 mmol, 2.03 equiv.) were added sequentially. The resulting mixture was stirred for 48 h before it was quenched by adding aqueous saturated NH₄Cl (2 mL). The organic layer was separated and the aqueous phase was extracted with EtOAc (3 × 6 mL). The combined organic phases were washed with brine (1 mL), and dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (33.3% EtOAc/hexanes) to give compound 22 as a colorless oil (64 mg, 79%).

• R_f = 0.40 (33% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +85.4 (c 0.50, CHCl₃);
• HRMS (ESI, m/z) for C₃₀H₄₈O₅Na⁺ [M+Na]⁺: calcd. 511.3394; found: 511.3379.
• ¹H NMR (400 MHz, CDCl₃) δ 5.56 (s, 1H), 5.02 (s, 2H), 4.98 (dd, J = 9.4, 1.7 Hz, 1H), 4.90–4.82 (m, 1H), 3.65–3.55 (m, 1H), 3.56 (s, 3H), 3.13–3.00 (m, 1H), 2.79–2.63 (m, 1H), 2.37 (ddd, J = 13.3, 8.2, 1.0 Hz, 1H), 2.32–2.15 (m, 2H), 2.12 (dd, J = 13.1, 4.1 Hz, 1H), 2.03–1.97 (m,1H), 2.01 (s, 3H), 1.94 (s, 3H), 1.70 (d, J = 1.4 Hz, 3H), 1.65 (d, J = 1.4 Hz, 3H), 1.59 (d, J = 1.4 Hz, 3H), 1.49–1.21 (m, 4H), 1.20 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.82 (t, J = 7.4 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 166.5, 166.0, 161.7, 135.7, 135.3, 132.2, 131.9, 131.7, 130.6, 109.8, 108.7, 99.1, 66.8, 57.9, 48.9, 45.2, 45.1, 34.3, 33.8, 30.5, 29.6, 21.9, 21.3, 17.9, 17.2, 16.5, 12.1, 11.0, 10.5.

4-(methoxymethoxy)-3,5-dimethyl-6-((2S,4E,6E,8S,12E,14S)-4,6,8,12,14-pentamethylhexadeca-4,6,12 -trien-2-yl)-2H-pyran-2-one (23)

To a cooled (−78 °C) stirred solution of compound 22 (40 mg, 0.0819 mmol, 1.0 equiv.) in anhydrous CS₂ (8.0 mL, 0.01 M), NaHMDS (61 μL, 0.123 mmol, 2.0 M in THF, 1.5 equiv.) was added. After stirring for 1 h, MeI (77 μL, 1.23 mmol, 15.0 equiv.) was added dropwise. The reaction mixture was allowed to warm to 0 °C and stirred for 3 h, at which point the starting material had been consumed, as verified by TLC analysis; the reaction was quenched by adding aqueous saturated NaHCO₃ (2 mL) slowly at 0 °C. The organic layer was separated and the aqueous phase was extracted with EA (3 × 5 mL). The combined organic phases were washed with brine (1 mL), and then dried over anhydrous Na₂SO₄. The solvent was removed in vacuo at 0 °C and the crude product was purified by flash chromatography on silica gel (25% EtOAc/hexanes) to give the intermediate compound S2 as a colorless oil.

• R_f = 0.50 (25% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +61.2 (c 0.50, CHCl₃);
• HRMS (ESI, m/z) for C₃₂H₅₀O₅S₂Na⁺ [M+Na]⁺: calcd. 601.2992; found: 601.2990.
• ¹H NMR (400 MHz, CDCl₃) δ 5.74–5.63 (m, 1H), 5.52 (s, 1H), 5.02 (s, 2H), 4.92 (d, J = 9.3 Hz, 1H), 4.82–4.74 (m, 1H), 3.56 (s, 3H), 3.12–2.98 (m, 1H), 2.61–2.43 (m, 2H), 2.51 (s, 3H), 2.35 (dd, J = 13.3, 8.0 Hz, 1H), 2.28–2.11 (m, 3H), 2.01 (s, 3H), 1.93 (s, 3H), 1.81–1.70 (m, 1H), 1.69 (d, J = 1.4 Hz, 3H), 1.61 (d, J = 1.4 Hz, 3H), 1.59–1.47 (m, 1H), 1.54 (d, J = 1.4 Hz, 3H), 1.40–1.09 (m, 2H), 1.18 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H), 0.81 (t, J = 7.4 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 215.2, 166.5, 166.0, 161.7, 135.6, 134.2, 132.4, 132.4, 131.5, 129.2, 109.8, 108.7, 99.1, 82.1, 57.9, 45.2, 44.5, 41.1, 34.3, 33.8, 30.5, 29.5, 21.6, 20.7, 18.9, 18.0, 17.9, 17.2, 16.7, 12.2, 11.0, 10.5.

To a stirred solution of the above compound S2 in anhydrous toluene (3 mL, 0.027 M) was added Bu₃SnH (0.15 mL, 0.573 mmol, 7.0 equiv.) and Et₃B (98 μL, 0.0983 mmol, 1 M in THF. 1.2 equiv.) sequentially. The reaction mixture was stirred for 12 h, at which point the starting material had been consumed, as verified by TLC analysis; and the reaction was quenched by adding H₂O (0.5 mL) and KF (71 mg, 1.23 mmol, 15.0 equiv.). The organic layer was separated and the aqueous phase was extracted with EA (3 × 3 mL). The combined organic phases were washed with brine (0.5 mL), and then dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (20% EtOAc/hexanes) to give compound 23 as a colorless oil (19 mg, 49% for two steps).

• R_f = 0.30 (12.5% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +78.0 (c 0.10, CHCl₃);
• HRMS (ESI, m/z) for C₃₀H₄₈O₄Na⁺ [M+Na]⁺: calcd. 495.3445; found: 495.3456.
• ¹H NMR (400 MHz, CDCl₃) δ 5.56 (s, 1H), 5.03 (s, 2H), 4.93–4.80 (m, 2H), 3.56 (s, 3H), 3.15–2.97 (m, 1H), 2.42–2.29 (m, 2H), 2.28–2.14 (m, 2H), 2.01 (s, 3H), 1.97–1.87 (m, 2H), 1.94 (s, 3H), 1.70 (d, J = 1.4 Hz, 3H), 1.61 (d, J = 1.4 Hz, 3H), 1.56 (d, J = 1.4 Hz, 3H), 1.42–1.08 (m, 6H), 1.20 (d, J = 6.8 Hz, 3H), 0.97–0.85 (m, 6H), 0.82 (t, J = 7.4 Hz, 3H).
• ¹³C NMR (101 MHz, CDCl₃) δ 166.5, 166.0, 161.8, 136.3, 133.8, 132.0, 131.8, 131.4, 130.9, 109.8, 108.7, 99.1, 57.9, 45.3, 40.0, 37.2, 34.1, 33.8, 32.5, 30.7, 26.0, 21.2, 21.2, 17.9, 17.3, 16.2, 12.1, 11.0, 10.5.

4-hydroxy-3,5-dimethyl-6-((2S,4E,6E,8S,12E,14S)-4,6,8,12,14-pentamethylhexadeca-4,6,12-trien-2-yl)-2H-pyran-2-one (1)

Compound 23 (10 mg, 0.0212 mmol, 1.0 equiv.) was dissolved in HCl/MeOH (21 mL, 0.015 M HCl in MeOH, 0.001 M) and stirred at 0 °C. The reaction was stirred and monitored by TLC. After 12 h, 23 was consumed, as verified by TLC analysis. The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel (50% EtOAc/hexanes) to give compound 1 as a colorless oil (6 mg, 67%).

• R_f = 0.40 (50% EtOAc/hexanes), UV and PMA stain.
• ${\left[\mathsf{\alpha }\right]}_{\mathbf{D}}^{\mathbf{20}}$ = +32.0 (c 0.10, CHCl₃);
• HRMS (ESI, m/z) for C₂₈H₄₄O₃H⁺ [M+H]⁺: calcd. 429.3363; found: 429.3364.
• ¹H NMR (400 MHz, Acetone-d6) δ 5.58 (s, 1H), 4.94–4.83 (m, 2H), 3.28–3.15 (m, 1H), 2.46–2.37 (m, 1H), 2.33 (ddd, J = 13.2, 8.6, 1.0 Hz, 1H), 2.29–2.23 (m, 1H), 2.19 (dd, J = 12.9, 6.5 Hz, 1H), 1.96 (s, 3H), 1.92–1.99 (m, 2H), 1.91 (s, 3H), 1.74 (d, J = 1.4 Hz, 3H), 1.63 (d, J = 1.4 Hz, 3H), 1.57 (d, J = 1.4 Hz, 3H), 1.44–1.10 (m, 6H), 1.17 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H), 0.82 (t, J = 7.4 Hz, 3H).
• ¹³C NMR (101 MHz, Acetone-d6) δ 165.3, 165.1, 161.5, 136.6, 134.5, 133.3, 132.2, 132.1, 131.9, 106.9, 98.4, 45.9, 40.5, 37.9, 34.8, 34.0, 33.1, 31.3, 26.6, 21.6, 21.4, 18.3, 18.0, 17.4, 16.3, 12.3, 10.0, 9.2.
• Natural alternapyrone¹: a colorless oil; ¹H NMR and ¹³C NMR data (see Table S1); APCI-TOFMS m/z: calculated for C₂₈H₄₄O₃H⁺ [M + H]⁺ 429.3363, found 429.3374

4. Conclusions

Alternapyrone consists of an α-pyrone moiety and a 6-alkenyl chain, with its absolute stereochemical configuration previously unknown. The stereochemical configuration was predicted using a biochemistry-guided prediction rule and verified through total synthesis. Key reactions include a retro Diels–Alder reaction to construct the pyrone skeleton, the Kagan–Molander coupling reaction to connect the alkenyl carbon chain, the Stille reaction to form the unstable conjugated diene, and the Barton–McCombie reaction for hydroxyl group removal. The assignment of the absolute configuration of alternapyrone provided strong evidence supporting the applicability of the biochemistry-guided prediction rule in predicting the stereostructures of fungal-reduced polyketide products.