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. Author manuscript; available in PMC: 2013 Oct 17.
Published in final edited form as: Chem Phys Lipids. 2012 Oct 17;165(7):794–801. doi: 10.1016/j.chemphyslip.2012.10.002

Practical multigram-scale synthesis of 4,6- and 4,8-sphingadienes, chemopreventive sphingoid bases

Hoe-Sup Byun 1, Robert Bittman 1,*
PMCID: PMC3734955  NIHMSID: NIHMS421486  PMID: 23085149

Abstract

Sphingadienes are chemopreventive agents that act by blocking signaling pathways that are activated in cancer. A practical synthesis of 4,6- and 4,8-sphingadienes on a scale of gram quantities is reported here in order to allow evaluation of the biological properties of these sphingolipids. The key steps in the preparation of 4,6-sphingadiene (1a) are an intramolecular cyclization of N-Boc derivative 5a to oxazolidinone derivative 6a, followed by conversion to carbamate intermediate 7a and base-mediated hydrolysis to afford the product without further purification. 4,8-Sphingadiene (1b) was prepared in a similar fashion; the requisite trans-γ,δ-unsaturated aldehyde 15 was prepared by an ester enolate Ireland-Claisen rearrangement.

Keywords: Lipid synthesis, Sphingadienine, Sphingolipid, Sphingoid base

1. Introduction

Sphingosine (trans-4-sphingenine) is the long-chain or sphingoid base that constitutes the backbone of sphingolipids. Sphingoid bases are structurally diverse, varying in the number of double bonds, alkyl chain length, and extent of hydroxylation or methyl branching. The predominant long-chain base in mammals is (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol (for reviews, see Pruett et al., 2008; Bittman, 2009). Sphingadienes, which are also called sphingadienines, contain two double bonds in the sphingoid base, and are commonly found in plants. The β-glucosylceramides (cerebrosides) of soybean, rice bran, and wheat grain often contain 4,8-sphingadiene (denoted as d18:24,8 in lipidomics nomenclature) as the major sphingoid base (Lynch et al., 1992; Sullards et al., 2000). The geometry of the double bond is E at C4–C5 and an E,Z mixture at C8,C9. A branched methyl group may be present; for example, the ceramide monohexosides of fungal cells contain 9-methyl-4,8-sphingadiene as the long-chain base (Levery, 2005; Barreto-Bergter et al., 2011). In contrast to plant cerebrosides, the sphingadienes of insects such as Drosophila Sply mutants (Fyrst et al., 2008) and moths such as Manduca sexta (Abeytunga et al., 2004) have a shorter aliphatic chain and conjugated double bonds at C4,C5 and C6,C7; these 4,6-sphingadienes typically have 14 and 16 carbons.

Sphingadienes have attracted a great deal of interest because they have potential uses in colon cancer treatment (for a recent review, see Fyrst and Saba, 2010). Soy glucosylceramides, in which the predominant sphingoid base is 4,8-sphingadiene, suppressed colon tumorigenesis in mice (Symolon et al., 2004). A monoglucosylceramide (β-glucosyl-N-2′-hydroxyarachidoyl-4,8-sphingadiene) from rice bran inhibited the induction of 1,2-dimethylhydrazine-induced aberrant crypt foci and β-catenin-accumulated crypt formation in F344 rats (Inamine et al., 2005). Moreover, aquatic plants containing sphingadienes have been used in oriental medicine (Tan et al., 2003; Sugawara et al., 2006). To assess whether the ability of soy sphingolipids to act as a chemopreventive agent is derived from the presence of the sphingadiene backbone, human colon cancer cells were treated with C14 (2S,3R,4E,6E)-4,6-sphingadiene and C18 (2S,3R,4E,8E)-4,8-sphingadiene (denoted as C14(4,6)- and C18(4,8)-sphingadiene, respectively). These sphingadienes prevented Akt translocation from the cytosol to the plasma membrane, and arrested the growth of human cancer cells in vitro more efficiently than soy glucosylceramides, suggesting that cerebroside’s tumor-preventing activity in vivo results from metabolism to sphingadienes (Fyrst et al., 2009). This study also showed that C14(4,6)-sphingadiene inhibited intestinal tumorigenesis in ApcMin/+ mice. Sphingadienes may have a longer half-life in intestinal epithelial cells than sphingosine because of a slower conversion to the phosphorylated sphingoid base (Fyrst et al., 2009). 4,8-Sphingadienes induced apoptosis in Caco-2 human colon cancer cells (Aida et al., 2004) and were cytotoxic to HL-60, RAW 264.7, HUVEC, and HEK-293 cell lines in vitro (Rozema et al., 2012a). In addition to blocking the phosphoinositide 3-kinase (PI3K)/Akt pathway (Fyrst et al., 2009), sphingadienes inhibited colon carcinogenesis by downregulating Wnt signaling through a protein phosphatase 2A/Akt/GSK3β-dependent mechanism (Kumar et al., 2012). 4,8-Sphingadiene also exerts an anti-inflammatory response by inhibiting the expression of pro-inflammatory agents (interleukin-8 and E-selectin) induced by tumor necrosis factor-α and lipopolysaccharide in human endothelial cells (Rozema et al., 2012b).

The studies described above implicate sphingadienes as potential therapeutic lipids. Thus sphingadiene is an important synthetic target. Indeed, many elegant syntheses of sphingadienes have been reported prior to the discovery that sphingadienes protect the intestine from inflammation and cancer (Murakami et al., 2000, 2005; Wang et al., 2000; Black and Kocienski, 2010; Moreno et al., 2011). Unfortunately, none of the reported syntheses can be readily scaled up. Furthermore, isolation of pure sphingadienes from natural sources is not practical on a large scale because they are obtained as a mixture with other sphingolipids, and thus the individual roles of each component in the mixture cannot be determined.

2. Experimental

2.1. Materials and analytical procedures

2.1.1. Chemicals

The sources of the chemicals were as follows: chlorotrimethyl-silane and potassium hydride (30 wt.% dispersion in mineral oil) from Acros (Morris Plains, NJ); pyridinium chlorochromate (PCC), p-toluenesulfonic acid monohydrate (p-TsOH), and decanal from Alfa Aesar (Pelham, NH); N-Boc-L-serine methyl ester, n-butyllithium (2.5 M solution in hexane), cerium(III) chloride, hexamethyldisilazane (TMS2NH), 2,2-dimethoxypropane, dimethyl methylphosphonate, sodium borohydride, and vinylmagnesium bromide (1.0 M solution in THF) from Aldrich (Milwaukee, WI). 3-tert-Butyloxycarbonyl (S)-4-carbomethoxy-2,2-dimethyl-3,4-oxazolidine ester ((S)-Garner ester, 2) was prepared from N-Boc-L-serine methyl ester as described previously (Garner and Park, 1987).

2.1.2. General methods

Air- and moisture-sensitive reactions were carried out under nitrogen in dried glassware. THF was distilled from sodium and benzophenone, and hexane was dried over sodium metal. TLC analyses was performed on precoated aluminum-backed or glass-backed silica gel 60 F254 plates (0.25-mm thick), and the compounds were visualized by charring with 10% H2SO4 in EtOH or by UV light (254 nm). Column chromatography was carried out with silica gel 60 (230–400 mesh) using the elution solvents indicated in the text. Suspended silica gel was removed by filtration through an Osmonics Cameo filter (Fisher Scientific, Pittsburgh, PA). The 1H and 13C NMR spectra were recorded on a Bruker Avance I spectrometer at 400 and 100 MHz, respectively, and were referenced to the residual CHCl3 at δ 7.24 (1H) and the central line of CDCl3 at δ 77.0 ppm (13C). The high-resolution mass spectrometric data were recorded at the CUNY mass spectrometry facility on an Agilent Technologies G6520A Q-TOF mass spectrometer using electrospray ionization. Optical rotations were measured on an Autopol III digital polarimeter at room temperature in the solvents stated.

2.2. Synthesis

2.2.1. N-tert-Butoxycarbonyl (4S)-4-[1′-oxo-(2′E,4′E)-dodecadienyl]-2,2-dimethyl-1,3-oxazolidine (4a)

A solution of dimethyl methylphosphonate (31.1 g, 250 mmol) in 50 mL of hexane and 120 mL of THF was cooled in an Et2O/dry ice bath (about −100 °C) for 1 h. To this solution was added 100 mL (250 mmol) of n-BuLi (2.5 M in hexane). After the reaction mixture was stirred for an additional 6 h, a solution of 2 (26.0 g, 100 mmol) in 50 mL of THF was added, and the reaction mixture was kept at −78 °C (acetone/dry ice bath) overnight. The reaction mixture was allowed to warm to 0 °C with stirring for 1 h. After the reaction was quenched with acetic acid (15 mL, 260 mmol), removal of the solvents under reduced pressure gave a residue that contained β-ketophosphonate 3 and an excess amount of dimethyl methylphosphonate. To this residue was added a solution of K2CO3 (125 g, 904 mmol) in 150 mL of H2O, followed by a solution of commercially available (E)-2-decenal (10.0 g, 64.8 mmol) in 150 mL of 2-propanol at 0 °C. After the mixture had been stirred at 0 °C for 4 h, the reaction mixture was gradually warmed to room temperature and stirred overnight. The reaction mixture was diluted with 100 mL of saturated aqueous NaCl solution, and the product was extracted with Et2O (3× 150 mL). The combined organic layers were washed with brine, dried (Na2SO4), and concentrated. Purification of the residue by column chromatography (elution with hexane/EtOAc 20:1, 15:1, and 10:1) gave 17.1 g (45%) of 4a (yield calculated from 2): [α]25D −37.2° (c 2.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.88 (t, 3H, J = 7.0 Hz), 1.09–1.31 (m, 10 H), 1.48 (s, 9H), 1.54 (s, 3H), 1.83 (s, 3H), 1.95 (q, 2H, J = 6.5 Hz), 3.82 (m, 2H), 4.43 (br s, 1H), 5.85 (m, 1H), 5.98 (m, 1H), 6.23 (d, 1H, J = 15.5 Hz), 7.30 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 14.1, 20.1, 22.6, 23.6, 28.4, 29.0, 29.1, 29.4, 29.5, 29.7, 32.2, 33.2, 33.3, (64.7), 65.0, (66.0), 66.3, (79.9), 80.0, (94.3), 95.0, 122.2, (128.8), 128.8, (143.1), 144.8, (147.1), 147.5 151.4, (152.2), (196.2), 197.1; HRMS (M+Na)+ calcd for m/z C22H37NNaO4+ 402.2620, found 402.2613. At ambient temperature, the 13C NMR spectrum was complicated by the presence of several doublet signals, indicating that the oxazolidine system undergoes a dynamic equilibrium. The parentheses indicate the other peak sets generated by the dynamic equilibrium of the oxazolidine system.

2.2.2. N-tert-Butoxycarbonyl (4S)-4-[1′-oxo-(2′E,6′E)-hexadecadienyl]-2,2-dimethyl-1,3-oxazolidine (4b)

The compound was prepared by the procedure described for the synthesis of 4a. The in situ preparation of β-ketophosphonate 3 (see Section 2.2.1) starting from 13.1 g (50.5 mmol) of 2 followed by reaction with 9.50 g (45.1 mmol) of aldehyde 15 gave 10.3 g (47%) of dienone 4b: [α]25D −36.1° (c 2.0, CHCl3); 1H NMR δ 0.88 (t, J = 7.0 Hz, 3H), 1.09–1.31 (m, 14H), 1.48 (s, 9H), 1.54 (s, 3H), 1.83 (s, 3H), 1.96 (m, 2H), 2.12–2.33 (m, 4H), 3.82 (m, 2H), 4.43 (br s, 1H), 5.35–5.51 (m, 2H), 6.23 (d, J = 15.5 Hz, 1H), 7.30 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 14.1, 20.1, 22.7, 23.6, 28.4, 29.0, 29.1, 29.4, 29.5, 29.7, 32.2, 33.2, 33.3, (64.7), 65.0, (66.0), 66.3, (79.9), 80.0, (94.3), 95.0, 122.2, (128.8), 128.8, (143.1), 144.8, (147.1), 147.5 151.4, (152.2), (196.2), 197.1; HRMS (M+Na)+ calcd for m/z C26H45NNaO4+ 458.3241, found 458.3234.

2.2.3. (4S,5R)-5-[(1′E,3′E)-Undecadienyl]-4-(hydroxymethyl)-2-oxazolidinone (7a)

To a mixture of 4a (8.48 g, 22.3 mmol) and CeCl3 (1.84 g, 7.47 mmol) in 250 mL of MeOH was added NaBH4 (0.95 g, 25.1 mmol) at −78 °C. The reaction mixture was gradually warmed to room temperature. Stirring was continued for 5 h, and the mixture was filtered through a pad of Celite, which was washed with 250 mL of Et2O. The filtrate was washed with brine, dried (Na2SO4), and concentrated. The residue was dried further under high vacuum overnight to give crude 5a. A solution of 5a in 50 mL of THF was added to a suspension of KH (181 mg, 4.49 mmol) in 50 mL of THF at 0 °C. After the reaction mixture was stirred overnight, the solvents were removed under reduced pressure in order to remove tert-butyl alcohol, which was formed by reaction of the C3-OH group with the N-Boc group. The residue was dissolved in dry THF and the mixture was stirred overnight. Because the Rf value of N-Boc derivative 5a is the same as that of oxazolidinone derivative 6a, the intramolecular base-catalyzed reaction (carbamate formation and loss of tert-butyl alcohol) was repeated three times to complete the cyclization reaction. In order to remove the isopropylidene group from 6a, p-TsOH (853 mg, 4.49 mmol) was added to a solution of crude 6a in 100 mL of THF/H2O (9:1). After the reaction mixture was stirred overnight, the solvents were removed under reduced pressure, and the resulting residue was diluted with Et2O (250 mL) and washed with brine and water. The organic layer was dried (Na2SO4) and concentrated. The product was purified by column chromatography on silica gel (elution with hexane/EtOAc 1:1, and then 1:2) to give 4.26 g (71%) of 7a : [α]25D −38.1° (c 2.2, CHCl3); 1H NMR (400 MHz, CD2Cl2) δ 0.88 (t, J = 6.5 Hz, 3H), 1.26 (m, 8H), 1.30–1.35 (m, 2H), 2.07 (q, J = 6.9 Hz, 2H), 3.62–3.77 (m, 2H), 3.91 (dd, J = 3.6, 11.3 Hz, 1H), 4.36 (t, J = 4.7 Hz, 1H), 5.14 (t, J = 8.2 Hz, 1H), 5.57–5.63 (m, 1H), 5.68–5.75 (m, 1H), 6.00–6.06 (m, 1H), 6.28 (dd, J = 10.3, 15.3 Hz, 1H); 13C NMR (100 MHz, CD2Cl2) δ 14.1, 22.6, 28.3, 29.1, 31.8, 32.6, 55.4, 62.5, 74.4, 129.1, 132.5, 136.3, 156.2; HRMS (M+Na)+ calcd for m/z C19H35NNaO4+ 364.2458, found 364.2461. In order to confirm the structure of 7a, (4S,5R)-N-acetyl-5-[(1′E,3′E)-undecadienyl]-4-(acetyloxymethyl)-2-oxazolidinone (7a′) was prepared from 7a by using an excess of Ac2O in pyridine: [α]25D −2.1° (c 1.8, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.5 Hz, 3H), 1.26 (m, 8H), 2.05 (s, 3H), 2.03–2.11 (m, 2H), 2.52 (s, 3H), 4.25 (dd, J = 2.1, 11.3 Hz, 1H), 4.36 (q, J = 4.1 Hz, 1H), 4.68 (dd, J = 4.1, 11.3 Hz, 1H), 4.82 (dd, J = 3.1, 7.2 Hz, 1H), 5.51 (dd, J = 7.2, 15.1 Hz, 1H), 5.85 (dt, J = 6.7, 15.1 Hz, 1H), 6.02 (dd, J = 10.3, 15.1 Hz, 1H), 6.32 (dd, J = 10.3, 15.1 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 14.1, 20.6, 22.6, 23.7, 28.9, 29.1, 31.7, 32.6, 58.3, 61.7, 76.8, 123.9, 126.2, 127.9, 135.5, 139.7, 153.0, 170.5; HRMS (M+Na)+ calcd for m/z C19H29NNaO5+ 374.1938, found 374.1940.

2.2.4. (4S,5R)-5-[(1′E,5′E)-Undecadienyl]-4-(hydroxymethyl)-2-oxazolidinone (7b)

This compound was prepared by the procedure described above by starting with 11.3 g (25.9 mmol) of 3b to give 6.30 g (75%) of 7b: [α]25D −37.0° (c 2.3, CHCl3); 1H NMR (400 MHz, CD2Cl2) δ 0.88 (t, J = 6.6 Hz, 3H), 1.26 (m, 14H), 1.96 (q, J = 6.7 Hz, 2H), 2.07–2.12 (m, 2H), 2.12–2.20 (m, 2H), 3.64 (m, 3H), 3.80–3.96 (m, 1H), 5.07 (t, J = 8.3 Hz, 1H), 5.36 (dt, J = 5.7, 15.2 Hz), 5.43 (dt, J = 6.3, 15.2 Hz), 5.63 (dd, J = 8.4, 15.4 Hz), 5.89 (dt, J = 6.2, 15.4 Hz), 6.74 (br s, 1H); 13C NMR (100 MHz, CD2Cl2) δ 14.2, 22.7, 29.2, 29.3, 29.50, 29.53, 29.58, 31.7, 31.9, 32.2, 32.6, 57.4, 61.6, 80.3, 122.4, 128.6, 131.5, 138.5, 160.5; HRMS (M+H)+ calcd for m/z C19H34NO3+ 324.2533, found 324.2530.

2.2.5. (2S,3R)-2-Amino-(4E,6E)-tetradecadiene-1,3-diol (1a)

A mixture of 7a (4.66 g, 17.4 mmol) in 100 mL of 2 M NaOH solution and 100 mL of EtOH was heated at 80 °C for 3 h. The reaction mixture was cooled to room temperature, concentrated under reduced pressure, and the product was extracted with Et2O (3× 20 mL). The organic layer was washed with brine, dried (K2CO3), and concentrated to give 4.20 g (100%) of 1a; [α]25D −2.5° (c 1.2, CHCl3); 1H NMR (400 MHz, 2 mg in 0.5 mL of CD2Cl2) δ 0.91 (t, J = 6.7 Hz, 3H), 1.20–1.43 (m, 8H), 2.79 (br s, 1H), 3.04 (br s, 3H), 3.48–3.66 (m, 2H), 4.09 (q, J = 6.1 Hz, 1H), 5.57 (dt, J = 6.2, 15.2 Hz, 1H), 5.71–5.80 (m, 1H), 6.08 (dd, J = 10.5, 15.2 Hz, 1H), 6.27 (dd, J = 10.5, 15.2 Hz, 1H); 1H NMR (400 MHz, 20 mg in 0.5 mL of pyridine-d5) δ 0.85 (t, J = 6.7 Hz, 3H), 1.26 (m, 8H), 1.30–1.35 (m, 2H), 2.05 (q, J = 7.0 Hz, 2H), 3.24 (q, J = 5.3 Hz, 0.55H), 3.38 (q, J = 6.0 Hz, 0.45 H), 3.99 (dd, J = 10.4, 6.4 Hz, 0.55H), 4.09–4.17 (m, 1H), 4.23 (dd, J = 10.4, 4.7 Hz, 0.45H), 4.64 (q, J = 5.5 Hz, 1H), 5.67–5.76 (m, 1H), 6.06–6.19 (m, 1H), 6.21–6.27 (m, 1H), 6.63 (dd, J = 10.5, 15.3 Hz, 1H); 13C NMR (CD2Cl2/CD3OD) δ 14.2, 22.9, 29.4, 29.6, 32.0, (58.83), 58.89, 64.69, (64.74), 73.2, 74.7, 130.7, 131.3, 131.7; HRMS (M+H)+ calcd for m/z C14H27NNaO2+ 264.1934, found 264.1932. In order to confirm the structure of 1a, (2S,3R)-2-acetylamido-(4E,6E)-tetradecadiene-1,3-diol (1c) was prepared by the reaction of 1a with 4-nitrophenyl acetate in THF/H2O (9:1): 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.6 Hz, 3H), 1.26 (m, 8H), 1.30–1.44 (m, 2H), 1.82 (br s, 2H), 2.04–2.09 (m, 2H), 2.05 (s, 3H), 3.59 (dd, J = 6.5, 11.6 Hz, 1H), 3.65–3.69 (m, 1H), 3.74 (dd, J = 2.9, 11.6 Hz, 1H), 4.81 (t, J = 6.5 Hz, 1H), 5.59 (dd, J = 7.9, 15.1 Hz, 1H), 5.82 (dt, J = 6.9, 15.1 Hz, 1H), 6.04 (dd, J = 10.5, 15.1 Hz, 1H), 6.31 (dd, J = 10.5, 15.1 Hz, 1H), 6.38 (br s, 1H); 13C NMR (100 MHz, CDCl3) 14.1, 22.6, 29.0, 19.12, 29.13, 31.8, 32.7, 60.1, 62.7, 79.5, 125.2, 128.3, 135.5, 138.9, 159.8; HRMS (M+H)+ calcd for m/z C16H31NO3+ 284.2220, found 284.2225.

2.2.6. (2S,3R)-2-Amino-(4E,8E)-octadecadiene-1,3-diol (1b)

This compound was prepared by the procedure described above for 1a by starting with 6.22 g (19.2 mmol) of 7b to give 5.51 g (96%) of 1b: [α]25D −2.7° (c 1.3, CHCl3); 1H NMR (400 MHz, pyridine-d5) δ 0.85 (t, J = 6.7 Hz, 3H), 1.26 (s, 8H), 1.30–1.35 (m, 2H), 2.05 (q, J = 7.0 Hz, 2H), 3.24 (q, J = 5.3 Hz, 0.55H), 3.38 (q, J = 6.0 Hz, 0.45 H), 3.99 (dd, J = 10.4, 6.4 Hz, 0.55H), 4.09–4.17 (m, 1H), 4.23 (dd, J = 10.4, 4.7 Hz, 0.45H), 4.64 (q, J = 5.5 Hz, 1H), 5.67–5.76 (m, 1H), 6.06–6.19 (m, 1H), 6.21–6.27 (m, 1H), 6.63 (dd, J = 10.5, 15.3 Hz); 13C NMR (100 MHz, CDCl3/CD3OD) δ 14.2, 22.9, 29.4, 29.6, 32.0, (58.83), 58.89, 64.69, (64.74), 73.2, 74.7, 130.7, 131.3, 131.7; HRMS (M+H)+ calcd for m/z C18H36NO2+ 298.2741, found 298.2743.

2.2.7. rac-3-Hydroxy-1-dodecene (8)

To a mixture of decanal (15.7 g, 0.100 mol) in 100 mL of THF was added slowly vinylmagnesium bromide (110 mL, 0.11 mol, 1 M solution in THF) at −78 °C. The reaction mixture was stirred −78 °C for 3 h, and then was allowed to stir at 0 °C for 1 h. After solid NH4Cl (6.0 g, 112 mmol) and 5 mL of water were added slowly, the mixture was filtered. The filtrate was diluted with 250 mL of EtOAc and washed with brine and water. The organic layer was dried (MgSO4) and concentrated. The product was purified by flash chromatography with 5% EtOAc/hexane as eluent to give 17.6 g (95%) of 8 as an oil: 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.6 Hz, 3H), 1.26 (m, 14H), 1.50–1.54 (m, 2H), 4.08 (q, J = 6.2 Hz, 1H), 5.09 (dt, J = 1.1, 10.4 Hz, 1H), 5.21 (dt, J = 1.4, 17.2 Hz, 1H), 5.84 (ddd, J = 6.2, 10.4, 17.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 14.1, 22.7, 25.3, 29.3, 29.53, 29.56, 31.9, 37.0, 73.2, 114.5, 141.3. The 1H and 13C NMR spectra are in agreement with the published spectra of a mixture of 8 and 8a (Cuzzupe et al., 2002; Itami et al., 2003).

2.2.8. rac-3-O-Acetyl-1-dodecene (10)

A solution of 8 (17.4 g, 94.4 mmol), acetic anhydride (10 mL, 105 mmol), and DMAP (600 mg, 4.9 mmol) in 25 mL of CH2Cl2 was stirred overnight at room temperature. After the mixture was concentrated under reduced pressure, the residue was dissolved in 250 mL of EtOAc and washed with water, aqueous saturated NaHCO3 solution, and brine. The organic layer was dried (Na2SO4) and concentrated. The residue was dried under high vacuum overnight to give 20.4 g (95%) of 10, which was used without further purification: 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.6 Hz, 3H), 1.26 (m, 14H), 1.57–1.62 (m, 2H), 2.06 (s, 3H), 5.15 (d, J = 10.4 Hz, 1H), 5.20 (q, J = 6.2 Hz, 1H), 5.23 (d, J = 7.4 Hz, 1H), 5.77 (ddd, J = 6.2, 7.4, 10.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 14.1, 21.2, 22.7, 25.0, 29.27, 29.34, 29.5, 31.9, 34.2, 74.9, 116.5, 136.6, 170.4. The NMR spectra agree with the published spectra (Keinan and Greenspoon, 1983).

2.2.9. Methyl 4(E)-tetradecenoate (13)

To a solution of (TMS)2NH (16.2 g, 100 mmol) in 40 mL of dry THF was added 40 mL (100 mmol) of n-BuLi (a 2.5 M solution in hexane) at −78 °C. After the mixture had been stirred for 1 h, a solution of 10 (20.4 g, 90.1 mmol) in 100 mL of THF was added slowly. The resulting mixture was stirred at −78 °C for 3 h, and then 13 mL (102 mmol) of TMSCl was added at −78 °C with stirring for 1 h in order to form enol silyl ether 11. The reaction mixture was allowed to warm to room temperature, and stirring was continued overnight. After concentration under reduced pressure, the residue was diluted with 250 mL of Et2O and washed with aqueous 1 M HCl solution and brine. The organic layer was dried (Na2SO4) and concentrated to give carboxylic acid 12. A mixture of crude 12, trimethyl orthoformate (13 mL, 0.119 mol), and p-toluenesulfonic acid (1.0 g, 5.0 mmol) in 50 mL of MeOH was stirred overnight. The reaction mixture was concentrated, and the residue was purified by flash chromatography with 5% EtOAc/hexane as eluent to give 17.6 g (81%) of 13: 1H NMR (400 MHz, CDCl3) δ 0.85 (t, J = 6.6 Hz, 3H), 1.26 (m, 14H), 1.91 (q, J = 4.4 Hz, 2H), 2.28 (t, J = 6.1 Hz, 2H), 2.32 (q, J = 7.1 Hz, 2H), 3.63 (s, 3H), 5.30–5.41 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 14.1, 22.7, 27.9, 29.1, 29.3, 29.42, 29.49, 29.53, 29.6, 31.9, 32.5, 34.2, 51.4, 127.8, 131.9, 173.7; 13C NMR (CDCl3) δ 14.0, 14.2 (OCH2CH3), 22.8, 29.1, 29.3, 29.4, 29.5, 31.9, 32.4, 127.9, 131.8, 173.1. The NMR spectra agree with the published spectra (Baer and Schmidt, 1988).

2.2.10. 4(E)-Tetradecen-1-ol (14)

To a suspension of 3.80 g (100 mmol) of LiAlH4 in 250 mL of THF was added a solution of 17.3 g (72.0 mmol) of 13 in 20 mL of THF at −78 °C. After the reaction mixture was stirred overnight at room temperature, the reaction was quenched by addition of 4 mL of 1 M NaOH solution. The mixture was diluted with 150 mL of CH2Cl2 and passed through a pad of Celite, which was washed with 250 mL of EtOAc. The filtrate was concentrated under reduced pressure. The residue was purified by chromatography on silica gel (elution with hexane/EtOAc 6:1) to give 13.6 g (89%) of 14: 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 6.6 Hz, 3H), 1.26 (m, 14H), 1.45 (br s, 1H), 1.63 (q, J = 6.7 Hz, 2H), 2.07 (q, J = 6.7 Hz, 2H), 3.65 (t, J = 6.5 Hz, 2H), 5.40–5.46 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 14.1, 28.9, 29.2, 29.3, 29.4, 29.50, 29.55, 29.57, 31.9, 32.4, 32.5, 62.6, 129.3, 131.3; 13C NMR (CDCl3) δ 14.0, 22.7, 28.9, 29.2, 29.3, 29.5, 29.6, 31.9, 32.5, 32.8, 62.6, 129.3, 131.3. The NMR spectra agree with the published spectra (Baer and Schmidt, 1988).

2.2.11. 4(E)-Tetradecenal (15)

To a mixture of pyridinium chlorochromate (26.0 g, 120 mmol) and silica gel (26 g) in 250 mL of CH2Cl2 was added a solution of 14 (12.8 g, 60.2 mmol) in 20 mL of CH2Cl2. After the mixture had been stirred for 3 h, it was diluted with 250 mL of Et2O and filtered through a pad of silica gel. The filtrate was concentrated and the residue was dissolved in Et2O and passed through a pad of silica gel to remove a colored impurity. The filtrate was concentrated to give 9.51 g (75%) of 15: 1H NMR δ 0.88 (t, J = 6.6 Hz, 3H), 1.26 (m, 14H), 1.96 (q, J = 6.7 Hz, 2H), 2.33 (q, J = 6.7 Hz, 2H), 2.49 (dt, J = 1.7, 7.0 Hz, 2H), 5.35–5.51 (m, 2H), 9.76 (t, J = 1.7 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 14.1, 22.7, 25.2, 29.1, 29.2, 29.3, 29.5, 31.9, 32.4, 32.5, 43.5, 127.5, 132.1, 202.5. 1H NMR (CDCl3) δ 0.88 (t, 3H, J = 7.4 Hz), 1.26–1.32 (m, 14H), 2.31–2.34 (m, 2H), 2.47–2.51 (m, 2H), 5.37–5.47 (m, 2H), 9.77 (s, 1H); 13C NMR (CDCl3) δ 14.0, 22.7, 25.2, 29.1, 29.3, 29.4, 29.5, 29.7, 31.9, 32.5, 43.5, 127.6, 132.1, 202.4; HRMS (M+H)+ calcd for m/z C14H27O 211.2062, found 211.2052. The NMR spectra agree with the published spectra (Baer and Schmidt, 1988).

3. Results and discussion

In earlier work from our laboratory, we reported the synthesis of ceramides with a 4E,6E-sphingadiene backbone (Struckhoff et al., 2004) on a scale of several hundred milligrams. The source of chirality at C-2 was a derivative of L-serine. The key reaction was alkylation of a β-ketosulfoxide with a long-chain allylic bromide (Chun et al., 2002). Subsequently, we outlined the synthesis of 4E,6E-sphingadiene (1a), again on a small scale, via the olefination of the same β-ketophosphonate intermediate (Fyrst et al., 2009). In the present work, we report an improved multigram-scale synthesis of sphingadiene 1a (Chart 1) from Garner ester 2 (Fig. 1). We found that the previous methods for the preparation of 1a could not be scaled up because the acid-mediated stepwise removal of the acetonide and Boc protecting groups from the diene system 5 resulted in byproducts formed via allylic rearrangement and dehydration, requiring that the intermediate be isolated by careful column chromatography after each step. As a result, a large quantity of starting ester 2 is required. Moreover, during acid hydrolysis of the Boc group, the erythro form of 1a partially undergoes conversion to its threo isomer, which cannot be removed from 1a without making derivatives in which the stereoisomers are separable. To circumvent these problems, we have now developed a protocol for a scalable synthesis of enantiomerically pure C14(4,6)-sphingadiene (1a) and have applied a related methodology for the preparation of C18(4,8)-sphingadiene (1b).

Chart 1.

Chart 1

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Structures of sphingadienes 1a and 1b.

Fig. 1.

Fig. 1

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Retrosynthetic route to 1a and 1b.

3.1. Retrosynthetic plan

Fig. 1 outlines the retrosynthetic analysis we envisioned, in which 1a and 1b can be accessed from oxazolidinone 7. The requisite cyclic carbamate can be obtained from the Boc-protected acetal 5, which in turn is formed by a diastereoselective reduction of enone 4. The enone can be prepared from L-serine derivative 2, which is available commercially.

3.2. Synthesis of 4E,6E-sphingadiene (1a) from Garner ester 2

Scheme 1 depicts the multigram synthesis of 1a from (S)-Garner ester 2. β-Ketophosphonate intermediate 3 was obtained by reaction of 2 (Garner and Park, 1987) with two equivalents of lithium dimethyl methylphosphonate (Koskinen and Krische, 1990; Byun and Bittman, 2010) in a dry ice/Et2O bath, followed by standing overnight at −78 °C. Dienone 4a was prepared by the Horner–Wadsworth–Emmons (HWE) reaction of 3 with the commercially available aldehyde (E)-2-decenal. The HWE reaction was carried out in water/2-propanol (1:1) in the presence of K2CO3 because under these conditions the reaction was found to proceed with high E-selectivity (Liu et al., 2010). In the presence of DBU or Cs2CO3, under anhydrous conditions, a long-chain analog of dienone 4a formed, but with a low optical rotation, suggesting that racemization may take place when strong bases are employed (Chun et al., 2002). In the presence of K2CO3 in aqueous alcohol, the reaction of CO32− with the acidic proton of β-ketophosphonate 3 in water produces a CO32−/HCO3− buffered medium, and racemization of dienone 4a may be avoided. The two-step sequence from the starting ester 2 gave 4a in 45% overall yield based on 2, and in 69% yield based on (E)-2-decenal. Diastereoselective reduction of ketone 4a with NaBH4/CeCl3 in methanol, as reported previously (Chun et al., 2002), gave an unseparable 5:1 mixture of erythro alcohol 5a together with the threo isomer (not shown). The erythro/threo selectivity was not improved when L-Selectride or DIBAL-H was used as the reducing agent; more bulky groups than 2,2-dimethyl-1,3-oxazolidine are apparently needed to improve the diastereoselectivity of the reduction (Kim et al., 2011). Since the minor amount of the threo diastereoisomer could not be removed by column chromatography (Chun et al., 2000, 2002), we decided to convert 5a to the cyclic carbamate intermediate 7a, where the minor stereoisomer could be separated. The conversion of 5a to 7a was achieved in two steps via formation of the key intermediate 6a (Triola et al., 2003). In the first step, the C3-hydroxyl group of crude 5a reacted with a catalytic amount of KH in THF, resulting in cleavage of the Boc group. The intramolecular cyclization from N-Boc derivative 5a to oxazolidinone derivative 6a could not be monitored by TLC because the Rf value of 5a was the same as that of 6a (Rf 0.6 in hexane/EtOAc 6:1); however, the conversion was confirmed by HRMS of the (M+H)+ ion of 6a: calcd for m/z C18H30NO3+ 308.2220, found 308.2216. The KH-catalyzed intramolecular carbamate formation reaction, with the loss of tert-butyl alcohol, was repeated three times, and each time tert-butanol was removed from the reaction mixture by rotary evaporation under reduced pressure, followed by addition of dry THF to the residue. In the second step, acid hydrolysis (p-TsOH in aqueous THF) of the acetonide group of 6a provided cyclic carbamate 7a in 71% overall yield from 4a. As shown in Fig. 2A, the presence of diastereomers in cyclic carbamate 7a was detected in the 1H NMR spectrum. At this stage, the threo isomer was removed by column chromatography, and the stereochemical purity of 7a was confirmed by its 1H and 13C NMR spectra. The chemical shift of the erythro isomer at an allylic position of 7a was δ 5.14 (t, J = 8.1 Hz), whereas that of threo isomer was δ 4.81 (t, J = 6.9 Hz) (see Fig. 2A). In the 13C NMR spectrum of 7a, the C-3 signal of the threo isomer appears at δ 79.70 ppm, whereas the C-3 signal of the erythro isomer is at δ 80.27 ppm. Carbamate 7a was fully characterized further by conversion to its diacetate derivative 7a′. In the final step, hydrolysis of 7a in ethanolic NaOH furnished the product 1a in almost quantitative yield; further purification was not required because 1a did not undergo elimination and epimerization reactions in basic media. In this procedure, only two column chromatographic purifications (one to obtain 4a and the other to obtain 7a) were needed to provide pure 1a starting from 2.

Scheme 1.

Scheme 1

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Synthesis of (4E,6E)-sphingadiene (1a).

Fig. 2.

Fig. 2

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Partial 1H NMR spectra of cyclic carbamate 7. (A) threo-enriched portion of 7a prior to further purification; (B) erythro-enriched portion of 7b after purification by column chromatography (note the minor signal at δ 4.75 ppm, indicating that most of the threo byproduct was removed).

3.3. Synthesis of 4(E)-tetradecenal (15)

The multigram synthesis of C18(4,8)-sphingadiene (1b) employed a similar strategy as described for 1a. Since the aldehyde required in the HWE reaction is not available commercially, it was first necessary to prepare a large quantity of 4(E)-tetradecenal (15). We initially attempted to prepare 15 via an orthoester Claisen rearrangement of allylic alcohol 8 (Baer and Schmidt, 1988). As shown in Scheme 2A, the reaction of decanal with vinylmagnesium bromide gave a mixture of 8 and 8a (Cuzzupe et al., 2002; Itami et al., 2003). After byproduct 8a was removed by column chromatography, pure allylic alcohol 8 was subjected to the orthoester Claisen rearrangement. The conversion to 9 proved difficult because byproduct 9a, the rearrangement product of 8a, could not be easily removed. Unfortunately, when the reaction was scaled up, the rearrangement gave a complex mixture, with multiple vinylic proton NMR signals. Therefore, an alternative approach became necessary in order to minimize the formation of products such as 9a, and we decided to employ the intramolecular Ireland-Claisen rearrangement (Ireland et al., 1976; for a review, see Enders et al., 1996) to generate carboxylic acid 12 and ester 13. As shown in Scheme 2B, the preparation of ester 13 commenced with the treatment of 8 with acetic anhydride in the presence of DMAP to furnish ester 10 (Keinan and Greenspoon, 1983) in 95% yield. In this step, the complete removal of the acetate derivative of 8a could be confirmed by the 1H NMR spectrum of 9. The reaction of 9 with LHMDS led to a lithium enolate intermediate, which on O-silylation with TMSCl delivered silyl enol ether 11. After the reaction mixture was brought to a neutral pH at room temperature, the Ireland-Claisen rearrangement provided γ,δ-alkenyl carboxylic acid 12. Without purification, 12 was converted to methyl ester 13 by treatment with trimethyl orthoformate and a catalytic amount of p-tosic acid in MeOH, giving 13 in 81% overall yield from 10. The E stereochemistry of 13 was confirmed by the 1H NMR spectrum of the olefinic signals: δ 5.35 (dt, J = 6.0, 15.3 Hz), 5.45 (dt, J = 6.4, 15.3 Hz). Because the attempted reduction of 13 with DIBAL provided a mixture of 13, 14, and 15, we employed the following two-step reaction sequence. Reduction of methyl ester 13 with LiAlH4 afforded primary alcohol 14 and PCC oxidation of 14 furnished the target aldehyde 15 in 63% overall yield. The E stereochemistry of aldehyde 15 was not changed during the reactions, as assessed by 1H NMR: δ 5.39 (dt, J = 5.9, 15.3 Hz), 5.45 (dt, J = 6.4, 15.3 Hz). Thus the preparation of 4(E)-tetradecenal (15) was achieved on a multigram scale by an intramolecular ester enolate Ireland-Claisen rearrangement followed by subsequent conversion reactions via the intermediate. Although three column chromatographic purifications (for 13, 14, and 15) were carried out in the conversion of 10–15, the polarity of each intermediate was markedly different; therefore, the chromatography was simple and rapid.

Scheme 2.

Scheme 2

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Synthesis of 4(E)-tetradecenal (15).

3.4. Synthesis of 4,8-sphingadiene (1b) from Garner ester 2

Similarly to the preparation of 1a, the multigram synthesis of sphingadiene 1b started with the reaction of (S)-Garner ester 2 with an excess of dimethyl methylphosphonate, as shown in Scheme 3. Deprotonation of β-ketophosphonate 3 with K2CO3 followed by the addition of γ,δ-unsaturated aldehyde 15 furnished the HWE product enone 4b in 47% overall yield from ester 2. Reduction of enone 4b, cyclization, and removal of the acetonide group with p-tosic acid in aqueous THF provided cyclic carbamate 7b in 75% yield. As shown in the partial 1H spectrum of 7b in Fig. 2B, the presence of the undesired threo enantiomer was readily detected by the 1H NMR signal at δ 4.75 (dd, J = 6.4, 7.0 Hz), and the decrease in the area of this signal provides an indication of the enrichment in the erythro isomer. The E stereochemistry of 7b was confirmed by the 1H NMR spectrum of the olefinic signals: δ 5.36 (dt, J = 5.7, 15.2 Hz), 5.43 (dt, J = 6.3, 15.2 Hz), 5.63 (dd, J = 8.4, 15.4 Hz), 5.89 (dt, J = 6.2, 15.4 Hz). The threo isomer was removed by flash chromatography (elution with a gradient of hexane/EtOAc 2:1 to 1:1). Then, basic hydrolysis of the carbamate group in 7b gave 1b in quantitative yield.

Scheme 3.

Scheme 3

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Synthesis of (4E,8E)-sphingadiene (1b).

4. Conclusions

We achieved a multigram preparation of C14(4,6)- and C18(4,8)-sphingadienes. A key reaction step is the introduction of a cyclic carbamate group into the sphingoid base to afford the intermediate 7a or 7b. In this intermediate, the unwanted threo isomer, which is generated during the reduction of the 3-keto group, can be readily removed from the mixture, and the absence of the threo byproduct can be confirmed by the 1H and 13C NMR spectra of 7a or 7b. In addition, no purification of 1a was needed after deprotection of the carbamate intermediate because 1a did not undergo elimination and epimerization reactions in basic media (ethanolic NaOH). We also report the synthesis of γ,δ-unsaturated aldehyde 15, which was required in the synthesis of C18(4,8)-sphingadiene (1b), by an Ireland-Claisen rearrangement.

Acknowledgments

This work was supported by National Institutes of Health grant HL-083187.

Abbreviations

C14(4,6)

sphingadiene

2S, 3R,4E,6E

2-aminotetradecadiene-1,3-diol

C(18)4,8

sphingadiene

2S, 3R,4E,8E

2-aminooctadecadiene-1,3-diol

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