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Synthesis 2013; 45(4): 511-517
DOI: 10.1055/s-0032-1317985
paper
© Georg Thieme Verlag Stuttgart · New York

Concise Syntheses of α-Galactosyl Ceramide, d-ribo-Phytosphingosine, and Ceramide

Yu-Fen Yen
,
R. C. Sawant
,
Shun-Yuan Luo*
Further Information

Publication History

Received: 19 November 2012

Accepted after revision: 13 December 2012

Publication Date:
08 January 2013 (online)

 
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Abstract

Total syntheses of α-galactosyl ceramide, d-ribo-phytosphingosine, and ceramide through an α-galactosyl phytosphingosine derivative as a common synthon were accomplished in overall yields of 26%, 15%, and 20% in nine, seven, and eight steps, respectively, starting from an acetonide-protected d-lyxose derivative. This short and efficient protocol involved protection and glycosylation of the acetonide-protected d-lyxose with d-galactosyl iodide as a key step. The resulting α-linked disaccharide was subsequently transformed into α-galactosyl ceramide, phytosphingosine, and ceramide.


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Key words

glycosylations - glycolipids - total synthesis

Glycolipids are essential components of cell membranes and play important roles in numerous biological processes. They can be recognized by the immune system and they can act as tumor-associated antigens. They can also be used in immunotherapy of individual forms of cancer, because α-configured glycolipids have been identified as potent stimulants of the immune system.[ 2 ] The isolation and structural characterization of α-galactosyl ceramide (1; α-GalCer), also known as KRN7000, were first reported by Natori and co-workers.[ 3 ] α-GalCer is the best-characterized antigen for CD1d-reactive T-cells in mice and humans.[ 4 ] To date, α-linked galactosyl ceramide has only been found in cancer[ 5 ] and fetal[ 6 ] cell lines. A greater understanding of lipid-specific T cells and the molecular mechanism of their immunogenicity should facilitate the development of lipid-based vaccines in the future.[ 7 ]

d-ribo -Phytosphingosine (2) is an important biomolecule first isolated in 1911 from mushrooms.[ 8 ] It serves as a metabolic precursor of important lipid mediators,[ 9 ] and is among the most important long-chain constituents of cell membranes.[2a] [10] It plays a critical role in numerous physiological processes.[ 11 ]

Ceramide (3) is another significant lipid molecule that acts as a modulator of cellular and whole-body metabolism.[ 12 ] Inhibition of the synthesis of ceramide has a broad spectrum of metabolic benefits and has promise in the development of therapeutic strategies for treating various metabolic diseases. The biological importance of α-GalCer (1), d-ribo -phytosphingosine (2), and ceramide (3) therefore makes them attractive targets for synthesis.

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Figure 1 The structures of α-galactosyl ceramide (1), d-ribo -phytosphingosine (2), and ceramide (3)

Recently, several efficient syntheses of α-GalCer and its analogues have been reported.[ 13 ] Du and Gervay-Hague achieved O-galactosylation of various acceptors with remarkable stereoselectivity.[ 14 ] d-Lyxose, which has all the requisite chiral centers in place, is the most suitable precursor for phytosphingosines.[ 15 ] Among the eight possible stereoisomers of phytosphingosine, (2S,3S,4R)-2-aminooctadecane-1,3,4-triol (2; Figure [1]) with d-ribo-stereochemistry is the most common. Because of the interest in their biological activities, numerous synthetic approaches to enantiomerically pure phytosphingosines have been reported.[ 16 ] The synthesis of ceramide is difficult, and few studies have been performed. Castillón and co-workers[ 17 ] developed a direct and efficient protocol for synthesizing α-configured glycolipids, including α-GalCer, which used ceramides as their stannyl derivatives in a tetrabutylammonium iodide-promoted glycosylation with glycosyl iodides. Panza and co-workers[ 18 ] reported an efficient synthesis of α-GalCer and related analogues through coupling of d-galactosyl bromide with a d-lyxose acceptor derived from d-mannose. For the development of glycolipids as a vaccine adjuvant, we were interested in establishing a short and general synthesis of α-GalCer and related analogues.

Our retrosynthetic analysis is shown in Scheme [1]. We proposed that α-GalCer (1) might be obtainable from the azido compound 4 through amide bond formation. The adduct 4 could be prepared from the protected sugar derivative 5 by azido displacement. Compound 5, in turn, should be obtainable by Wittig olefination of disaccharide 6, obtained through selective glycosylation of an acetonide-protected d-lyxose derivative with a glycosyl iodide. Retrosynthetic analyses for d-ribo-phytosphingosine (2) suggested that it might be prepared from adduct 4 through a reductive cleavage of the sugar moiety and deprotection. An amide bond formation reaction of 2 should then give ceramide (3).

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Scheme 1 Retrosynthetic plan for α-galactosyl ceramide (1), d-ribo-phytosphingosine (2), and ceramide (3)

The formation of the glycosidic bond is the key step in the synthesis of α-galactosyl ceramide (1). Various glycosyl donors, including glycosyl trichloroacetimidates, fluorides, phosphates, and sulfides, have been reported.[ 19 ] However, glycosylations with these donors suffer from low yields, poor selectivities, and difficulties in separation of the α/β mixtures.[ 20 ] Therefore, α-directing protecting groups must be used in forming the essential α-glycosidic linkage. For example, galactosyl donors protected with a 4,6-O-benzylidene group or a 4,6-di-tert-butylsilylene group have been successfully used to obtain high yields of desired glycosylated products.[ 21 ]

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Scheme 2 Preparation of α-galactosyl ceramide 1. Reagents and conditions: (a) 2,3-O-isopropylidene-d-lyxofuranose (8), TBAI, DIPEA, 4 Å MS, toluene, 65 °C; (b) LiHMDS, Me(CH2)12P+Ph3 Br – , THF, 0 °C; (c) H2, 10% Pd/C, MeOH, r.t.; (d) Tf2O, py, CH2Cl2, 0 °C then (Me2N)2C=NH2 + N3 –; (e) 1. Ph3P, py, THF, H2O, 60 °C, overnight, 2. Me(CH2)24CO2H, 1H-1,2,3-benzotriazol-1-ol, EDC, Et3N, CH2Cl2; (f) 0.4 M HCl, CH2Cl2–MeOH, r.t.; (g) H2 (60 psi), Pd(OH)2/C, MeOH–CHCl3.

We began with a regio- and stereoselective synthesis of the key disaccharide 9 (Scheme [2]). The 2- and 3-hydroxyl groups of the d-lyxose derivative 8 were selectively protected as the acetonide by treatment with anhydrous acetone containing a catalytic amount of sulfuric acid.[ 21 ] A solution of 2,3,4,6-tetra-O-benzyl-d-galactopyranosyl iodide (7)[ 22 ] (one equivalent), prepared by treatment of 1-O-acetyl-2,3,4,6-tetra-O-benzyl-d-galactopyranose with iodo(trimethyl)silane, was dried by careful azeotropic distillation and then treated with N,N-diisopropylethylamine (DIPEA, 1 equiv), and tetrabutylammonium iodide (3 equiv) in toluene for one hour at 65 °C. This gave the α-linked disaccharide 9 exclusively in 78% yield over the two steps. Therefore, coupling between glycosyl iodide 7 and the 1,5-diol 8 took place regio- and stereoselectively at the primary hydroxyl group, leaving the hemiacetal hydroxy group in the 1-position free for further reaction. The selectivity of this reaction eliminated two steps that would otherwise have been necessary to block the anomeric hydroxy group and remove it after glycosylation. Furthermore, in our method we used relatively cheap reagents, such as iodo(trimethyl)silane, tetrabutylammonium iodide, and N,N-diisopropylethylamine, whereas in their synthesis, Panza et al.[ 18 ] used oxalyl bromide and tris(pyrrolidin-1-yl)phosphine oxide, which are expensive and scarce.

By using the conditions adapted from those of Kulkarni and Gervay-Hague,[ 23 ] we succeeded in glycosylating the acetonide-protected d-lyxose acceptor with galactosyl iodide with α-stereoselective formation of compound 9. The free hemiacetal in disaccharide 9 permitted Wittig olefination to form the cis-compound 10 as the major product. Of several reaction conditions that we examined in attempts to perform this Wittig reaction, the in situ generation of phosphorane[ 24 ] by slow addition of the base to a well-stirred suspension of 9 and the phosphonium salt at 0 °C showed the best performance in producing the desired cis-product 10.

The double bond in compound 10 was successfully hydrogenated to give the saturated alcohol 11. Triflation of alcohol 11 by triflic anhydride, followed by a displacement reaction with sodium azide in N,N-dimethylformamide did not, however, give the desired azido compound in a high yield. Even at an elevated temperature (100 °C), the starting material was not consumed completely, and purification of the product was difficult.[ 18 ] Optimization studies showed that the best results for the azide-mediated inversion were achieved by using tetramethylguanidinium azide,[ 25 ] which gave compound 12 in a high yield.[ 26 ] The azide 12 is a crucial intermediate, because it provides direct access to α-GalCer and related analogues. Reduction of the azide group to the corresponding amine followed by amide bond formation gave amide 13. The isopropylidene group was hydrolyzed under acidic conditions to give diol 14. Finally, the benzyl groups were removed by hydrogenolysis over palladium(II) hydroxide on carbon to give the target compound 1 in a quantitative yield.

To synthesize d-ribo -phytosphingosine and ceramide (Scheme [3]), precursor 12 was treated with 1 M aqueous sulfuric acid in an attempt to hydrolyze the galactosyl unit; however, this reaction was sluggish. Treatment with 75% sulfuric acid in 1,4-dioxane resulted only in hydrolysis of the isopropylidene group. Treatment with 4 M hydrochloric acid in refluxing tetrahydrofuran gave a mixture of compounds that included phytosphingosine. However, when we used 4 M hydrochloric acid in a sealed vial at 100 °C, we obtained the azide 15 [ 27 ] in 65% yield. Hydrogenation of azide 15 in the presence of a palladium catalyst gave d-ribo -phytosphingosine (2)[ 28 ] in 70% yield. Finally, a Staudinger reaction and amide bond formation reaction of azide compound 15 gave ceramide (3) in 53% yield over the two steps.

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Scheme 3 Preparation of phytosphingosine (2) and ceramide (3). Reagents and conditions: (a) 8 M aq HCl, THF, 65%; (b) H2, Pd/C, MeOH–EtOAc, 70% (c) 1. py, Ph3P, THF; 2. Me(CH2)24CO2H, EDC, 1H-1,2,3-benzotriazol-1-ol, Et3N, DMF, 53% (two steps).

In conclusion, we have developed a protocol for the total synthesis of α-GalCer, phytosphingosine, and ceramide. Although our synthesis resembles that reported by Panza and co-workers,[ 18 ] there is a key difference in that our synthesis involves a regioselective protection and glycosylation reaction of the acetonide-protected d-lyxose. Our short and efficient route for the synthesis of α-GalCer is expected to provide access to other structurally related glycolipids, permitting exploration of their immunostimulating activities and other biological properties. The phytosphingolipid chains can easily be modified by using various Wittig salts, and the analogues can be modified by using various starting glycosyl iodides. The key step is the formation of the α-disaccharide linkage with the glycosyl iodide; this is followed by synthesis of the common synthon 12, which permits total syntheses of α-GalCer and its analogues through simple synthetic routes. The overall yields of α-GalCer, d-ribo -phytosphingosine, and ceramide were 26%, 15%, and 20%, respectively, in nine, seven, and eight steps, respectively. Derivatives of α-GalCer are currently being prepared and investigations on their biological properties will be reported in the near future.

All reactions were conducted under dry N2 in flame-dried glassware. Toluene, THF, and CH2Cl2 were purified and dried in a safe purification system containing activated Al2O3. Anhyd DMF, pyridine, and MeOH were used as purchased. All reagents obtained from commercial sources were used without purification unless otherwise mentioned. Flash column chromatography was carried out on silica gel 60 (230–400 mesh; Merck). TLC was performed on glass plates coated with silica gel 60 F254 (0.25 mm, Merck); detection was performed by spraying with a soln of Ce(NH4)2(NO3)6 (0.5 g), (NH4)6Mo7O24 (24 g), and H2SO4 (28 mL) in H2O (500 mL) with subsequent heating on a hot plate. Melting points were determined with a capillary apparatus. Optical rotations were measured at 589 nm (Na) at ~25 °C on an Autopol V polarimeter. 1H, 13C NMR, DEPT, 1H–1H COSY, 1H–13C COSY, and NOESY spectra were recorded with Oxford 400 MHz and Varian Unity Inova 600 MHz instruments. Chemical shifts are reported in ppm relative to TMS on the basis of the CDCl3 lock signal at 7.24 ppm. IR spectra were recorded on an FT-IR spectrometer by using KBr plates or solns in CHCl3. Mass spectra were analyzed on a Finnigan LTQ-Orbitrap­ XL instrument with an ESI source (Thermo Electron, Bremen­, Germany).


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2,3-O-Isopropylidene-5-O-(2,3,4,6-tetra-O-benzyl-α-d-galactopyranosyl)-d-lyxofuranose (9)

TMSI (86 mg, 0.43 mmol, 1.25 equiv) was added to a soln of galactopyranoside 7 (200 mg, 0.34 mmol, 1 equiv) in anhyd CH2Cl2 (3.4 mL) at 0 °C under N2, and the mixture was stirred for 30 min. The reaction was quenched by addition of anhyd toluene (3 mL), and the mixture was dried by azeotropic distillation (×3). The slightly yellow residue of the iodo derivative was dissolved in toluene (1 mL) and kept under N2.

A mixture of 2,3-O-isopropylidene-d-lyxofuranose (8; 71 mg, 0.37 mmol, 1.1 equiv), DIPEA (59 μL, 0.34 mmol, 1 equiv), TBAI (376 mg, 1.02 mmol, 3 equiv), and 4 Å MS (100 mg) in anhyd toluene (1 mL) was stirred for 10 min at 65 °C under N2. The soln of the iodo derivative in toluene (1.0 mL) was added to the reaction flask from a cannula, and the mixture was stirred at 65 °C for 1 h. The reaction was stopped by addition of EtOAc (10 mL), and the mixture was cooled to 0 °C. The white precipitate and the MS were removed by filtration through Celite and the filtrate was extracted with aq Na2S2O3 (3 × 10 mL) and brine (10 mL). The organic layers were combined, dried (MgSO4), filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel) to afford the desired disaccharide 9; yield: 188 mg (78%, two steps); Rf  = 0.30 (EtOAc–hexanes, 1:2); [α]D 28 +34.5 (c 1.3, CHCl3).

IR (CHCl3): 3436, 3030, 1454, 1096 cm–1.

1H NMR (400 MHz, CDCl3): δ = 7.40–7.28 (m, 20 H, ArH), 5.32 (d, J = 2.8 Hz, 1 H, H-1), 4.94 (d, J = 11.6 Hz, 1 H, CH2Ph), 4.90 (d, J = 3.6 Hz, 1 H, H-1′), 4.84 (d, J = 11.6 Hz, 1 H, CH2Ph), 4.82 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.76–4.74 (m, 1 H, H-2), 4.74 (d, J = 11.2 Hz, 1 H, CH2Ph), 4.68 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.57 (d, J = 11.6 Hz, 1 H, CH2Ph), 4.56 (d, J = 5.6 Hz, 1 H, H-3), 4.45 (d, J = 11.6 Hz, 1 H, CH2Ph), 4.41–4.38 (m, 1 H, H-4), 4.38 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.06–3.96 (m, 4 H, H-2′, H-3′, H-4′, H-5′), 3.85–3.83 (m, 2 H, H-5a, H-5b), 3.52–3.51 (m, 2 H, H-6a′, H-6b′), 2.5 (s, 1 H, OH), 1.40 (s, 3 H, CH3), 1.28 (s, 3 H, CH3).

13C NMR (150 MHz, CDCl3): δ = 138.8 (C), 138.6 (C), 138.5 (C), 137.8 (C), 128.33 (CH × 2), 128.28 (CH × 2), 128.25 (CH × 2), 128.2 (CH), 128.16 (CH × 2), 128.1 (CH × 2), 127.9 (CH), 127.8 (CH × 2), 127.66 (CH), 127.61 (CH), 127.5 (CH), 127.37 (CH), 127.35 (CH × 2), 112.4 (C), 101.0 (CH), 97.9 (CH), 85.4 (CH), 79.9 (CH), 78.9 (CH), 78.6 (CH), 76.4 (CH), 74.9 (CH), 74.7 (CH2), 73.3 (CH2), 73.2 (CH2), 72.9 (CH2), 69.2 (CH), 68.9 (CH2), 66.3 (CH2), 26.0 (CH3), 24.8 (CH3).

HRMS (FAB): m/z [M + Na]+ calcd for C42H48NaO10: 735.3145; found: 735.3129.


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(2R)-2-{(4S,5R)-2,2-Dimethyl-5-[(1Z)-tetradec-1-en-1-yl]-1,3-dioxolan-4-yl}-2-hydroxyethyl 2,3,4,6-tetra-O-benzyl-α-d-galactopyranoside (10)

A mixture of disaccharide 9 (3.45 g, 4.84 mmol) and Me(CH2)12P+Ph3 Br– (5.09 g, 9.68 mmol) in anhyd THF (35 mL) was cooled to 0 °C under N2. A 1.0 M soln LiHMDS in THF (9.68 mL, 9.68 mmol) was added, and the mixture was stirred for 6 h at 0 °C. H2O (35 mL) was then added to quench the reaction, and the mixture was extracted with EtOAc (3 × 20 mL). The organic layers were combined, washed with brine, dried (MgSO4), filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel) to give a colorless oil; yield: 3.66 g (86%); Rf  = 0.50 (EtOAc–hexanes, 1:3); [α]D 29 +9.5 (c 1.3, CHCl3).

IR (CHCl3): 3492, 2925, 1605, 1454, 1099 cm–1.

1H NMR (600 MHz, CDCl3): δ = 7.39–7.24 (m, 20 H, ArH), 5.69–5.62 (m, 2 H, H-5, H-6), 4.95 (t, J = 7.8 Hz, 1 H, H-4), 4.92 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.82 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.81 (d, J = 3.6 Hz, 1 H, H-1′), 4.80 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.73 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.67 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.55 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.47 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.39 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.12 (dd, J = 6.6, 3.0 Hz, 1 H, H-3), 4.05–4.02 (m, 2 H, H-2′, H-5′), 3.96–3.93 (m, 2 H, H-3′, H-4′), 3.77 (br s, 1 H, H-2), 3.65 (dd, J = 10.8, 4.2 Hz, 1 H, H-1a), 3.53–3.48 (m, 3 H, H-1b, H-6a′, H-6b′), 2.73 (d, J = 5.4 Hz, 1 H, 2-OH), 2.13–1.98 (m, 2 H, H-7a, H-7b), 1.50 (s, 3 H, CH3), 1.36 (s, 3 H, CH3), 1.30–1.24 (m, 20 H, CH2), 0.88 (t, J = 6.8 Hz, 3 H, CH3).

13C NMR (150 MHz, CDCl3): δ = 138.7 (C), 138.5 (C), 138.4 (C), 137.9 (C), 135.4 (CH), 128.33 (CH × 3), 128.31 (CH × 2), 128.2 (CH × 3), 127.9 (CH × 2), 127.72 (CH × 2), 127.65 (CH × 2), 127.54 (CH), 127.45 (CH), 127.4 (CH × 2), 125.0 (CH), 108.4 (C), 98.0 (CH), 79.0 (CH), 77.4 (CH), 76.3 (CH), 74.8 (CH), 74.7 (CH2), 73.42 (CH2), 73.37 (CH2), 73.0 (CH), 72.9 (CH2), 70.0 (CH2), 69.4 (CH), 68.9 (CH2), 68.6 (CH), 31.9 (CH2), 29.64 (CH2 × 3), 29.62 (CH2 × 2), 29.58 (CH2), 29.49 (CH2), 29.47 (CH2), 29.32 (CH2), 29.25 (CH2), 27.7 (CH2), 27.1 (CH3), 25.0 (CH3), 22.7 (CH2), 14.1 (CH3).

HRMS (FAB): m/z [M + Na]+ calcd for C55H74NaO9: 901.5231; found: 901.5220.


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(2R)-2-[(4S,5R)-2,2-Dimethyl-5-tetradecyl-1,3-dioxolan-4-yl]-2-hydroxyethyl 2,3,4,6-tetra-O-benzyl-α-d-galactopyranoside (11)

A mixture of olefin 10 (400 mg, 0.45 mmol), 10% Pd/C (130 mg), and MeOH (5 mL) was bubbled with N2 for 5 min. The flask was then fitted with a H2-filled balloon and the suspended soln was stirred for 2.5 h. The H2 was displaced with N2, and the mixture was filtered through Celite. The filtrate was concentrated in vacuo to give a crude product that was purified by column chromatography (silica gel) to give a colorless oil; yield: 334 mg (85%); Rf = 0.40 (EtOAc–hexanes, 1:4); [α]D 29 +19.7 (c 1.4, CHCl3).

IR (CHCl3): 3492, 2924, 1497, 1099 cm–1.

1H NMR (600 MHz, CDCl3): δ = 7.39–7.24 (m, 20 H, ArH), 4.93 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.84 (d, J = 3.6 Hz, 1 H, H-1′), 4.82 (d, J = 12.6 Hz, 1 H, CH2Ph), 4.81 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.73 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.67 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.56 (d, J = 10.8 Hz, 1 H, CH2Ph), 4.47 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.39 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.14–4.10 (m, 1 H, H-3), 4.07 (dd, J = 6.6, 3.0 Hz, 1 H, H-4), 4.04 (dd, J = 10.2, 3.6 Hz, 1 H, H-2′), 4.02 (t, J = 6.6 Hz, 1 H, H-5′), 3.96 (d, J = 2.4 Hz, 1 H, H-4′), 3.93 (dd, J = 10.2, 3.0 Hz, 1 H, H-3′), 3.84–3.80 (m, 1 H, H-2), 3.69 (dd, J = 10.8, 6.6 Hz, 1 H, H-1a), 3.55 (dd, J = 10.2, 5.4 Hz, 1 H, H-1b), 3.50–3.48 (m, 2 H, H-6a′, H-6b′), 2.63 (d, J = 6.6 Hz, 1 H, 2-OH), 1.54–1.53 (m, 2 H, CH2), 1.47 (s, 3 H, CH3), 1.34 (s, 3 H, CH3), 1.31–1.25 (m, 24 H, CH2), 0.88 (t, J = 7.2 Hz, 3 H, CH3).

13C NMR (150 MHz, CDCl3): δ = 138.7 (C), 138.5 (C), 138.4 (C), 137.8 (C), 128.34 (CH × 2), 128.31 (CH × 4), 128.19 (CH × 2), 128.17 (CH × 2), 127.9 (CH × 2), 127.7 (CH × 2), 127.66 (CH × 2), 127.54 (CH), 127.45 (CH), 127.4 (CH × 2), 107.8 (C), 98.1 (CH), 79.0 (CH), 77.4 (CH), 76.7 (CH), 76.3 (CH), 74.8 (CH), 74.7 (CH2), 73.4 (CH2), 73.0 (CH2), 70.3 (CH2), 69.5 (CH), 68.9 (CH2), 68.2 (CH), 31.9 (CH2), 29.7 (CH2 × 5), 29.62 (CH2 × 3), 29.58 (CH2 × 2), 29.32 (CH2), 27.2 (CH3), 26.9 (CH2), 25.1 (CH3), 22.7 (CH2), 14.1 (CH3).

HRMS (ESI): m/z [M + Na]+ calcd for C55H76NaO9: 903.5387; found: 903.5379.


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(2S)-2-Azido-2-[(4S,5R)-2,2-dimethyl-5-tetradecyl-1,3-dioxolan-4-yl]ethyl 2,3,4,6-tetra-O-benzyl-α-d-galactopyranoside (12)

A mixture of alcohol 11 (442 mg, 0.50 mmol) and pyridine (100 μL, 1.25 mmol) was dissolved in CH2Cl2 (5 mL) under N2, and the flask was immersed in an ice bath. Tf2O (126 μL, 0.75 mmol) was added dropwise to the soln and the mixture was stirred for 3 h at 0 °C. A soln of (Me2N)2C=NH2 + N3 – (0.32 g, 2.00 mmol) and CH2Cl2 (1 mL) was injected into the flask, and the mixture was stirred for overnight at 0 °C. H2O (10 mL) was added and the mixture was extracted with EtOAc (3 × 10 mL). The organic layers were combined, dried (MgSO4), filtered, and concentrated in vacuo. The residue, which was purified by column chromatography (silica gel) to give a colorless oil; yield: 353 mg (78%); Rf = 0.60 (EtOAc–hexanes, 1:5); [α]D 29 +30.7 (c 1.1, CHCl3).

IR (CHCl3): 2924, 2099, 1496, 1100 cm–1.

1H NMR (600 MHz, CDCl3): δ = 7.39–7.24 (m, 20 H, ArH), 4.95 (d, J = 10.8 Hz, 1 H, CH2Ph), 4.94 (d, J = 4.2 Hz, 1 H, H-1′), 4.84 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.79 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.72 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.71 (d, J = 12.6 Hz, 1 H, CH2Ph), 4.56 (d, J = 10.8 Hz, 1 H, CH2Ph), 4.48 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.39 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.10–4.01 (m, 4 H, H-1b, H-3, H-4, H-2′), 4.00–3.97 (m, 2 H, H-3′, H-5′), 3.93 (s, 1 H, H-4′), 3.72 (dd, J = 10.8, 6.6 Hz, 1 H, H-1a), 3.53–3.45 (m, 3 H, H-2, H-6a′, H-6b′), 1.55–1.52 (m, 2 H, CH2), 1.36 (s, 3 H, CH3), 1.30–1.26 (m, 27 H, CH3, CH2), 0.88 (t, J = 6.6 Hz, 3 H, CH3).

13C NMR (150 MHz, CDCl3): δ = 138.8 (C × 2), 138.5 (C), 137.9 (C), 128.3 (CH × 2), 128.27 (CH × 2), 128.25 (CH × 2), 128.2 (CH × 4), 127.7 (CH × 2), 127.62 (CH), 127.58 (CH × 2), 127.53 (CH), 127.51 (CH × 2), 127.4 (CH × 2), 108.1 (C), 98.8 (CH), 78.6 (CH), 77.7 (CH), 76.5 (CH), 75.3 (CH), 75.2 (CH), 74.7 (CH2), 73.4 (CH2), 73.3 (CH2), 72.8 (CH2), 69.8 (CH), 69.5 (CH2), 69.1 (CH2), 59.7 (CH), 31.9 (CH2), 29.7 (CH2 × 3), 29.62 (CH2 × 2), 29.59 (CH2), 29.57 (CH2), 29.5 (CH2), 29.33 (CH2), 29.26 (CH2), 28.1 (CH3), 26.5 (CH2), 25.7 (CH3), 22.7 (CH2), 14.1 (CH3).

HRMS (ESI): m/z [M + Na]+ calcd for C55H73N3NaO8: 926.5290; found: 926.5275.


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N-{(1S)-1-[(4S,5R)-2,2-Dimethyl-5-tetradecyl-1,3-dioxolan-4-yl]-2-[(2,3,4,6-tetra-O-benzyl-α-d-galactopyranosyl)oxy]ethyl}hexacosanamide (13)

Pyridine (1.5 mL) and H2O (1.5 mL) were added sequentially to a soln of the azido compound 12 (0.50 g, 0.55 mmol) in THF (7 mL) at r.t. and then the flask was warmed to 60 °C. Ph3P (290 mg, 1.10 mmol) was added, and the mixture was stirred for 12 h then gradually cooled to r.t. and concentrated in vacuo. The resulting crude amine was dissolved in CH2Cl2 (7 mL) at r.t. and treated by sequential addition of EDC·HCl (192 mg, 0.99 mmol), Me(CH2)24CO2H (286 mg, 0.715 mmol), and 1H-1,2,3-benzotriazol-1-ol (136 mg, 0.99 mmol). The mixture was stirred continuously for 12 h. H2O was added and the mixture was extracted with CH2Cl2 (2 × 10 mL). The organic layers were combined, dried (MgSO4), filtered, and concentrated in vacuo. The residue was purified by column chromatography (silica gel) to give the amide 13 as a white solid; yield: 590 mg (85%); mp 82–83 °C; Rf = 0.50 (EtOAc–hexanes, 1:5); [α]D 30 +40.4 (c 1.0, CHCl3).

IR (CHCl3): 3316, 2918, 1648 cm–1.

1H NMR (600 MHz, CDCl3): δ = 7.40–7.24 (m, 20 H, ArH), 6.36 (d, J = 8.4 Hz, 1 H, NH), 4.93 (d, J = 10.8 Hz, 1 H, CH2Ph), 4.90 (d, J = 3.6 Hz, 1 H, H-1′), 4.81 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.80 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.74 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.66 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.58 (d, J = 10.8 Hz, 1 H, CH2Ph), 4.48 (d, J = 12.6 Hz, 1 H, CH2Ph), 4.37 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.12–4.03 (m, 4 H, H-1a, H-2, H-2′, H-3′), 3.99–3.97 (m, 1 H, H-5′), 3.93–3.90 (m, 3 H, H-3, H-4, H-4′), 3.61 (dd, J = 11.4, 2.4 Hz, 1 H, H-1b), 3.55 (dd, J = 9.0, 7.2 Hz, 1 H, H-6a′), 3.36 (dd, J = 9.0, 5.4 Hz, 1 H, H-6b′), 2.09–1.96 (m, 2 H), 1.55–1.42 (m, 4 H, CH2), 1.40 (s, 3 H, CH3), 1.31 (s, 3 H, CH3), 1.30–1.23 (m, 68 H, CH2), 0.89–0.83 (m, 6 H, CH3 × 2).

13C NMR (150 MHz, CDCl3): δ = 172.4 (C), 138.6 (C), 138.3 (C), 138.2 (C), 137.5 (C), 128.40 (CH × 2), 128.37 (CH × 3), 128.35 (CH × 3), 128.2 (CH × 2), 127.94 (CH × 2), 127.87 (CH × 3), 127.8 (CH), 127.7 (CH), 127.6 (CH), 127.5 (CH × 2), 107.8 (C), 99.7 (CH), 78.8 (CH), 77.8 (CH), 76.7 (CH), 75.3 (CH), 74.62 (CH2), 74.57 (CH), 73.54 (CH2), 73.47 (CH2), 72.9 (CH2), 70.6 (CH2), 69.8 (CH), 69.5 (CH2), 48.6 (CH), 36.7 (CH2), 31.9 (CH2 × 2), 29.7 (CH2 × 20), 29.59 (CH2 × 2), 29.56 (CH2 × 2), 29.5 (CH2), 29.4 (CH2), 29.3 (CH2 × 2), 28.8 (CH2), 28.2 (CH3), 26.6 (CH2), 26.0 (CH3), 25.6 (CH2), 22.7 (CH2 × 2), 14.1 (CH3 × 2).

HRMS (ESI): m/z [M + H]+ calcd for C81H128NO9: 1258.9589; found: 1258.9602.


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N-((1S,2S,3R)-2,3-Dihydroxy-1-{[(2,3,4,6-tetra-O-benzyl-α-d-galactopyranosyl)oxy]methyl}heptadecyl)hexacosanamide (14)

4 M aq HCl (200 μL) was added to a soln of amide 13 (0.20 g, 0.16 mmol) in 5:1 CH2Cl2–MeOH (2 mL) at 0 °C, and the mixture was warmed to r.t. and stirred for 12 h. The reaction was quenched with sat. aq NaHCO3 (3 mL), and the mixture was extracted with CH2Cl2 (2 × 5 mL). The organic layers were combined, dried (MgSO4), filtered, and concentrated in vacuo to give a residue that was purified by column chromatography (silica gel) to give a white solid; yield: 138 mg (71%); mp 75 °C; Rf = 0.33 (EtOAc–hexanes, 1:3); [α]D 29 +33.3 (c 1.9, CHCl3).

IR (CHCl3): 3332, 2920, 1620 cm–1.

1H NMR (600 MHz, CDCl3): δ = 7.38–7.25 (m, 20 H, ArH), 6.45 (d, J = 7.8 Hz, 1 H, NH), 4.91 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.87 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.84 (d, J = 4.2 Hz, 1 H, H-1′), 4.77 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.74 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.67 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.56 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.47 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.38 (d, J = 11.4 Hz, 1 H, CH2Ph), 4.22–4.19 (m, 1 H, H-2), 4.04 (dd, J = 10.2, 3.6 Hz, 1 H, H-2′), 3.96 (d, 1 H, H-4′), 3.89–3.84 (m, 4 H, H-1a, H-1b, H-3′, H-5′), 3.51–3.44 (m, 4 H, H-2, H-3, H-6a′, H-6b′), 2.30 (s, 1 H, OH), 2.12 (t, 2 H, CH2), 1.80 (s, 1 H, OH), 1.59–1.56 (m, 4 H, CH2), 1.47–1.42 (m, 2 H, CH2), 1.36–1.25 (m, 66 H, CH2), 0.88 (t, 3 H, CH3), 0.84 (t, 3 H, CH3).

13C NMR (150 MHz, CDCl3): δ = 173.2 (C), 138.32 (C), 138.27 (C), 137.8 (C), 137.5 (C), 128.44 (CH × 2), 128.42 (CH × 2), 128.39 (CH × 2), 128.23 (CH × 2), 128.17 (CH × 2), 128.1 (CH × 2), 127.94 (CH × 3), 127.88 (CH), 127.7 (CH), 127.6 (CH), 127.4 (CH × 2), 99.1 (CH), 79.2 (CH), 76.1 (CH), 76.0 (CH), 74.7 (CH2), 74.3 (CH), 74.2 (CH2), 73.6 (CH2), 73.2 (CH), 72.7 (CH2), 69.9 (CH2), 69.9 (CH), 68.9 (CH2), 49.5 (CH), 36.7 (CH2), 33.2 (CH2), 31.9 (CH2), 29.7 (CH2 × 25), 29.5 (CH2), 29.4 (CH2), 29.34 (CH2 × 2), 29.29 (CH2), 25.9 (CH2), 25.7 (CH2), 22.7 (CH2 × 2), 14.1 (CH3 × 2).

HRMS (ESI): m/z [M + H]+ calcd for C78H124NO9: 1218.9276; found: 1218.9311.


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N-{(1S,2S,3R)-1-[(α-d-Galactopyranosyloxy)methyl]-2,3-dihydroxyheptadecyl}hexacosanamide (1; α-Galactosyl Ceramide)

The protected compound 14 (200 mg) was dissolved in 3:1 MeOH–CHCl3 (2 mL) at r.t. and Pd(OH)2/C (200 mg, Degussa type) was added. The vessel was purged with H2, and the mixture was stirred at 60 psi at r.t. for 1 d. The resulting soln was filtered through Celite, and the filtrate was concentrated in vacuo to give a white solid; yield: 140 mg (quant); mp 182–185 °C; Rf = 0.60 (MeOH–CHCl3, 1:4); [α]D 27 +38.2 [c 0.05, CHCl3–MeOH (1:1)].

IR (KBr): 3426, 2918, 2851, 1645 cm–1.

1H NMR (600 MHz, pyridine-d 5): δ = 8.55 (d, J = 8.4 Hz, 1 H), 5.54 (d, J = 3.6 Hz, 1 H), 5.23 (m, 1 H), 4.65–4.25 (m, 10 H), 2.42 (t, 2 H), 2.32–2.21 (m, 1 H), 1.92–1.57 (m, 5 H), 1.29–1.22 (m, 66 H), 0.84 (t, 6 H).

13C NMR (150 MHz, CDCl3): δ = 173.3 (C), 101.4 (CH), 76.5 (CH), 73.0 (CH), 72.4 (CH), 71.5 (CH), 70.9 (CH), 70.2 (CH), 68.5 (CH2), 62.6 (CH2), 51.4 (CH), 36.8 (CH2), 34.2 (CH2), 32.1 (CH2), 30.3–29.3 (CH2), 26.5 (CH2), 26.4 (CH2), 22.9 (CH2), 14.3 (CH3).

HRMS (ESI): m/z [M + H]+ calcd for C50H100NO9: 858.7398; found: 858.7408.


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(2S,3S,4R)-2-Azidooctadecane-1,3,4-triol (15)

A soln of azide 12 (170 mg, 0.19 mmol) in 8 M aq HCl (1.5 mL) and THF (1.5 mL) was placed in a sealable vial and warmed to 100 °C with stirring for 1 d. The reaction was quenched with sat. aq NaHCO3, and the mixture was extracted with EtOAc (3 × 5 mL). The organic layers were combined, dried (MgSO4), filtered, and concentrated in vacuo, and the residue was purified by column chromatography (silica gel) to give a white solid: yield: 42 mg (65%); mp 97 °C; Rf = 0.14 (EtOAc–hexanes, 1:2); [α]D 27 +4.47 (c 0.85, MeOH).

IR (KBr): 3343, 2918, 2848, 2118, 1463 cm–1.

1H NMR (600 MHz, CD3OD): δ = 3.92 (dd, J = 10.8, 3.0 Hz, 1 H, H-1a), 3.75 (dd, J = 11.4, 7.8 Hz, 1 H, H-1b), 3.61–3.58 (m, 1 H, H-2), 3.54–3.50 (m, 2 H, H-3, H-4), 1.70–1.66 (m, 1 H), 1.57–1.54 (m, 1 H), 1.41–1.25 (m, 24 H, CH2), 0.90 (t, J = 7.2 Hz, 3 H, CH3).

13C NMR (150 MHz, CD3OD): δ = 76.0 (CH), 72.8 (CH), 66.7 (CH), 62.5 (CH2), 33.9 (CH2), 33.1 (CH2), 30.81 (CH2), 30.80 (4 CH2), 30.77 (2 CH2), 30.76 (CH2), 30.5 (CH2), 26.7 (CH2), 23.7 (CH2), 14.5 (CH3).

HRMS (ESI): m/z [M + Na]+ calcd for C18H37N3NaO3: 366.2727; found: 366.2723.


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d-ribo-Phytosphingosine (2)

A mixture of triol 15 (71 mg, 0.21 mmol), 10% Pd/C (30 mg), and a 1:1 mixture of MeOH and EtOAc (2 mL) was bubbled with N2 for 5 min. The flask was fitted with a H2-filled balloon and the suspension was stirred for 1 d. The H2 was displaced with N2, and the mixture was filtered through Celite. The filtrate was concentrated in vacuo to give a crude product as a white solid that was used without further purification; yield: 46 mg (70%); mp 96–98 °C (Lit.9 98.5–101.5 °C); Rf = 0.20 (CHCl3–MeOH–NH4OH, 80:20:1); [α]D 27 +5.16 (c 1.2, MeOH).

IR (KBr): 3359, 2918, 2850, 1468 cm–1.

1H NMR (600 MHz, CD3OD): δ = 3.76 (dd, J = 10.8, 4.2 Hz, 1 H, H-1a), 3.57 (dd, J = 10.8, 6.6 Hz, 1 H, H-1b), 3.52 (ddd, J = 8.4, 8.0, 3.0 Hz, 1 H, H-4), 3.35 (dd, J = 8.4, 6.0 Hz, 1 H, H-3), 2.96 (ddd, J = 6.6, 6.0, 4.2 Hz, 1 H, H-2), 1.76–1.73 (m, 1 H), 1.57–1.54 (m, 1 H), 1.41–1.29 (m, 24 H, CH2), 0.90 (t, J = 7.2 Hz, 3 H, CH3).

13C NMR (150 MHz, CD3OD): δ = 76.4 (CH), 74.4 (CH), 64.0 (CH2), 55.8 (CH), 34.8 (CH2), 33.1 (CH2), 31.0 (CH2), 30.8 (4 CH2), 30.5 (CH2), 26.6 (CH2), 23.7 (CH2), 14.5 (CH3).

HRMS (ESI): m/z [M + H]+ calcd for C18H40NO3: 318.3003; found: 318.3007.


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N-[(1S,2S,3R)-2,3-Dihydroxy-1-(hydroxymethyl)heptadecyl]hexacosanamide (3; Ceramide)

Pyridine (0.33 mL) was added to a soln of azide 15 (72 mg, 0.21 mmol) and Ph3P (110 mg, 0.42 mmol) in THF (1 mL). The flask was warmed to 60 °C, and the mixture was stirred for 12 h then gradually cooled down to r.t. Me(CH2 ) 24CO2H (108 mg, 0.27 mmol), 1H-1,2,3-benzotriazol-1-ol (51 mg, 0.38 mmol), EDC·HCl (72.5 mg, 0.38 mmol), and Et3N(30 μL, 0.21 mmol) were added sequentially, and the mixture was stirred for 12 h. The mixture was then filtered and washed with EtOAc (2 × 10 mL) and MeOH (2 × 10 mL) to give a white solid; yield: 77 mg (53%); mp 106–109 °C.

IR (KBr): 3315, 2918, 2850, 1643 cm–1

13C NMR (75 MHz, solid): δ = 182.7, 74.5, 72.4, 57.2, 55.1, 40.2 (2 C), 33.5 (32 C), 27.6, 25.0 (2 C), 15.2 (2 C).

HRMS (ESI): m/z [M – H]+ calcd for C44H88NO4: 694.6708; found: 694.6713.


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Acknowledgment

The authors thank the National Science Council of Taiwan (NSC99-2113-M-005-013-MY2 and NSC101-2113-M-005-006-MY2) and National Chung Hsing University for financial support. R.C.S. acknowledges the receipt of a doctoral fellowship from National Chung Hsing University. The authors thank Professor Der-Lii M. Tzou and Dr. Medel Manuel L. Zulueta of Academia Sinica, Taipei 115, Taiwan, for performing the solid-state NMR and for their helpful suggestions with regard to the writing of manuscript.

Supporting Information

    for this article is available online at http://www.thieme-connect.com.accesdistant.sorbonne-universite.fr/ejournals/toc/synthesis.
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Figure 1 The structures of α-galactosyl ceramide (1), d-ribo -phytosphingosine (2), and ceramide (3)
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Scheme 1 Retrosynthetic plan for α-galactosyl ceramide (1), d-ribo-phytosphingosine (2), and ceramide (3)
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Scheme 2 Preparation of α-galactosyl ceramide 1. Reagents and conditions: (a) 2,3-O-isopropylidene-d-lyxofuranose (8), TBAI, DIPEA, 4 Å MS, toluene, 65 °C; (b) LiHMDS, Me(CH2)12P+Ph3 Br – , THF, 0 °C; (c) H2, 10% Pd/C, MeOH, r.t.; (d) Tf2O, py, CH2Cl2, 0 °C then (Me2N)2C=NH2 + N3 –; (e) 1. Ph3P, py, THF, H2O, 60 °C, overnight, 2. Me(CH2)24CO2H, 1H-1,2,3-benzotriazol-1-ol, EDC, Et3N, CH2Cl2; (f) 0.4 M HCl, CH2Cl2–MeOH, r.t.; (g) H2 (60 psi), Pd(OH)2/C, MeOH–CHCl3.
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Scheme 3 Preparation of phytosphingosine (2) and ceramide (3). Reagents and conditions: (a) 8 M aq HCl, THF, 65%; (b) H2, Pd/C, MeOH–EtOAc, 70% (c) 1. py, Ph3P, THF; 2. Me(CH2)24CO2H, EDC, 1H-1,2,3-benzotriazol-1-ol, Et3N, DMF, 53% (two steps).