An Efficient and Concise Synthesis of α-Galactosylceramide

Abstract

A concise and stereoselective synthesis of α-galactosylceramide (α-GalCer) is described. The key features of the synthetic strategy are the use of a phytosphingosine in which the amine is masked as a tetrachlorophthalimide and the diol as an isopropylidene acetal, and the galactosyl donor is protected as a 4,6-benzylidene to improve the α selectivity of the glycosylation reaction. The pattern of protecting groups on the donor and the acceptor have proven to give an excellent match of reactivity, allowing the glycosylation reaction to take place stereoselectively. The overall synthesis gave α-GalCer in good yields and in few steps.

Key words

glycosylation - phytosphingosine - protecting groups - galactosylceramide - glycolipids - asymmetric synthesis

Publication History

Received: 14 September 2020

Accepted after revision: 20 October 2020

Accepted Manuscript online:
20 October 2020

Article published online:
05 November 2020

• References and Notes

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• 10 (2S,3S,4R)-3,4-O-Isopropylidene-2-(tetrachlorophthalimido)octadecan-1-ol (3) Camphorsulfonic acid (0.3 g, 1.29 mmol) was added to a mixture of compound 2 (3.0 g, 5.14 mmol) in 2,2-dimethoxypropane (60 mL), and the mixture was stirred at r.t. for 30 min. The mixture was diluted with EtOAc(100 mL) and washed with aq NaHCO₃ (2 × 100 mL) and brine, then dried (MgSO₄) and concentrated under vacuum. The crude product was redissolved in 10:1 CH₂Cl₂–MeOH (66 mL) and 60% aq AcOH (0.6 mL) was added. The mixture was warmed at 60 °C for 90 min then diluted with EtOAc (100 mL), washed with aq NaHCO₃ (2 × 100 mL) and brine, dried (MgSO₄), and concentrated under vacuum. The crude product was purified by dry column vacuum chromatography²² (cyclohexane–EtOAc, 10:0 to 8:2) to give a yellow solid; yield: 2.57 g (80%); mp 86–88 °C, [α]_D ²⁰ –5.7 (c 1.0 CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ = 4.96 (dd, J = 10.0, 5.4 Hz, 1 H), 4.48 (ddd, J = 10.0, 8.6, 5.1 Hz, 1 H), 4.18–3.75 (m, 3 H), 2.05 (br s, 1 H), 1.67–1.42 (m, 5 H), 1.37 (s, 3 H), 1.35–1.06 (m, 24 H), 0.89 (t, J = 6.9 Hz, 3 H). ¹³C NMR (100 MHz, CDCl₃): δ = 163.8, 140.5, 129.9, 127.1, 108.5, 77.6, 74.1, 61.4, 52.9, 31.9, 29.7, 29.6, 29.54, 29.5, 29.42, 29.36, 29.0, 28.2, 26.2, 25.8, 22.7, 14.1. HRMS (ESI): m/z [M – H]^– calcd for C₂₉H₄₀Cl₄NO₅: 624.16311; found: 624.16376.
• 11 Thimon C, Panza L, Morin C. Synlett 2003; 1399
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• 13 (2S,3S,4R)-3,4-O-Isopropylidene-2-(tetrachlorophthalimido)octadecyl 2,3-Di-O-benzyl-4,6-O-benzylidene-α-d-galactopyranoside (5) Triflic anhydride (1.7 mL, 10 mmol) was added to a solution of dimethyl disulfide (1 mL, 11.3 mmol) in anhyd CH₂Cl₂ (7.5 mL) at −10 °C, and the mixture was stirred for 30 min to give a 1.0 M solution of Me₂S₂–Tf₂O. Compound 3 (1.19 g, 2.15 mmol), compound 4 (0.89 g, 1.44 mmol), and 2,6-di-tert-butyl-4-methylpyridine (0.59 g, 2.88 mmol) were dissolved in anhyd THF (13 mL) under argon, and 4 Å molecular sieves were added. The mixture was stirred at r.t. for 1 h, then cooled to −10 °C in a salt–ice bath before adding the 1.0 M Me₂S₂–Tf₂O (2.9 mL) and stirring for 30 min. The reaction was quenched with Et₃N (0.7 mL) and the mixture was diluted with CH₂Cl₂ (20 mL). The organic layer was washed with H₂O (2 × 40 mL) and brine, dried (MgSO₄), and concentrated. The residue was purified by flash chromatography [silica gel, cyclohexane–EtOAc (9:1)] to give a white solid; yield: 1.19 g (78%; on changing the ratio of donor 2 to acceptor 3 to 1:1.5, the yield increased to 90%); mp 87–89 °C, [α]_D ²⁰ +15.9 (c 1.0 CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ = 7.54–7.46 (m, 2 H), 7.45–7.26 (m, 8 H), 7.23–7.14 (m, 3 H), 6.98 (dd, J = 6.6, 3.0 Hz, 2 H), 5.47 (s, 1 H), 4.98 (dd, J = 10.3, 5.3 Hz, 1 H), 4.90 (d, J = 3.3 Hz, 1 H), 4.78–4.56 (m, 3 H), 4.36 (dd, J = 12.5 Hz, 2 H), 4.29–3.88 (m, 7 H), 3.83 (dd, J = 10.1, 3.3 Hz, 1 H), 3.69 (s, 1 H), 1.60 (m, 2 H), 1.51 (s, 3 H), 1.39 (s, 3 H), 1.35–0.95 (m, 24 H), 0.91 (t, J = 6.9 Hz, 3 H). ¹³C NMR (100 MHz, CDCl₃): δ = 140.1, 138.8, 138.2, 137.8, 129.4, 128.8, 128.3, 128.08, 128.06, 127.7, 127.6, 127.3, 127.1, 126.3, 126.1, 108.6, 101.0, 99.1, 77.6, 76.2, 75.9, 74.9, 73.5, 72.6, 69.4, 66.4, 63.1, 51.4, 31.9, 29.7, 29.6, 29.52, 29.47, 29.4, 29.0, 28.4, 26.2, 25.9, 22.7, 14.1. HRMS (ESI): m/z [M + Na]⁺ calcd for C₅₆H₆₇Cl₄NNaO₁₀: 1078.33873; found: 1078.33862.
• 14 (2S,3S,4R)-2-(Hexacosanoylamino)-3,4-O-isopropylideneoctadecyl 2,3-Di-O-benzyl-4,6-O-benzylidene-α-d-galactopyranoside (7) Compound 5 (1.0 g, 0.95 mmol) was dissolved in 1:1 EtOH–THF (10 mL) and ethane-1,2-diamine (0.32 mL) was added. The mixture was warmed at 60 °C for 3 h, and then the solvent was removed under vacuum. The crude product was purified on a short pad of silica, eluting with cyclohexane–EtOAc (7:3 then 5:5). Amine 6 was obtained in quantitative yield and directly used for the next step. Amine 6 (0.74 g, 0.95 mmol) was dissolved in CH₂Cl₂ (22 mL), and the solution was added to a solution previously prepared by dissolving hexacosanoic acid (0.45 g, 1.1 mmol), DIPEA (0.37 g, 2.8 mmol), EDCI (0.36 g, 1.89 mmol), and HOBt (0.26 g, 1.89 mmol) in DMF (11 mL). The resulting solution was stirred overnight at 40 °C and then allowed to cool to r.t. The solution was diluted with Et₂O and washed with 1 N HCl, sat. aq NaHCO₃, H₂O, and brine, then dried (MgSO₄) and concentrated under vacuum. The crude product was purified by crystallization from EtOAc and collected by filtration to give a white solid; yield: 0.97 g (88%); mp 110–112 °C, [α]_D ²⁰ +73.8 (c 1.0 CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ = 7.56 (dd, J = 7.6, 1.9 Hz, 2 H), 7.48–7.26 (m, 13 H), 5.81 (d, J = 9.0 Hz, 1 H), 5.52 (s, 1 H), 5.18 (d, J = 3.5 Hz, 1 H), 4.91 (d, J = 11.2 Hz, 1 H), 4.80 (s, 2 H), 4.69 (d, J = 11.2 Hz, 1 H), 4.30–4.26 (m, 2 H), 4.15–3.93 (m, 6 H), 3.88 (dd, J = 11.3, 3.1 Hz, 1 H), 3.77–3.62 (m, 2 H), 2.03 (dt, J = 12.0, 7.3 Hz, 2 H), 1.98–1.45 (m, 4 H), 1.43 (s, 3 H), 1.36 (s, 3 H), 1.37–1.28 (m, 68 H), 0.91 (t, J = 6.8 Hz, 6 H).¹³C NMR (100 MHz, CDCl₃): δ = 172.4, 138.6, 138.4, 137.8, 128.9, 128.43, 128.35, 128.14, 128.09, 127.9, 127.8, 127.7, 127.6, 126.3, 107.9, 101.1, 99.1, 77.7, 76.1, 75.9, 75.8, 74.3, 74.0, 71.5, 69.5, 67.8, 62.8, 48.8, 36.9, 31.9, 29.8, 29.72, 29.68, 29.62, 29.5, 29.4, 28.8, 28.0, 26.6, 25.9, 25.8, 22.7, 14.1. HRMS (ESI): m/z [M + H]⁺ calcd C₇₄H₁₂₀NO₉: 1166.89631; found: 1166.89575.

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• 19 (2S,3S,4R)-2-Hexacosanoylamino-3,4-dihydroxyoctadecyl 2,3-Di-O-benzyl-α-d-galactopyranoside (8) To a solution of 7 (0.95 g, 0.81 mmol) in CH₂Cl₂ (228 mL) and MeOH (45 mL) was added 4 M HCl in 1,4-dioxane (4.5 mL) at 0 °C, and the mixture was stirred at r.t. for 90 min. Et₃N (5 mL) was slowly added and then the mixture was washed with H₂O (2 × 100 mL) and brine, dried (MgSO₄), and concentrated under vacuum. The residue was purified by flash chromatography [silica gel, PE–i-PrOH (9:1)] to give a white solid; yield: 0.68 g (80%); mp 76–78 °C, [α]_D ²⁰ +49.9 (c 1.0 CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ = 7.43–7.30 (m, 10 H), 6.40 (d, J = 8.3 Hz, 1 H), 4.93 (d, J = 3.6 Hz, 1 H), 4.87 (d, J = 11.7 Hz, 1 H), 4.78 (d, J = 11.5 Hz, 1 H), 4.74 (s, 1 H), 4.70 (d, J = 11.7 Hz, 1 H), 4.28 (dd, J = 8.1, 3.5 Hz, 1 H), 4.12 (d, J = 2.6 Hz, 1 H), 3.98–3.72 (m, 7 H), 3.51 (t, J = 5.2 Hz, 2 H), 2.62–2.10 (br m, 4 H), 2.19 (t, J = 7.9 Hz, 2 H), 1.63 (m, 3 H), 1.54–1.09 (m, 69 H), 0.90 (t, J = 6.8 Hz, 6 H). ¹³C NMR (100 MHz, CDCl₃): δ = 173.4, 137.7, 137.6, 128.62, 128.57, 128.13, 128.11, 128.06, 127.8, 98.7, 77.9, 76.2, 75.2, 74.2, 73.2, 72.4, 69.9, 68.4, 62.8, 49.6, 36.81, 31.9, 29.7, 29.6, 29.44, 29.38, 29.34, 25.8, 22.7, 14.1. HRMS (ESI): m/z [M + H]⁺ calcd for C₆₄H₁₁₂NO₉: 1038.83371; found: 1038.83401.
• 20 (2S,3S,4R)-2-Hexacosanoylamino-3,4-dihydroxyoctadecyl-α-d-galactopyranoside (1) A mixture of compound 8 (430 mg, 0.41 mmol) and Pearlman’s catalyst (86 mg) in EtOH (15 mL) and THF (15 mL) was stirred under H₂ for 3 h. The catalyst was removed by filtration through a Celite pad, and the product was recovered by washing with warm EtOH. Evaporation of the solvent and crystallization from EtOH–H₂O (92:8) gave a white solid; yield: 301 mg (85%). Spectroscopic data were consistent with the reported values for α-GalCer (see ref. 21).
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