Unless otherwise noted, all materials and solvents were used as received from Adamas-beta® without further purification. NMR spectra were recorded on the Varian Mercury 500-MHz spectrometers. Chemical shifts are reported in parts per million (ppm) relative to MeOH-d ₄ , CDCl₃, or TMS signature chemical shifts. NMR data are presented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublet, and m = multiplet and/or multiple resonances), coupling constant in hertz (Hz), and integration. ¹³C data were derived from the HSQC spectrum. All NMR signals were assigned on the basis of ¹H NMR, COSY, and HSQC experiments. Mass spectra were recorded on a Shimadzu LCMS-IT-TOF mass spectrometer or a Bruker IT-TOF mass spectrometer. TLC analysis was performed on silica gel 60 F254 (Huang Hai Inc.) with detection by UV absorption (254 nm) when applicable and by spraying with a solution of (NH₄)₆Mo₇O₂₄·H₂O (25 g/L) in 5% sulfuric acid in ethanol, followed by charring. All reactions were carried out under an argon atmosphere.

(2S,3S,4R)-2-Azido-1-(((2,3-dimethylbutan-2-yl)dimethylsilyl)oxy) octadecane-3,4-diol ( 11 ). Compound 10 (2.2 g) and imidazole (2 equiv, 872 mg) were dissolved in anhydrous DMF, followed by dropwise addition of TDSCl (1.2 equiv, 1.51 ml) at 0°C. Stirring was continued until TLC (hexane/EtOAc, 1/1, v/v) indicated disappearance of starting material (∼30 min) and then quenched with methanol. The reaction mixture was diluted with EtOAc and then washed with NaHCO₃ and brine. The organic layer was dried (Na₂SO₄) and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexanes and EtOAc (from 50/10 to 30/10, v/v) to afford compound 11 (2.292 g, 60%). HRMS (ESI⁺) calcd for C₂₆H₅₅N₃NaO₃Si (M⁺+Na) 508.4013, found 508.4021. ¹H NMR (500 MHz, CDCl₃) δ 4.00 (dd, J = 10.7, 4.4 Hz, 1H, H-1^A), 3.91 (dd, J = 10.5, 4.8 Hz, 1H, H-1^B), 3.70 (S, 2H, H-3, H-4), 3.52 (d, J = 5.0 Hz, 1H, H-2), 1.19–1.40 (m, 27H, -C₁₃ H ₂₆-CH₃, TDS-CH), 0.93–0.81 (m, 15H, -C₁₃H₂₆-CH ₃, TDS -CH ₃), and 0.16 (s, 6H, -Si-CH ₃). ¹³C NMR (126 MHz, CDCl₃) δ 74.71, 72.51, 63.13, 63.13, 62.99, 62.55, 33.98, 32.07, 29.58, 25.62, 24.74, 22.55, 22.55, 20.06, 18.44, 18.30, and 14.05.

(2S,3S,4R)-2-azido-1-hydroxyoctadecane-3,4-diyl dibenzoate ( 7 ). DAMP (0.3 equiv, 204 mg) was added to a solution of 11 (2.7 g) in anhydrous pyridine (28 ml) and then cooled to 0°C. BzCl (8 equiv, 5.17 ml) was added drop-wise. Stirring was continued until TLC (hexane/EtOAc, 11/1, v/v) indicated disappearance of starting material (∼12 h). CH₃OH was added, and stirring was continued for 30 min. The mixture was concentrated under reduced pressure. The residue was diluted with CH₂Cl₂, washed with NaHCO₃ and brine, dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexanes and EtOAc to afford a pure product (3.43 g, 89%). The pure product (3.8 g) was dissolved in anhydrous THF, and HF-pyridine (20 equiv, 10 ml) was added drop-wise at 50°C. The reaction mixture was stirred at 50°C for 12 h until TLC analysis (hexane/EtOAc, 1/1, v/v) indicated completion of the reaction and then quenched with saturated NaHCO_3. The mixture was concentrated under reduced pressure, and then, the residue was diluted with EtOAc and washed with brine. The organic layer was collected, dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexanes and EtOAc (from 15/1 to 12/1, v/v) to afford compound 7 (2.53 g, 84%). HRMS (ESI⁺) calcd for C₃₂H₄₅N₃NaO₅ (M⁺+Na) 574.3359, found 574.3367. ¹H NMR (500 MHz, CDCl₃) δ 8.07–7.37 (m, 10H, CH aromatics), 5.60–5.49 (m, 2H, H-3, H-4), 3.99 (d, J = 8.9 Hz, 1H, H-1^A), 3.85–3.76 (m, 2H, H-1^B, H-2), 2.00–1.82 (m, 2H, -CH ₂-C₁₂H₂₄-CH₃), 1.23 (d, J = 10.2 Hz, 24H, -CH₂-C₁₂ H ₂₄-CH₃), and 0.87 (t, J = 6.9 Hz, 3H, -CH₂-C₁₂H₂₄-CH ₃). ¹³C NMR (126 MHz, CDCl₃) δ 133.67, 133.19, 130.01, 129.77, 129.52, 128.55, 128.30, 72.87, 63.10, 61.64, 29.40, 29.40, 25.50, 22.57, and 13.77.

p-Tolyl 2,3-di-O-benzoyl-4,6-di-O-di-tert-butylsilylidene-1-thio-D-galactopyranoside ( 6 ). Compound 6 was prepared according to previously reported procedures. HRMS (ESI⁺) calcd for C₃₅H₄₂O₇SNaSi (M⁺+Na) 657.2421, found 657.2429. ¹H NMR (500 MHz, CDCl₃) δ 8.14–7.35 (m, 14H, CH aromatics), 5.91 (t, J = 10.0 Hz, 1H, H-2), 5.19 (d, J = 9.8 Hz, 1H, H-3), 4.93–4.83 (m, 2H, H-1, H-4), 4.30 (q, J = 10.0 Hz, 2H, H-6), 3.62 (s, 1H, H-5), 2.31 (s, 3H, CH ₃ of STol), 1.15 (s, 9H, CH ₃ of DTBS), and 0.95 (s, 9H, CH ₃ of DTBS). ¹³C NMR (126 MHz, CDCl₃) δ 133.74, 133.25, 133.25, 133.25, 130.32, 130.07, 129.83, 129.58, 128.36, 128.36, 128.36, 87.83, 87.83, 75.37, 74.88, 70.25, 68.05, 68.05, 67.07, 27.27, 27.27, 27.27, 27.27, and 20.92.

ɑ-D-Galactopyranoside,(2S,3S,4R)-2-azido-3,4-bis(benzoyloxy) octadecyl 4, 6-O-[bis(1,1-dimethylethyl)silylene]-,2,3-dibenzoate ( 5 ).

Compounds 7 (1 equiv, 890 mg) and 6 (1.5 equiv, 1.54 g) were dissolved in CH₂Cl₂, dried by co-evaporation, pumped for about 1 h on cold nitrile, and then dissolved in anhydrous CH₂Cl₂. Freshly activated powdered molecular sieves (4 Å) were added, and the mixture was stirred for 10 min at room temperature, and then, NIS(2 equiv, 1.15 g) was added and cooled to 0°C. TfOH (0.3 equiv, 42.8 ul) was added, and the resulting reaction mixture was stirred for 6 h at room temperature and then quenched by the addition of TEA. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexanes and EtOAc (from 10/1 to 80/10, v/v) to afford 5 (957 mg, 56%).

Compound 7 (100 mg) was dissolved in 2.7 ml of acetone and 300 μL of water cooled at 0°C, and NBS was added (2 equiv, 56 mg). The resulting reaction mixture was stirred for 30 min at room temperature before being quenched with saturated NaHCO₃ and saturated Na₂S₂O₃ solution. Acetone was removed under reduced pressure, and the remaining aqueous mixture was extracted with EtOAc. Combined extracts were sequentially washed with water and brine. The organic layer was collected, dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexanes and EtOAc (from 60/10 to 40/10, v/v) to afford pure product (55 mg, 66%). The pure product (86 mg) and 2,2,2-trifluoro-N-phenylethanimidoyl chloride (1.5 equiv, 39.6 ul) were dissolved in CH₂Cl₂ and cooled to 0°C; then, NaH (1.5 equiv, 9.76 mg) was added. The resulting reaction mixture was stirred for 30 min at room temperature. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexanes and EtOAc (from 10/1 to 15/1, v/v) to afford N-phenyl donor (102 mg, 90%). Compound 6 (1 equiv, 65 mg) and N-phenyl donor (1.5 equiv, 124 mg) were dissolved in CH₂Cl₂, dried by co-evaporation, pumped for about 1 h on cold nitrile, and then dissolved in anhydrous CH₂Cl₂. Freshly activated powdered molecular sieves (4 Å) were added, and the mixture was stirred for 10 min at room temperature and then cooled to 0°C. TfOH (0.3 equiv, 42.8 ul) was added, and the resulting reaction mixture was stirred for 40 min at 0°C and then quenched by the addition of TEA. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexanes and EtOAc (from 10/1 to 80/10, v/v) to afford 5 (78 mg, 62%). HRMS (ESI⁺) calcd for C₆₀H₇₉N₃NaO₁₂Si (M⁺+Na) 1084.5433, found 1084.5425. ¹H NMR (500 MHz, CDCl₃) δ 8.03–7.27 (m, 20H, CH aromatics), 5.79 (d, J = 10.8 Hz, 1H, H-2), 5.54 (d, J = 10.5 Hz, 1H, H-3), 5.48 (d, J = 5.9 Hz, 2H, H-9, H-10), 5.31 (d, J = 3.6 Hz, 1H, H-1), 4.87 (s, 1H, H-4), 4.29–4.14 (m, 2H, H-6), 3.96 (s, 1H, H-8), 3.88 (s, 1H, H-5), 3.67 (t, J = 9.5 Hz, 2H, H-7), 1.80 (s, 2H, -CH ₂-C₁₂H₂₄-CH₃), 1.27–1.09 (m, 24H, -CH₂-C₁₂ H ₂₄-CH₃), and 0.87 (t, J = 6.6 Hz, 3H, -CH₂-C₁₂H₂₄-CH ₃). ¹³C NMR (126 MHz, CDCl₃) δ 133.25, 133.01, 132.76, 129.59, 128.37, 128.12, 128.12, 97.11, 72.69, 70.74, 70.49, 67.89, 67.56, 67.07, 66.59, 60.97, 29.44, 29.22, 27.03, and 26.78.

[(1S,2S,3R)-1-[[[2,3-Bis-O-(Dibenzoyloxy)-ɑ-D-galactopyranosyl]oxy]methyl]-2,3-bis(Dibenzoyloxy)heptadecyl]hexacosanamide ( 14 ). Compound 5 (1.5 g) was dissolved in CH₂Cl₂/MeOH (1:1, 50 ml) and stirred with a tablespoon Pd/C and 1M HCl (1.2 equiv) under H₂ atmosphere at room temperature for 8 h. After the disappearance of starting material monitored by TLC, the reaction mixture was filtered through a 0.22-um nylon microporous membrane. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexanes and EtOAc (from 60/10 to 30/10, v/v) to give a pure product. Equimolar hydrochloric acid was added when the pure product was concentrated (1.16 g, 80%). Ceric acid (1.5 equiv, 330 mg), EDC (7 equiv, 734 mg), and HOBT (7 equiv, 524 mg) were dissolved in anhydrous THF/DMF (1:1, 10 ml) and then cooled to 0°C. DAMP (0.1 equiv, 6.77 mg) was added, and the resulting reaction mixture was stirred for 30 min at room temperature. The pure product (1 equiv, 574 mg) was dissolved in anhydrous THF (5 ml), DIPEA (3 equiv, 275 ul) was added, the step 1 reaction mixture was added dropwise into the step 2 reaction mixture, and the reaction flask in step 1 was washed with 2 ml of THF, stirring the reaction overnight at room temperature. Then, it was quenched with water and concentrated under reduced pressure. The residue was diluted with CH₂Cl₂, washed with NaHCO₃, water, and brine. The organic layer was collected, dried (Na₂SO₄), and filtered, and the filtrate was concentrated under reduced pressure. The residue (784 mg) was diluted in anhydrous THF (15 ml). HF-Pyridine (20 equiv, 998 ul) was added dropwise, and the resulting reaction mixture was stirred for 4 h at room temperature and then quenched with NaHCO₃. The mixture was concentrated under reduced pressure until only the aqueous layer remained. The residue was diluted with EtOAc and washed with brine. The organic layer was collected, dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexanes and EtOAc (from 60/10 to 30/10, v/v) to afford 14 (353 mg, 50% for two steps). HRMS (ESI⁺) calcd for C₇₈H₁₁₅NNaO₁₃ (M⁺+Na) 1296.8368, found 1296.8362. ¹H NMR (500 MHz, CDCl₃) δ 8.03–7.21 (m, 20H, CH aromatics), 7.26 (s, 1H, -NH), 5.81 (d, J = 9.9 Hz, 1H, H-9), 5.59 (s, 2H, H-2, H-3), 5.27–5.14 (m, 2H, H-1, H-10), 4.56 (t, J = 10.3 Hz, 1H, H-8), 4.37 (s, 1H, H-4), 4.33 (t, J = 5.9 Hz, 1H, H-5), 4.06 (dd, J = 12.6, 3.5 Hz, 1H, H-7^A), 4.01 (s, 2H, H-6), 3.60 (d, J = 12.4 Hz, 1H, H-7^B), 2.31 (t, J = 7.5 Hz, 2H, -CH ₂-C₂₂H₄₄-CH₃), 1.92 (d, J = 8.5 Hz, 2H, -CH ₂-C₁₂H₂₄-CH₃), 1.17 (m, 68H, -CH₂-C₁₂ H ₂₄-CH₃, -CH₂-C₂₂ H ₄₄-CH₃), and 0.87 (t, J = 6.9 Hz, 6H, -CH₂-C₁₂H₂₄-CH ₃, -CH₂-C₂₂H₄₄-CH ₃). ¹³C NMR (126 MHz, CDCl₃) δ 133.43, 133.19, 133.19, 133.19, 133.19, 129.77, 129.77, 129.77, 129.77, 129.77, 129.52, 129.52, 128.55, 128.30, 128.30, 128.30, 128.30, 128.30, 128.30, 99.49, 74.58, 72.63, 71.65, 71.16, 70.92, 69.21, 68.72, 64.12, 62.37, 48.69, 36.49, 29.40, 29.40, 27.45, and 13.77.

α-Galactosylceramide. Compound 14 (1 equiv, 100 mg) was dissolved in anhydrous MeOH/DCM (1:1, 2 ml), and 10 mg of solid sodium metal was added and dissolved in 1 ml of anhydrous methanol; the resulting reaction mixture was stirred for 4 h at room temperature. Cationic resin was added to neutralize, filtered, and then concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel EtOAc/pyridine to afford KRN7000 (48.5 mg, 72%). HRMS (ESI⁺) calcd for C₅₀H₉₉NNaO₉ [M + Na]⁺ 880.7320, found 880.7329. ¹H NMR (500 MHz, C₅H₅N) δ 8.56 (d, J = 8.4 Hz, 1H, -NH), 5.60 (d, J = 4.0 Hz, 1H, H-1), 5.29 (d, J = 8.7 Hz, 1H, H-8), 4.69 (q, J = 5.5 Hz, 2H, H-2, H-7^A), 4.60–4.50 (m, 2H, H-4, H-5), 4.49–4.38 (m, 3H, H-3, H-6, H-7^B), 4.34 (d, J = 8.4 Hz, 2H, H-9, H-10), 1.93 (s, 2H, -CH ₂-C₂₂H₄₄-CH₃), 1.83 (dd, J = 9.5, 5.5 Hz, 2H, -CH ₂-C₁₂H₂₄-CH₃), 1.75–1.07 (m, 68H, -CH₂-C₁₂ H ₂₄-CH₃, -CH₂-C₂₂ H ₄₄-CH₃), and 0.88 (t, J = 6.6 Hz, 6H, -CH₂-C₁₂H₂₄-CH ₃, -CH₂-C₂₂H₄₄-CH ₃). ¹³C NMR (126 MHz, C₅H₅N) δ 101.29, 76.39, 72.72, 72.24, 71.26, 70.77, 70.04, 68.33, 62.47, 51.23, 34.14, 26.08, 22.42, and 13.87.

[(1S,2S,3R)-1-[[[6-O-[(4-Methylphenyl)sulfonyl]-2,3-bis-O-(Dibenzoyloxy)-ɑ-D-galactopyranosyl]oxy]methyl]-2,3-bis(Dibenzoyloxy)heptadecyl]hexacosanamide ( 15 ). Compound 14 (1.0 equiv, 250 mg, 196 umol) was dissolved in anhydrous CH₂Cl₂ (8 ml), and TsCl (3 equiv, 112 mg, 588 umol) was added, followed by Et₃N (137 ul, 5 equiv, 981 umol) at 0°C. The reaction mixture was raised up to room temperature and stirred for 5 h. Saturated aqueous NH₄Cl was slowly added to neutralize. Two layers were separated, and the aqueous layer was extracted with CH₂Cl₂. The organic extracts were combined and dried over anhydrous Na₂SO₄, and then concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel hexanes/EtOAc (from 50/10 to 30/10, v/v) to afford 15 (174.9 mg, 62%, white solid). HRMS (ESI⁺) calcd for C₈₅H₁₂₂NO₁₅S (M⁺+H) 1428.8457, found 1428.8462. ¹H NMR (500 MHz, CDCl₃) δ 8.03–7.13 (m, 24H, CH aromatics), 6.61 (d, J = 9.4 Hz, 1H, -NH), 5.68–5.54 (m, 3H, H-2, H-3, H-9), 5.34 (td, J = 6.6, 3.1 Hz, 1H, H-10), 5.07 (d, J = 3.6 Hz, 1H, H-1), 4.63 (tt, J = 8.6, 3.7 Hz, 1H, H-8), 4.33 (s, 1H, H-5), 4.26 (m, J = 4.8 Hz, 1H, H-4), 4.17 (dq, J = 9.9, 4.8 Hz, 2H, H-6), 3.79 (dd, J = 10.9, 3.2 Hz, 1H, H-7^A), 3.56 (dd, J = 10.9, 4.0 Hz, 1H, H-7^B), 2.42 (s, 3H, CH ₃ of Ts), 2.24 (q, J = 7.0 Hz, 2H, -CH ₂-C₂₂H₄₄-CH₃), 1.86 (q, J = 7.0 Hz, 2H, -CH ₂-C₁₂H₂₄-CH₃), 1.35–1.16 (m, 68H, -CH₂-C₁₂ H ₂₄-CH₃, -CH₂-C₂₂ H ₄₄-CH₃), and 0.92–0.83 (m, 6H, -CH₂-C₁₂H₂₄-CH ₃, -CH₂-C₂₂H₄₄-CH ₃). ¹³C NMR (126 MHz, CDCl₃) δ 133.19, 133.19, 132.94, 130.01, 129.77, 129.77, 129.77, 129.77, 129.52, 128.55, 128.30, 128.30, 128.06, 128.06, 128.06, 97.53, 73.85, 72.14, 70.18, 68.11, 67.99, 67.99, 67.50, 67.25, 48.21, 36.49, 29.40, 29.40, 28.43, 21.34, and 13.77.

[(1S,2S,3R)-1-[[[6-Azido-6-deoxy-2,3-bis-O-(Dibenzoyloxy)-β-D-galactopyranosyl]oxy]methyl].

-2,3-bis(Dibenzoyloxy)heptadecyl]hexacosanamide ( 4 ). Compound 15 (1 equiv, 108 mg) was dissolved in anhydrous DMF (4 ml), followed by the addition of NaN₃ (3 equiv, 14.7 mg). The reaction mixture was stirred at 80°C overnight and cooled to room temperature. A measure of 40 ml of H₂O was added, and two layers were separated. The aqueous layer was extracted with CH₂Cl₂. The organic extracts were combined and dried over anhydrous Na₂SO₄, and then concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel hexanes/EtOAc (from 50/10 to 30/10, v/v) to afford 4 (85 mg, 86%). HRMS (ESI⁺) calcd for C₇₈H₁₁₄N₄NaO₁₂S (M⁺+Na) 1321.8433, found 1321.8439. ¹H NMR (500 MHz, C₅H₅N) δ 8.24–7.26 (m, 20H, CH aromatics), 7.82 (d, J = 5.8 Hz, 1H, -NH), 6.44 (dd, J = 10.8, 3.9 Hz, 1H, H-2), 6.27 (d, J = 4.7 Hz, 1H, H-9), 6.16–6.05 (m, 2H, H-3, H-10), 5.73 (d, J = 3.8 Hz, 1H, H-1), 5.44 (d, J = 8.7 Hz, 1H, H-8), 4.81 (s, 1H, H-4), 4.60 (dd, J = 16.5, 6.4 Hz, 2H, H-5, H-7^A), 4.15–3.99 (m, 2H, H-6^A, H-7^B), 3.62 (dd, J = 12.7, 4.6 Hz, 1H, H-6^B), 2.50 (t, J = 7.3 Hz, 2H, -CH ₂-C₂₂H₄₄-CH₃), 2.29 (td, J = 10.3, 5.5 Hz, 2H, -CH ₂-C₁₂H₂₄-CH₃), 1.38–1.18 (m, 68H, -CH₂-C₁₂ H ₂₄-CH₃, -CH₂-C₂₂ H ₄₄-CH₃), and 0.95–0.75 (m, 6H, -CH₂-C₁₂H₂₄-CH ₃, -CH₂-C₂₂H₄₄-CH ₃). ¹³C NMR (126 MHz, C₅H₅N) δ 133.50, 133.25, 133.25, 129.84, 129.59, 128.86, 128.62, 128.62, 128.62, 128.37, 97.85, 73.92, 73.92, 72.21, 72.21, 70.74, 69.28, 67.93, 67.57, 51.57, 49.25, 36.31, 31.91, 29.47, 29.47, 28.74, 22.39, and 13.84.

N-[(1S,2S,3R)-1-[[(6-Azido-6-deoxy-ɑ-D-galactopyranosyl)oxy]methyl]-2,3-dihydroxyheptadecyl]hexacosanamide ( 1 ). Compound 4 (1 equiv, 118 mg) was dissolved in anhydrous MeOH/pyridine (1:1, 4 ml), 10 mg of solid sodium metal was added and dissolved in 1 ml of anhydrous methanol, and the resulting reaction mixture was stirred for 4 h at room temperature. Cationic resin was added to neutralize, was filtered, and then concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel EtOAc/pyridine (from 100/1 to 90/1, v/v) to afford 1 (43.3 mg, 54%). HRMS (MALDI-TOF) calcd for C₅₀H₉₈N₄NaO₈ [M + Na]⁺ 905.7385, found 905.7391. ¹H NMR (500 MHz, C₅H₅N) δ 8.57 (d, J = 8.8 Hz, 1H, -NH), 5.55 (d, J = 3.9 Hz, 1H, H-1), 5.31 (s, 1H, H-8), 4.73 (dd, J = 10.8, 5.5 Hz, 1H, H-7^A), 4.61 (dd, J = 10.0, 4.0 Hz, 1H, H-2), 4.42–4.31 (m, 4H, H-3, H-5, H-7^A, H-10), 4.26 (d, J = 3.3 Hz, 1H, H-4), 3.99 (dd, J = 12.7, 8.2 Hz, 1H, H-6^A), 3.62 (dd, J = 12.6, 4.7 Hz, 1H, H-6^B), 2.47 (d, J = 4.0 Hz, 2H, -CH ₂-C₂₂H₄₄-CH₃), 1.68–1.63 (m, 2H, -CH ₂-C₁₂H₂₄-CH₃), 1.35–1.14 (m, 68H, -CH₂-C₁₂ H ₂₄-CH₃, -CH₂-C₂₂ H ₄₄-CH₃), and 0.86 (dt, J = 17.2, 6.9 Hz, 6H, -CH₂-C₁₂H₂₄-CH ₃, -CH₂-C₂₂H₄₄-CH ₃). ¹³C NMR (126 MHz, C₅H₅N) δ 100.95, 76.29, 72.14, 70.67, 70.43, 69.45, 68.47, 68.11, 51.87, 51.38, 50.65, 36.24, 31.85, 30.38, 29.40, 29.40, 22.32, 18.90, and 13.53.

N-[(1S,2S,3R)-1-[[(6-Amino-6-deoxy-ɑ-D-galactopyranosyl)oxy]methyl]-2,3-dihydroxyheptadecyl]hexacosanamide ( 2 ). Compound 1 (1 equiv, 26 mg) was dissolved in CH₂Cl₂/MeOH (1:1, 2 ml) and was stirred with a tablespoon of Pd/C under H₂ atmosphere at room temperature for 5 h. After the disappearance of starting material monitored by TLC, the reaction mixture was filtered through a 0.22-um nylon microporous membrane. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexanes/EtOAc (from 50/10 to 10/10, v/v, added 2% TEA) to afford 2 (21.9 mg, 87%). HRMS (ESI⁺) calcd for C₅₀H₁₀₁N₂O₈ (M⁺+H) 857.7480, found 857.7481. ¹H NMR (500 MHz, C₅H₅N) δ 8.56 (d, J = 8.5 Hz, 1H, -NH), 5.27 (d, J = 5.0 Hz, 2H, H-1, H-4), 5.18 (s, 1H, H-8), 4.84 (d, J = 5.4 Hz, 1H, H-5), 4.76 (dd, J = 10.4, 4.2 Hz, 2H, H-6), 4.59 (s, 1H, H-2), 4.43–4.34 (m, 3H, H-3, H-7^A, H-9), 4.25 (d, J = 8.9 Hz, 1H, H-10), 4.19 (d, J = 9.4 Hz, 1H, H-7^B), 2.40 (t, J = 7.4 Hz, 2H, -CH ₂-C₂₂H₄₄-CH₃), 1.91 (d, J = 9.7 Hz, 2H, -CH ₂-C₁₂H₂₄-CH₃), 1.36–1.14 (m, 68H, -CH₂-C₁₂ H ₂₄-CH₃, -CH₂-C₂₂ H ₄₄-CH₃), and 0.87 (d, J = 7.5 Hz, 6H, -CH₂-C₁₂H₂₄-CH ₃, -CH₂-C₂₂H₄₄-CH ₃ ). ¹³C NMR (126 MHz, C₅H₅N) δ 98.10, 82.64, 78.25, 75.96, 72.45, 70.78, 69.64, 69.29, 51.89, 36.61, 31.87, 29.76, 29.58, 26.24, 22.55, and 13.95.

[(1S,2S,3R)-1-[[(6-(Pent-4-ynamidomethyl)-6-deoxy-ɑ-Dgalactopyranosyl)oxy]methyl]-2,3 dihydroxyheptadecyl]hexacosanamide ( 3 ). Compound 2 (1 equiv, 12.6 mg) was dissolved in anhydrous DMF (2 ml) and was stirred with 4-pentynoic acid (5 equiv, 14.3 mg) after NHS activation. HATU (1.5 equiv, 8.38 mg) and TEA (10 equiv, 20.5 ul) were added, stirring the reaction overnight at room temperature. Then, it was quenched with methanol and concentrated in vacuo, and the residue was purified by SePhadexLH-20 using CH₂Cl₂/methanol (1:2, v/v) to afford 3 (12.8 mg, 93%). HRMS (ESI⁺) calcd for C₅₅H₁₀₄N₂NaO₉ (M⁺+Na) 959.7742, found 959.7746. ¹H NMR (500 MHz, C₅H₅N) δ 9.06 (s, 1H, -NH), 8.61 (d, J = 8.5 Hz, 1H, -NH), 5.51 (d, J = 4.1 Hz, 1H, H-1), 5.25 (s, 1H, H-8), 4.62 (dd, J = 16.2, 9.4 Hz, 2H, H-2, H-7^A), 4.50 (t, J = 6.5 Hz, 1H, H-5), 4.32 (d, J = 14.2 Hz, 5H, H-3, H-4, H-7^B, H-9, H-10), 4.28–4.15 (m, 1H, H-6^A), 3.94 (d, J = 6.7 Hz, 1H, H-6^B), 3.00–2.55 (m, 4H, H-11, H-12), 2.48 (q, J = 7.9 Hz, 2H, -CH ₂-C₂₂H₄₄-CH₃), 1.93 (s, 2H, -CH ₂-C₁₂H₂₄-CH₃), 1.88–1.04 (m, 68H, -CH₂-C₁₂ H ₂₄-CH₃, -CH₂-C₂₂ H ₄₄-CH₃), and 0.88 (t, J = 6.8 Hz, 9H, -CH₂-C₁₂H₂₄-CH ₃, -CH₂-C₂₂H₄₄-CH ₃ ). ¹³C NMR (126 MHz, C₅H₅N) δ 101.05, 76.45, 72.23, 70.83, 70.30, 69.77, 68.02, 50.97, 41.14, 40.87, 36.39, 35.16, 33.93, 32.00, 30.42, 29.72, 29.54, 26.03, 22.51, 14.78, and 13.90.

Bald/C mice were purchased from Sibeifu Co., LTD. The mice were kept under specific pathogen-free conditions and used at 7 weeks of age. KRN7000 and synthesized glycolipidases (1.0 mg) were dissolved in dimethyl sulfoxide (DMSO, 1.0 ml) at 80°C. After 30 min at 80°C, the solutions were cooled to room temperature and diluted to 200 μg/ml with phosphate-buffered saline (PBS) containing 0.5% Tween 20 (polymethylene sorbitan monolaurate). The obtained solutions were diluted to 10 μg/ml with PBS just before injecting into the mice. Each glycolipid solution (10 μg/ml, 200 μL) was administered intravenously. Peripheral blood was collected from the retro-orbital plexus of mice at 3, 6, 12, 24, 36, 48, and 60 h using capillary tubes, and plasma was prepared. The animal protocol involved in this research was approved (HNUAUCC-2021–00048).

The cytokine concentrations in plasma were measured by using the mouse ELISA kit (Dekewe company) for IFN-γ and IL-4.