Phosphorylated glycosphingolipids essential for cholesterol mobilization in C. elegans

Supplementary Note Phosphorylated glycosphingolipids essential for cholesterol mobilization in C. elegans Sebastian Boland, Ulrike Schmidt, Vyacheslav Zagoriy, Julio L. Sampaio, Raphael Fritsche, Regina Czerwonka, Tilo Lübken, Jakob Reimann, Sider Penkov, Hans-Joachim Knölker & Teymuras V. Kurzchalia Inventory 1. Experimental procedures for the syntheses of mmpegc-c22 and d17iso-glccer p. 2 and corresponding NMR-spectra p. 17 2. Supplementary References p. 38 1

1. Experimental procedures for the synthesis of mmpegc-c22 (1) and d17iso-glccer (11) and corresponding NMR-spectra General: All reactions were carried out using dry solvents in oven-dried glassware under an argon atmosphere, unless stated otherwise. Silica gel 60 M, 0.04 0.063 mm, was purchased from Macherey-Nagel. NMR spectra were recorded on Bruker AC 300-P, Bruker DRX 500 and Avance III 600 spectrometers, chemical shifts δ are reported in ppm with the solvent signal as internal standard. Coupling constants were determined assuming first-order spin-spin coupling. The multiplicity of the carbon signals was determined with the help of DEPT 135 spectra. Assignment of the 1 H NMR and 13 C NMR signals was achieved using the 2D NMR methods COSY, HSQC, HMBC, and NOESY. The homonuclear 1 H- 1 H shift-correlated 2D NMR spectrum ( 1 H- 1 H-COSY) of the isolated compound was recorded on a Bruker AV-III NMR spectrometer equipped with a Bruker 5 mm Z gradient probe (PABBI) operating at 600.16 MHz using the cosygpqf pulse sequence with spectral widths of 6009 Hz (10 ppm). The spectrum was collected with 2048 320 data points with 32 scans per experiment (NS) and a relaxation delay (d1) of 2.0 s at 295.0 K (21.8 C). In t2, data were zero filled to 4096 data points and multiplied with a sine window function before Fourier transformation. In t1, 1024 points were linear forward predicted using 128 coefficients, zero filled to 4096 data points, and a sine window function was applied before 2nd Fourier transformation. The baseline in both directions was adjusted and a background was subtracted. Chemical shifts (δ) are given in ppm relative to TMS. The residual solvent signal of CHD 2 OD (3.380 ppm) was used as internal reference. The COSY spectrum of the synthesized compound was collected with 2048 360 data points with 16 scans and was further processed the same way. GC-MS were recorded on an Agilent Technologies 6890N GC / 5973N Mass Selective Detector (electron impact, 70 ev). ESI-MS were recorded on a Bruker-Esquire mass spectrometer with an ion trap detector using MeOH+0.1% NH 4 OAc as solvent, positive and negative ions were detected. HRMS were recorded on a Waters Xevo G2-S QTof, positive and negative ions were detected. Elemental analyses have been carried out on a Heka-Tech EA 3000 Euro Vector CHNS elemental analyzer. Optical rotations were measured on a Perkin Elmer 341 polarimeter at a wavelength of 589 nm (Sodium D line) using a 1.00 dm cell with a total volume of 1.0 ml. 2

Reaction schemes: The synthesis of the compounds 1 and 11 was performed according to the following schemes: 3

Syntheses: (R)-2-(tert-Butyldimethylsilyloxy)docos-4-en-1-ol (15a) and (R)-1-(tert-butyldimethylsilyloxy)docos-4- en-2-ol (15b) The mixture of isomers 8 was obtained by Wittig reaction of lactol 13 and phosphonium salt 14, both being literature known compounds. Lactol 13 1 was prepared from (R)-3-hydroxy-γ-butyrolactone (Alfa Aesar, product-no.: L20255, purity: 90+%, ee 99+%) via TBS-protection and reduction, phosphonium salt 14 2 was synthesized using 1-iodooctadecane (Sigma Aldrich, product-no.: 251984, purity: 95%) as starting material. Over a period of 30 min a solution of n-buli (1.5 ml, 2.4 mmol, 1.6 M in hexane) was added at 0 C to a solution of octadecylphosphonium iodide 14 (1.48 g, 2.30 mmol) in THF (2.0 ml), and the mixture was allowed to stir 30 min at this temperature. A solution of lactol 13 (207 mg, 0.95 mmol) in THF (0.5 ml) was added dropwise to the reaction mixture over 30 min. The reaction mixture was stirred overnight and allowed to warm slowly to room temperature. After addition of NH 4 Cl (5 ml, aq., sat.) the reaction mixture was vigorously stirred for 30 min. H 2 O (5 ml) and EtOAc (5 ml) were added. The layers were separated and the aqueous layer was extracted with EtOAc five times. The combined organic layers were washed with brine, dried over MgSO 4, and the solvent was removed. The residue was purified by column chromatography on silica gel (isohexane/etoac 20:1) to obtain compound 15 (311 mg, 0.68 mmol, 72%) as a mixture of isomers (ratio: ~1:1). ESI-MS (10 V): m/z = 455.4 [M+H] +, 472.5 [M+NH 4 ] +. GC-MS: 15a: m/z (%) = 423 (12), 397 (32), 175 (48), 117 (100), 75 (64) 15b: m/z (%) = 397 (46), 175 (35), 105 (65), 75 (100). (4R)-4-Icosyl-2-phenyl-1,3-dioxolane (16) Pd/C (10%, 30.4 mg, 10 wt%) was added to a mixture of (R)-2-(tert-butyldimethylsilyloxy)docos-4- en-1-ol (15a) and (R)-1-(tert-butyldimethylsilyloxy)docos-4-en-2-ol (15b) (306 mg, 0.67 mmol) in EtOAc (15 ml). The reaction mixture was stirred for 14.5 h at room temperature under a hydrogen atmosphere. The reaction mixture was filtered through a short pad of celite (EtOAc). After removal of the solvent, the residue was purified by column chromatography on silica gel (isohexane/etoac 40:1) to obtain (R)-2-(tert-butyldimethylsilyloxy)docosan-1-ol and (R)-1-(tert-butyldimethylsilyloxy)docosan-2-ol (298 mg, 0.65 mmol, 97%) as mixture. TBAF (7.62 ml, 7.62 mmol, 1 M in THF) was added to the mixture of the silyl ethers (1.16 g, 2.55 mmol) in THF (15 ml) at room temperature and the reaction was allowed to stir for 1.5 h. H 2 O (15 ml) was added and the mixture was extracted with Et 2 O three times. The combined organic layers were washed with brine and dried over MgSO 4. Removal of the solvent afforded crude (R)- docosane-1,2-diol (0.955 g). 4

The crude diol was dissolved in toluene (50 ml). Benzaldehyde (294 mg, 2.77 mmol) and PPTS (10.4 mg, 41 µmol) were added, and the two-necked flask containing the reaction mixture was equipped with a dropping funnel with pressure compensator filled with molecular sieves 4 Å, topped by a Dimroth condenser. The reaction was heated at reflux for 5 h. Additional PPTS (27.0 mg, 107 µmol) was added and the mixture was heated for further 16 h. The solvent was removed and the residue was purified by column chromatography on silica gel (isohexane/etoac 20:1) to afford (4R)- 4-icosyl-2-phenyl-1,3-dioxolane (16) (792 mg, 1.84 mmol, 72% over two steps) as mixture of two diastereoisomers. 1 H NMR (500 MHz, CDCl 3 (7.25)): (ppm) = 0.87 (t, J = 6.9 Hz, 3 H), 1.20 1.40 (m, 34 H), 1.41 1.65 (m, 3 H), 1.70 1.80 (m, 1 H), 3.60 (t, J = 7.0 Hz) and 3.66 (t, J = 7.2 Hz, 1 H), 4.09 (t, J = 7.1 Hz) and 4.18 4.26 (m, 2 H), 5.79 (s) and 5.91 (s, 1 H), 7.34 7.38 (m, 3 H), 7.45 7.49 (m, 2 H). 13 C NMR (125 MHz, CDCl 3 (76.99)): (ppm) = 14.12 (CH 3 ), 22.68 (CH 2 ), 25.74 and 25.78 (CH 2 ), 29.35 (CH 2 ), 29.45 29.70 (14 CH 2 ), 31.91 (CH 2 ), 33.30 and 33.40 (CH 2 ), 70.14 and 70.80 (CH 2 ), 76.55 and 77.57 (CH), 103.03 and 103.92 (CH), 126.36 and 126.64 (2 CH), 128.32 (2 CH), 129.02 and 129.23 (CH), 137.87 and 138.50 (C). ESI-MS: m/z = 431.4 [M+H] +. Elemental Analysis: C 29 H 50 O 2. Calcd.: C: 80.87%; H: 11.70%. Found: C: 80.83%; H: 11.89%. (R)-2-(Benzyloxy)docosanoic acid (8) DIBAL-H (350 µl, 0.35 mmol, 1 M in PhMe) was added dropwise at 0 C to a solution of acetal 16 (99 mg, 0.23 mmol) in CH 2 Cl 2 (0.7 ml) and the reaction mixture was allowed to stir for 30 min. Methanol (1 ml) was added and the reaction was allowed to warm to room temperature. Rochelle salt (5 ml, aq., sat.)was added, the mixture was stirred for 30 min and extracted with CH 2 Cl 2 four times. The combined organic layers were dried over MgSO 4 and the solvent was evaporated. The residue was purified by column chromatography on silica gel (isohexane/ethyl acetate 6:1) providing (R)-2-(benzyloxy)docosan-1-ol (91 mg, 0.21 mmol, 92%). In the dark PIDA (846 mg, 2.63 mmol), TEMPO (10.3 mg, 66 µmol), and H 2 O (2 ml) were added at 0 C to a solution of (R)-2-(benzyloxy)docosan-1-ol (284 mg, 0.66 mmol) in CH 2 Cl 2 (6 ml). The mixture was stirred overnight and allowed to warm slowly to room temperature. The reaction mixture was diluted with CH 2 Cl 2 (60 ml). NH 4 Cl (9 ml, aq., sat.) and H 2 O (50 ml) were added and the layers were separated. The aqueous layer was extracted with CH 2 Cl 2 five times, the combined organic layers were washed with brine, dried over MgSO 4, and the solvent was evaporated. Purification of the residue by column chromatography on silica gel (isohexane/etoac 6:1 to 1:1) afforded (R)-2-(benzyloxy)docosanoic acid (8) (271 mg, 0.61 mmol, 92%). 5

1 H NMR (500 MHz, CDCl 3 (7.25)): (ppm) = 0.87 (t, J = 7.0 Hz, 3 H), 1.21 1.31 (m, 34 H), 1.38 1.45 (m, 2 H), 1.78 1.82 (m, 2 H), 4.00 (t, J = 5.9 Hz, 1 H), 4.52 (d, J AB = 11.5 Hz, 1 H), 4.68 (d, J AB = 11.5 Hz, 1 H), 7.30 7.38 (m, 5 H). 13 C NMR (125 MHz, CDCl 3 (77.00)): (ppm) = 14.12 (CH 3 ), 22.69 (CH 2 ), 24.82 (CH 2 ), 29.22 29.70 (15 CH 2 ), 31.92 (CH 2 ), 32.26 (CH 2 ), 72.64 (CH 2 ), 77.65 (CH), 128.12 (2 CH), 128.25 (CH), 128.60 (2 CH), 136.76 (C), 175.12 (COOH). ESI-MS: m/z ( 25 V) = 445.3 [M H] ; 891.5 [2M H]. D 20 = +13 (c = 0.12, MeOH). Elemental analysis: C 29 H 50 O 3 Calcd.: C: 77.97%; H: 11.28%; Found: C: 77.61%; H: 11.66%. (12-Methyltridecyl)triphenylphosphonium bromide (17) Magnesium shavings (1.81 g, 74.6 mmol) were activated by heating for 5 min together with two small crumbs of iodine. THF (5 ml) was added. Freshly distilled isopropyl bromide (7.0 ml, 75.0 mmol) and THF (13 ml) were continuously added, maintaining a gentle boiling of the reaction mixture. THF (40 ml) was added and the suspension was heated at reflux for 3 h. In a second flask LiCl (23 mg, 0.54 mmol) and CuCl 2 (32 mg, 0.24 mmol) were suspended in THF (1.2 ml) and stirred for 20 min. The freshly prepared Grignard reagent was added at 78 C to a solution of 11-bromoundecan-1-ol (4.99 g, 19.9 mmol, Sigma Aldrich, product no.: 184136, purity: 98%) in THF (48 ml). The mixture was allowed to stir at this temperature for 10 min, and then the freshly prepared solution of the catalyst (CuLi 2 Cl 4 in THF) was added. The reaction mixture was stirred overnight and allowed to warm slowly to room temperature. It was poured into NH 4 Cl (100 ml, aq., sat.) and the mixture was extracted with EtOAc four times. The combined organic layers were dried over MgSO 4 and the solvent was evaporated. The residue was purified by column chromatography on silica gel (isohexane/ethyl acetate 6:1 to 3:1) providing 12-methyltridecan-1-ol (3.89 g, 18.1 mmol, 91%). CBr 4 (4.93 g, 14.9 mmol) was portionwise added at 0 C to a solution of 12-methyltridecan-1-ol (2.90 g, 13.5 mmol) and PPh 3 (4.63 g, 17.6 mmol) in CH 2 Cl 2 (60 ml). The solution turned intense yellow. After 2.5 h of stirring at this temperature, MeOH (2 ml) was added and the reaction mixture was stirred for additional 3 h. The solvent was removed and the residue was purified by column chromatography on silica gel (isohexane/etoac 100:1) to afford 1-bromo-12-methyltridecane (3.68 g, 13.2 mmol, 98%). 1-Bromo-12-methyltridecane (3.32 g, 12.0 mmol) and PPh 3 (6.29 g, 24.0 mmol) were dissolved in MeCN (17 ml) and the reaction mixture was heated at reflux for 13 h. The mixture was extracted with isohexane five times to remove most of the remaining PPh 3. MeCN was evaporated and the viscid residue was heated three times with isohexane (3 x 50 ml) followed by decantation, to remove leftover PPh 3. The product was dried in vacuo to afford (12-methyltridecyl)- triphenylphosphonium bromide (17) (6.39 g, 11.9 mmol, 99%). 6

1 H NMR (500 MHz, CDCl 3 (7.25)): (ppm) = 0.83 (d, J = 6.6 Hz, 6 H), 1.08 1.13 (m, 2 H), 1.15 1.25 (m, 14 H), 1.49 (non, J = 6.6 Hz, 1 H), 1.55 1.65 (m, 4 H), 3.72 3.80 (m, 2 H), 7.67 7.70 (m, 6 H), 7.76 7.78 (m, 3 H), 7.79 7.85 (m, 6 H). 13 C NMR (125 MHz, CDCl 3 (77.00)): (ppm) = 22.61 (2 CH 3 ), 22.61 (d, J = 5.9 Hz, CH 2 ), 22.72 (d, J = 49.2 Hz, CH 2 ), 27.34 (CH 2 ), 27.90 (CH), 29.14 (CH 2 ), 29.21 (CH 2 ), 29.49 (CH 2 ), 29.53 (CH 2 ), 29.60 (CH 2 ) 29.85 (CH 2 ), 30.37 (d, J = 15.2 Hz, CH 2 ), 38.98 (CH 2 ), 118.40 (d, J = 85.4 Hz, 3 C), 130.43 (d, J = 12.3 Hz, 6 CH), 133.65 (d, J = 9.9 Hz, 6 CH), 134.92 (d, J = 3.1 Hz, 3 CH). ESI-MS: m/z = 459.4 [M Br] +. Elemental analysis: C 32 H 44 BrP Calcd.: C: 71.23%; H: 8.22%; Found: C: 71.00%; H: 8.42%. (R,Z)-N-Boc-2,2-dimethyl-4-(13-methyltetradec-1-enyl)oxazolidine (6) NaHMDS (2 M in THF, 4.0 ml, 8.0 mmol) was added dropwise over a period of 20 min to a solution of phosphonium salt 17 (4.07 g, 7.54 mmol) in THF (34 ml) at 78 C. The mixture was stirred at 23 C for 50 min, cooled to 78 C, and transferred via transfer cannula over a period of 10 min to a second flask, filled with Garner s aldehyde (1.40 g, 6.09 mmol) and THF (7 ml) at 78 C. The cooling bath was removed and the mixture was stirred for 40 min. NH 4 Cl (16 ml, aq., sat.), H 2 O (11 ml), and EtOAc (22 ml) were added and the mixture was stirred for 30 min. The layers were separated and the aqueous layer was extracted with EtOAc five times. The combined organic layers were washed with brine, dried over MgSO 4, and the solvent was evaporated. Purification of the residue by column chromatography on silica gel (isohexane/etoac 20:1 to 10:1) afforded (R,Z)-N- Boc-2,2-dimethyl-4-(13-methyltetradec-1-enyl)oxazolidine (6) (2.36 g, 5.76 mmol, 95%). 1 H NMR (500 MHz, CDCl 3 (7.25)): (ppm) = 0.85 (d, J = 6.6 Hz, 6 H), 1.11 1.17 (m, 2 H), 1.24 1.35 (br s, 16 H), 1.35 1.61 (m, 16 H), 2.00 2.15 (m, 2 H), 3.63 (dd, J = 17.3, 6.5 Hz, 1 H), 4.04 (dd, J = 17.3, 12.6 Hz, 1 H), 4.55 4.70 (m, 1 H), 5.35 5.50 (m, 2 H). 13 C NMR (125 MHz, CDCl 3 (76.99)): (ppm) = 22.65 (2 CH 3 ), 24.1 (br CH 3, HSQC), 25.1 (br CH 3, HSQC), 27.40 (CH 2 ), 27.45 (CH 2 ), 27.95 (CH), 28.48 (3 CH 3 ), 29.31 (CH 2 ), 29.50 (CH 2 ), 29.60 (CH 2 ), 29.64 (CH 2 ), 29.69 (CH 2 ), 29.72 (CH 2 ), 29.92 (CH 2 ), 39.04 (CH 2 ), 54.57 (CH), 69.04 (CH 2 ), 79.16 (br C), 93.96 (br C), 130.0 (br 2 CH, HSQC), 152.00 (C=O). ESI-MS: m/z = 410.3 [M+H] +, 432.4 [M+Na] +. ] 20 D = +51 (c = 0.11, MeOH). Elemental analysis: C 25 H 47 NO 3. Calcd.: C: 73.30%; H: 11.56%; N: 3.42%; Found: C: 73.48%; H: 11.44%; N: 3.50%. 7

(S)-N-Boc-2,2-dimethyl-4-((1S,2R)-1,2-bis(benzyloxy)-13-methyltetradecyl)oxazolidine (18) A solution of OsO 4 (9.9 mg, 3.9 µmol) in tbuoh (260 µl) was added dropwise at 0 C to a solution of olefin 6 (262 mg, 0.64 mmol), NMO (121 mg, 1.03 mmol), and DABCO (8.9 mg, 7.9 µmol) in tbuoh (1.35 ml) and H 2 O (1.35 ml). The mixture was stirred at this temperature for 21 h. NaHSO 3 (6.5 ml, aq., sat.) was added and the mixture was stirred at 0 C for 30 min. The mixture was partitioned between H 2 O and EtOAc, the layers were separated, and the aqueous layer was extracted with EtOAc six times. The combined organic layers were dried over MgSO 4, and the solvent was evaporated. Purification of the residue by column chromatography on silica gel (isohexane/etoac 1:0 to 3:2) afforded (S)-N-Boc-2,2-dimethyl-4-((1R,2S)-1,2-dihydroxy-13- methyltetradecyl)oxazolidine (74 mg, 0.17 mmol, 26%) as less polar fraction and the desired (S)-N- Boc-2,2-dimethyl-4-((1S,2R)-1,2-dihydroxy-13-methyltetradecyl)oxazolidine (182 mg, 0.41 mmol, 64%) as more polar fraction. BnBr (0.50 ml, 4.21 mmol) was added to a solution of (S)-N-Boc-2,2-dimethyl-4-((1S,2R)-1,2- dihydroxy-13-methyltetradecyl)oxazolidine (470 mg, 1.06 mmol) in DMF (4.8 ml) at 0 C. NaH (106 mg, 2.64 mmol, 60% in mineral oil) and KI (35.6 mg, 0.21 mmol) were added. The reaction mixture was stirred at 0 C for 25 min and at 23 C for 21 h. The mixture was partitioned between H 2 O and EtOAc, the layers were separated and the aqueous layer was extracted with EtOAc three times. The combined organic layers were washed with brine, dried over MgSO 4, and the solvent was evaporated. Purification of the residue by column chromatography on silica gel (isohexane/etoac 40:1 to 25:1) afforded (S)-N-Boc-2,2-dimethyl-4-((1R,2S)-1,2-bis(benzyloxy)-13-methyltetradecyl)- oxazolidine (18) (640 mg, 1.03 mmol, 97%). 1 H NMR (500 MHz, CDCl 3 (7.25)): (ppm) = 0.85 (d, J = 6.7 Hz, 6 H), 1.12 1.16 (m, 2 H), 1.19 1.30 (m, 16 H), 1.40 1.60 (m, 18 H), 3.45 3.51 (m, 1 H), 3.92 3.95 (m, 1 H), 4.02 4.13 (m, 2 H), 4.23 4.28 (m, 1 H), 4.49 (d, J AB = 11.6 Hz, 1 H), 4.58 4.74 (m, 3 H), 7.26 7.28 (m, 2 H), 7.30 7.36 (m, 8 H). 13 C NMR (125 MHz, CDCl 3 (76.99)): (ppm) = 22.65 (2 CH 3 ), 25.44 (CH 3 ), 26.59 (CH 3 ), 27.42 (CH 2 ), 27.95 (CH), 28.35 (CH 3 ), 28.44 (2 CH 3 ), 29.61 29.94 (7 CH 2 ), 30.87 (CH 2 ), 39.05 (CH 2 ), 57.85 (CH), 64.27 (CH 2 ), 72.50 (CH 2 ), 73.70 (CH 2 ), 78.86 (CH), 79.99 (CH), 80.10 (C), 93.36 (C), 127.52 128.29 (10 CH), 138.71 (2 C), 152.72 (C). ESI-MS: m/z (10 V) = 624.6 [M+H] +. ] 20 D = 7 (c = 0.07, MeOH). Elemental analysis: C 39 H 61 NO 5. Calcd.: C: 75.08%; H: 9.85%; N: 2.25%; Found: C: 74.73%; H: 9.98%; N: 2.50%. 8

(2S,3S,4R)-2-Amino-3,4-bis(benzyloxy)-15-methylhexadecan-1-ol (7) ptsoh H 2 O (10.7 mg, 56 µmol) was added at 23 C to a solution of (S)-N-Boc-2,2-dimethyl-4- ((1R,2S)-1,2-bis(benzyloxy)-13-methyltetradecyl)oxazolidine (18) (352 mg, 0.564 mmol) in MeOH (4.4 ml) and the reaction mixture was stirred for 21 h. The reaction was quenched with NaHCO 3 (aq., sat.), the mixture was partitioned between EtOAc and H 2 O, and the layers were separated. The aqueous layer was extracted with EtOAc three times and the combined organic layers were washed with brine, dried over MgSO 4, and the solvent was evaporated. Purification of the residue by column chromatography on silica gel (isohexane/etoac 15:1 to 3:1) afforded (2S,3S,4R)-2-(N-Boc-amino)- 3,4-bis(benzyloxy)-15-methylhexadecanol (313 mg, 0.536 mmol, 95%). KOH (2.22 g, 39.6 mmol) and H 2 O (2 ml) were added to a solution of (2S,3S,4R)-2-(N-Boc-amino)- 3,4-dibenzyloxy-15-methylhexadecanol (466 mg, 0.797 mmol) in EtOH (20 ml) and the reaction was stirred at reflux for 17 h. The reaction was quenched with NH 4 Cl (aq., sat.), partitioned between EtOAc and H 2 O, and the layers were separated. The organic layer was washed with NH 4 Cl (aq., sat.). The combined aqueous layers were extracted with EtOAc three times. The combined organic layers were washed with brine, dried over MgSO 4. Evaporation of the solvent afforded (2S,3S,4R)- 2-amino-3,4-bis(benzyloxy)-15-methylhexadecanol (7) (387 mg, quant.) which was used without further purification. 1 H NMR (500 MHz, CDCl 3 (7.25)): (ppm) = 0.85 (d, J = 6.6 Hz, 6 H), 1.12 1.16 (m, 2 H), 1.25 (br s, 14 H), 1.30 1.35 (m, 1 H), 1.44 1.54 (m, 2 H), 1.55 1.60 (m, 1 H), 1.65 1.70 (m, 1 H), 2.19 (br s, 3 H), 3.05 (q, J = 5.3 Hz, 1 H), 3.55 3.61 (m, 2 H), 3.70 3.75, m, 2 H), 4.566 (d, J AB = 11.4 Hz, 1 H), 4.569 (d, J AB = 11.4 Hz, 1 H), 4.63 (d, J AB = 11.4 Hz, 1 H), 4.73 (d, J AB = 11.4 Hz, 1 H), 7.26 7.36 (m, 10 H). 13 C NMR (125 MHz, CDCl 3 (76.99)): (ppm) = 22.65 (2 CH 3 ), 25.75 (CH 2 ), 27.42 (CH 2 ), 27.96 (CH), 29.60 (CH 2 ), 29.62 (CH 2 ), 29.66 (CH 2 ), 29.71 (CH 2 ), 29.77 (CH 2 ), 29.94 (CH 2 ), 30.58 (CH 2 ), 39.05 (CH 2 ), 53.52 (CH), 64.46 (CH 2 ), 72.45 (CH 2 ), 73.43 (CH 2 ), 79.47 (CH), 83.07 (CH), 127.77 (CH), 127.87 (CH), 127.94 (2 CH), 127.96 (2 CH), 128.42 (2 CH), 128.50 (2 CH), 138.06 (C), 138.14 (C). ESI-MS: m/z (10 V) = 484.6 [M+H] +, 967.3 [2M+H] +. Ceramide 9 ((R)-2-(Benzyloxy)-N-((2S,3S,4R)-3,4-bis(benzyloxy)-1-hydroxy-15-methylhexadecan-2- yl)docosanamide) HOBt (39.6 mg, 0.293 mmol) and EDC HCl (55.7 mg, 0.291 mmol) were added to a solution of (2S,3S,4R)-2-Amino-3,4-bis(benzyloxy)-15-methylhexadecanol (7) (104 mg, 0.215 mmol) and (R)-2- (benzyloxy)docosanoic acid (8) (91.1 mg, 0.206 mmol) in CH 2 Cl 2 (2.5 ml) at 10 C. The reaction 9

mixture was stirred for 6.5 h, while it was slowly warmed to room temperature. HCl (1 M, aq., 10 ml) was added and the mixture was partitioned between CH 2 Cl 2 and H 2 O. The aqueous layer was extracted with CH 2 Cl 2 two times and the combined organic layers were washed with brine, dried over MgSO 4, and the solvent was evaporated. Purification of the residue by column chromatography on silica gel (isohexane/etoac 6:1 to 2:1) afforded (R)-2-(benzyloxy)-N-((2S,3S,4R)-3,4- bis(benzyloxy)-1-hydroxy-15-methylhexadecan-2-yl)docosanamide (9) (150 mg, 0.164 mmol, 80%). 1 H NMR (600 MHz, CDCl 3 (7.25)): (ppm) = 0.85 (d, J = 6.7 Hz, 6 H), 0.87 (t, J = 6.8 Hz, 3 H), 1.12 1.15 (m, 2 H), 1.19 1.40 (m, 51 H), 1.41 1.47 (m, 1 H), 1.50 (non, J = 6.7 Hz, 1 H), 1.55 1.64 (m, 2 H), 1.67 1.74 (m, 2 H), 2.86 (br s, 1 H), 3.63 3.65 (m, 2 H), 3.69 (t, J = 3.9 Hz, 1 H), 3.83 (dd, J = 7.4, 4.1 Hz, 1 H), 3.90 (dd, J = 11.4, 3.0 Hz, 1 H), 4.15 4.18 (m, 1 H), 4.46 (d, J AB = 11.3 Hz, 1 H), 4.50 (d, J AB = 11.3 Hz, 1 H), 4.54 (d, J AB = 11.4 Hz, 1 H), 4.55 (d, J AB = 11.3 Hz, 1 H), 4.63 (d, J AB = 11.4 Hz, 1 H), 4.68 (d, J AB = 11.3 Hz, 1 H), 7.24 7.35 (m, 16 H). 13 C NMR (151 MHz, CDCl 3 (76.99)): (ppm) = 14.11 (CH 3 ), 22.66 (2 CH 3 ), 22.68 (CH 2 ), 25.09 (CH 2 ), 26.02 (CH 2 ), 27.43 (CH 2 ), 27.97 (CH), 29.35 (CH 2 ), 29.40 (CH 2 ), 29.55 29.71 (18 CH 2 ), 29.96 (CH 2 ), 30.58 (CH 2 ), 31.92 (CH 2 ), 32.99 (CH 2 ), 39.07 (CH 2 ), 50.31 (CH), 62.68 (CH 2 ), 72.79 (CH 2 ), 72.85 (CH 2 ), 73.28 (CH 2 ), 79.07 (CH), 80.25 (CH), 81.69 (CH), 127.76 (CH), 127.88 (2 CH), 127.99 (3 CH), 128.09 (CH), 128.16 (2 CH), 128.42 (2 CH), 128.55 (2 CH), 128.61 (2 CH), 137.26 (C), 137.63 (C), 138.09 (C), 172.49 (C=O). ESI-MS: m/z (10 V) = 913.0 [M+H] +, 934.9 [M+Na] +. ] 20 D = 3 (c = 0.10, CHCl 3 ). Elemental analysis: C 60 H 97 NO 5. Calcd: C: 78.98%; H: 10.72%; N: 1.54%; Found: C: 79.02%; H: 10.72%; N: 1.50%. O-(O 2,O 3,O 4 -Tribenzyl-O 6 -triisopropylsilyl- -D-glucopyranosyl) trichloroacetimidate 19 NaH (60%, 18.1 mg, 0.453 mmol) was added at 23 C to a solution of O 2,O 3,O 4 -tribenzyl-o 6 - triisopropylsilyl-d-glucopyranose (268 mg, 0.441 mmol), synthesized adapting a literature known procedure 3, in CH 2 Cl 2 (2 ml), and the mixture was stirred for 20 min. Cl 3 CCN (155 µl, 1.55 mmol) was added dropwise. The solution slowly turned greenish brown. The mixture was stirred for 3 h. The solvent was removed in vacuo and purification of the residue by column chromatography on silica gel (isohexane/et 2 O 5:1 to 1:1) afforded the desired activated glucosyl building block 16 (169 mg, 0.225 mmol, 51%) and its slightly more polar epimer O-(O 2,O 3,O 4 -tribenzyl-o 6 -triisopropylsilyl-β- D-glucopyranosyl) trichloroacetimidate (74 mg, 0.098 mmol, 22%). 1 H NMR (300 MHz, CDCl 3 (7.25)): (ppm) = 0.99 1.12 (m, 21 H), 3.66 3.80 (m, 2 H), 3.85 3.91 (m, 1 H), 3.92 4.00 (m, 2 H), 4.06 (t, J = 9.3 Hz, 1 H), 4.64 4.95 (m, 6 H), 6.53 (d, J = 3.4 Hz, 1 H), 7.23 7.38 (m, 15 H). 10

ESI-MS: m/z (25 V) = 606.5 [M LG+NH 3 ] +, 772.5 [M+Na] +, 1194.6 [2(M LG)+NH 3 H] + ; LG = OC(NH)CCl 3. Glucosylceramide 10 ((R)-2-(Benzyloxy)-N-((2S,3S,4R)-3,4-bis(benzyloxy)-1-((O 2,O 3,O 4 -tribenzyl- O 6 -triisopropylsilyl-β-d-glucopyranosyl)oxy)-15-methylhexadecan-2-yl)docosanamide) BF 3 Et 2 O (2 µl, 16 µmol) was added to a solution of ceramide 9 (48.3 mg, 53 µmol), MS (4 Å, 89 mg) and trichloroacetimidate 19 (61.4 mg, 82 µmol) in CH 2 Cl 2 (2.5 ml) at 28 C and the mixture was stirred at this temperature for 4 h. The mixture was partitioned between CH 2 Cl 2 and H 2 O. The aqueous layer was extracted with CH 2 Cl 2 three times and the combined organic layers were washed with brine, dried over MgSO 4, and the solvent was evaporated. Purification of the residue by column chromatography on silica gel (isohexane/etoac 1:0 to 6:1) afforded the glucosylceramide 10 (58.5 mg, 39 µmol, 74%). 1 H NMR (600 MHz, CDCl 3 (7.25)): (ppm) = 0.85 (d, J = 6.7 Hz, 6 H (2 H 3-16 LCB )), 0.87 (t, J = 6.8 Hz, 3 H (H 3-22 FA )), 1.00 1.10 (m, 21 H (6 H 3 -TIPS Glc, 3 H-TIPS Glc )), 1.12 1.16 (m, 2 H (H 2-14 LCB ), 1.16 1.39 (m, 51 H (H 2 -[4 21] FA, H 2 -[7 13] LCB, H A -6 LCB )), 1.50 (non, J = 6.7 Hz, 1 H (H-15 LCB )), 1.44 1.72 (m, 5 H (H 2-3 FA, H 2-5 LCB, H B -6 LCB )), 3.21 (ddd, J = 9.4, 3.8, 1.5 Hz, 1 H (H-5 Glc )), 3.32 (dd, J = 9.0, 7.8 Hz, 1 H (H-2 Glc )), 3.45 3.48 (m, 1 H (H-4 LCB )), 3.54 (t, J = 9.0 Hz, 1 H (H-3 Glc )), 3.59 (t, J = 9.2 Hz, 1 H (H-4 Glc )), 3.71 3.75 (m, 2 H (H-2 FA, H A -1 LCB )), 3.78 (dd, J = 6.7, 2.8 Hz, 1 H (H-3 LCB )), 3.86 (dd, J AB = 11.1 Hz, J = 4.0 Hz, 1 H (H A -6 Glc )), 3.90 (dd, J AB = 11.1 Hz, J = 1.0 Hz, 1 H (H B -6 Glc ), 4.18 (dd, J AB = 10.4 Hz, J = 6.7 Hz, 1 H (H B -1 LCB )), 4.30 (d, J AB = 11.9 Hz, 1 H (H A -OBn FA )), 4.32 (d, J = 7.8 Hz, 1 H (H-1 Glc )), 4.34 4.38 (m, 2 H (H-2 LCB, H A -OBn C-4 LCB )), 4.49 (d, J AB = 11.5 Hz, 1 H (H B -OBn C-4 LCB )), 4.56 (d, J AB = 11.9 Hz, 1 H (H B -OBn FA )), 4.608 (d, J AB = 11.5 Hz, 1 H (H A -OBn C-3 LCB )), 4.615 (d, J AB = 11.2 Hz, 1 H (H A -OBn C-2 Glc )), 4.66 (d, J AB = 11.0 Hz, 1 H (H A -OBn C-4 Glc )), 4.72 (d, J AB = 11.5 Hz, 1 H (H B -OBn C-3 LCB )), 4.75 (d, J AB = 10.9 Hz, 1 H (H A -OBn C-3 Glc )), 4.81 4.85 (m, 3 H (H B -OBn C-2 Glc, H B -OBn C-3 Glc, H B -OBn C-4 Glc )), 6.89 (d, J = 9.0 Hz, 1 H (H N LCB )), 7.20 7.32 (m, 30 H (6 5 H- OBn)). 13 C NMR (151 MHz, CDCl 3 (77.00)): (ppm) = 11.92 (3 CH (TIPS Glc )), 14.11 (CH 3 (C-22 FA )), 17.96 and 17.98 (6 CH 3 (TIPS Glc )), 22.66 (2 CH 3 (C-16 LCB )), 22.68 (CH 2 (C-21 FA )), 25.15 (CH 2 (C-4 FA )), 26.11 (CH 2 (C-6 LCB )), 27.45 (CH 2 (C-13 LCB )), 27.97 (CH (C-15 LCB ), 29.36 (CH 2 ), 29.39 (CH 2 ), 29.59 29.97 (20 CH 2 ), 31.92 (CH 2 (C-20 FA )), 33.08 (CH 2 (C-3 FA )), 39.07 (CH 2 (C-14 LCB ), 49.69 (CH (C- 2 LCB )), 62.33 (CH 2 (C-6 Glc )), 68.50 (CH 2 (C-1 LCB )), 71.81 (CH 2 (OBn C-4 LCB )), 72.14 (CH 2 (OBn FA ), 73.46 (CH 2 (OBn C-3 LCB )), 74.75 (CH 2 (OBn C-2 Glc )), 74.95 (CH 2 (OBn C-4 Glc )), 75.72 (CH 2 (OBn C- 11

3 Glc )), 76.09 (CH (C-5 Glc )),77.47 (CH (C-4 Glc )), 79.08 (CH (C-3 LCB )), 79.94 (CH (C-2 FA )), 80.12 (CH (C-4 LCB )), 82.45 (CH (C-2 Glc )), 84.62 (CH (C-3 Glc )), 103.73 (CH (C-1 Glc )), 127.44 (CH), 127.49 (CH), 127.53 (CH), 127.58 (CH), 127.64 (2 CH), 127.72 (CH), 127.78 (2 CH), 127.92 (7 CH), 127.99 (2 CH), 128.26 (2 CH), 128.27 (2 CH), 128.32 (4 CH), 128.42 (2 CH), 128.47 (2 CH), 137.35 (C (OBn FA )), 138.30 (C), 138.37 (C), 138.46 (C), 138.54 (C), 138.56 (C), 172.61 (C=O (C-1 FA )). ESI-MS: m/z (10 V) = 912.9 [M sugar moiety+h] +, 1501.0 [M+H] +. ] 20 D = +7 (c = 0.12, CHCl 3 ). Elemental analysis: C 96 H 145 NO 10 Si. Calcd.: C: 76.80%; H: 9.74%; N: 0.93%; Found: C: 77.06%; H: 9.72%; N: 0.79%. Glucosylceramide d17iso-glc-cer (11) ((R)-2-(Hydroxy)-N-((2S,3S,4R)-3,4-dihydroxy-1-((β-D-glucopyranosyl)oxy)-15-methylhexadecan-2-yl)docosanamide) TBAF (1 M in THF, 45 µl, 45 µmol) was added at 50 C to a solution of Glucosylceramide 10 (45.1 mg, 30 µmol) in THF (0.9 ml). The solution turned immediately from colorless to yellow. The mixture was stirred for 40 min and then partitioned between EtOAc and H 2 O. The organic layer was washed with water two times. The combined aqueous layers were extracted with EtOAc two times and the combined organic layers were washed with brine, dried over MgSO 4, and the solvent was evaporated. Purification of the residue by column chromatography on silica gel (isohexane/etoac 1:0 to 4:1) afforded (R)-2-(benzyloxy)-N-((2S,3S,4R)-3,4-bisbenzyloxy-1-((O 2,O 3,O 4 -tribenzyl-β-dglucopyranosyl)oxy)-15-methylhexadecan-2-yl)docosanamide (34.0 mg, 25 µmol, 84%). A round-bottom flask filled with (R)-2-(benzyloxy)-N-((2S,3S,4R)-3,4-bisbenzyloxy-1-((O 2,O 3,O 4 - tribenzyl-β-d-glucopyranosyl)oxy)-15-methylhexadecan-2-yl)docosanamide (30.8 mg, 23 µmol) and Pd/C (10%, 3.7 mg) was evacuated and set under H 2 (balloon). EtOAc (0.4 ml) and EtOH (0.8 ml) were added and the mixture was stirred at room temperature for 17 h. The solvent was evaporated and the residue was purified by column chromatography on silica gel (CH 2 Cl 2 /isohexane/meoh 90:10:2 to 90:5:10) to afford the glucosylceramide d17iso-glc-cer (11) (16.7 mg, 21 µmol, 91%). 1 H NMR (600 MHz, CD 3 OD (3.32)): (ppm) = 0.89 (d, J = 6.6 Hz, 6 H (2 H 3-16 LCB )), 0.91 (t, J = 6.9 Hz, 3 H (H 3-22 FA )), 1.17 1.21 (m, 2 H (H 2-14 LCB )), 1.30 (br s, 51 H), 1.40 1.46 (m, 2 H), 1.54 (non, J = 6.6 Hz, 1 H (H-15 LCB )), 1.50 1.66 (m, 2 H), 1.73 1.78 (m, 1 H), 3.179 (dd, J = 9.2, 7.8 Hz, 1 H (H-2 Glc )), 3.28 3.29 (m, 2 H (H-5 Glc, H-2 FA )), 3.35 3.38 (m, 1 H (H-3 Glc )), 3.53 (ddd, J = 9.7, 5.9, 2.5 Hz, 1 H (H-4 LCB )), 3.63 (t, J = 6.1 Hz, 1 H (H-3 LCB )), 3.66 3.69 (m, 1 H (H A -6 Glc )), 3.81 (dd, J AB = 10.5 Hz, J =3.8 Hz, 1 H (H A -1 LCB )), 3.88 (dd, J AB = 12.0 Hz, J = 1.6 Hz, 1 H (H B -6 Glc )), 4.02 (dd, J = 7.6; 3.9 Hz, 1 H (H-4 Glc )), 4.07 (dd, 12

J AB = 10.4 Hz, J = 6.2 Hz, 1 H (H B -1 LCB )), 4.26 (td, J = 6.3, 3.8 Hz, 1 H (H-2 LCB )), 4.29 (d, J = 7.8 Hz, 1 H (H-1 Glc )). 13 C NMR (HSQC-projection) (151 MHz, CD 3 OD (49.0)): (ppm) = 14.4 (CH 3 (C-22 FA )), 23.1 (2 CH 3 (C-16 LCB )), 23.8 (CH 2 ), 26.2 (CH 2 ), 27.0 (CH 2 ), 27.2 (CH 2 ), 28.8 (CH 2 ), 29.3 (CH (C-15 LCB )), 30 34 (20 CH 2 ), 33.3 (CH 2 ), 35.4 (CH 2 ), 35.9 (CH 2 ), 40.3 (CH 2 (C-14 LCB )), 51.8 (CH (C-2 LCB )), 62.9 (CH 2 (C-6 Glc )), 70.1 (CH 2 (C-1 LCB )), 71.6 (CH (C-5 Glc )), 73.1 (2 CH (C-4 Glc, C-4 LCB )), 75.1 (CH (C-2 Glc )), 75.6 (CH (C-3 LCB )), 78.0 (CH (C-3 Glc )), 78.1 (CH (C-2 FA )), 105.0 (CH (C-1 Glc )). ESI-MS: m/z (10 V) = 827.1 [M+Na] +. ] D 20 = +11 (c = 0.06, MeOH). HRMS: C 45 H 89 NO 10. [M+H] + : calcd.: 804.6559, found: 804.6572. Phosphoglucosylceramide 12 ((R)-2-(Benzyloxy)-N-((2S,3S,4R)-3,4-bisbenzyloxy-1-((O 2,O 3,O 4 - tribenzyl-o 6 -(O-(2-(N-benzyloxcarbonyl-N-methyl)amino)ethyl)-O (2-cyanoethyl)-phosphato)-β-Dglucopyranosyl)oxy)-15-methylhexadecan-2-yl)docosanamide) TBAF (1 M in THF, 45 µl, 45 µmol) was added at 50 C to a solution of Glucosylceramide 10 (45.1 mg, 30 µmol) in THF (0.9 ml). The solution turned immediately from colorless to yellow. The mixture was stirred for 40 min and then partitioned between EtOAc and H 2 O. The organic layer was washed with water two times. The combined aqueous layers were extracted with EtOAc two times and the combined organic layers were washed with brine, dried over MgSO 4, and the solvent was evaporated. Purification of the residue by column chromatography on silica gel (isohexane/etoac 1:0 to 4:1) afforded (R)-2-(benzyloxy)-N-((2S,3S,4R)-3,4-bisbenzyloxy-1-((O 2,O 3,O 4 -tribenzyl-β-dglucopyranosyl)oxy)-15-methylhexadecan-2-yl)docosanamide (34.0 mg, 25 µmol, 84%). A round-bottom flask filled with (R)-2-(benzyloxy)-N-((2S,3S,4R)-3,4-bisbenzyloxy-1-((O 2,O 3,O 4 - tribenzyl-β-d-glucopyranosyl)oxy)-15-methylhexadecan-2-yl)docosanamide (49.8 mg, 37 µmol), 2- cyanoethyl 2-(N-benzyloxycarbonyl-N-methylamino)ethyl diisopropylphosphoramidite (92.2 mg, 225 µmol), synthesized adapting a literature known procedure 4, and tetrazole (15.3 mg, 218 µmol) was evacuated for 2 h. CH 2 Cl 2 (2.5 ml) was added at room temperature and the mixture was stirred for 1.5 h. After completion of the coupling reaction (TLC), mcpba (77%, 48.9 mg, 218 µmol) was added and the mixture was stirred for 25 min. A small portion of silica gel was added and the solvent was removed in vacuo. The so adsorbed residue was purified by column chromatography on silica gel (isohexane/etoac 1:1 (+1% NEt 3 ) to 1:2.5) which afforded the phosphoglucosylceramide 12 (55.8 mg, 33 µmol, 90%) as mixture of a pair of epimers at the phosphorous stereogenic center. 13

1 H NMR (600 MHz, CDCl 3 (7.25)): (ppm) = 0.85 (d, J = 6.6 Hz, 6 H (2 H 3-16 LCB )), 0.87 (t, J = 7.0 Hz, 3 H (H-22 FA )), 1.12 1.15 (m, 2 H (H-14 LCB )), 1.20 1.34 (m, 51 H (H 2 -[4-21] FA, H 2 -[7-13] LCB, H A - 6 LCB )), 1.41 1.53 (m, 1 H (H B -6 LCB )), 1.50 (non, J = 6.6 Hz, 1 H (H-15 LCB )), 1.56 1.66 (m, 4 H (H 2-3 FA, H 2-5 LCB )), 2.32 2.44 (m, 2 H (H 2-2 HG )), 2.89 and 2.90 (2 s, 3 H (H 3-3 HG )), 3.34 3.37 (m, 2 H (H-2 Glc, H-5 Glc )), 3.42 3.49 (m, 3 H (H 2-2 HG, H-4 Glc )), 3.50 3.51 (m, 1 H (H-4 LCB )), 3.57 (t, J = 8.9 Hz, 1 H (H-3 Glc )), 3.77 3.79 (m, 3 H (H A -1 LCB, H-2 FA, H-3 LCB ), 3.90 4.27 (m, 7 H (H 2-1 HG, H 2-1 HG, H B -1 LCB, H 2-6 Glc )), 4.33 4.36 (m, 2 H (H-1 Glc, H A -OBn C-3 LCB )), 4.41 4.43 (m, 2 H (H-2 LCB, H A -OBn C-2 FA )), 4.51 4.53 (m, 2 H (H B -OBn C-2 FA, H B -OBn C-3 LCB )), 4.56 4.58 (m, 2 H (H A -OBn C- 4 Glc, H A -OBn C-4 LCB )), 4.636 and 4.642 (2 d, J = 11.2; 11.2 Hz, 1 H (H A -OBn C-2 Glc )), 4.70 4.72 (m, 1 H (H B -OBn C-4 LCB )), 4.73 (d, J = 11.0 Hz, 1 H (H A -OBn C-3 Glc ), 4.83 4.88 (m, 3 H (H B -OBn C- 2 Glc, H B -OBn C-3 Glc, H B -OBn C-4 Glc )), 5.07 and 5.08 (2 s, 2 H (H 2 -Cbz HG )), 7.00 (d, J = 8.0 Hz, 1 H (NH LCB )), 7.22 7.31 (m, 35 H (6 5 H-OBn, 5 H-Cbz)). 13 C NMR (151 MHz, CDCl 3 (77.00)): (ppm) = 14.12 (CH 3 (C-22 FA )), 19.28 and 19.32 (2 d, J = 6.5 Hz, CH 2 (C-2 HG )), 22.67 (2 CH 3 (2 C-16 LCB )), 22.69 (CH 2 ), 25.14 (CH 2 ), 25.95 (CH 2 ), 27.45 (CH 2 ), 27.97 (CH (C-15 LCB )), 29.36 29.98 (m, 22 CH 2 ), 31.92 (CH 2 ), 33.02 (br, CH 2 ), 35.38 and 35.88 (br, CH 3 (C-3 HG ), 39.07 (CH 2 (C-14 LCB )), 48.60 and 49.24 (2 d, J = 6.1; 6.7 Hz, CH 2 (C-2 HG )), 49.55 and 49.58 (2 s, CH (C-2 LCB )), 61.83 and 61.95 (2 d, J = 4.9; 6.4 Hz, CH 2 (C-1 HG )), 65.86 65.90 (m, CH 2 (C- 6 Glc )), 66.62 66.75 (m, CH 2 (C-1 HG )), 67.18 and 67.34 (2 br s, CH 2 (CH 2 -Cbz)), 69.24 and 69.34 (2 s, CH 2 (C-1 LCB )), 71.80 (CH 2 (OBn C-4 LCB )), 72.22 and 72.25 (2 s, CH 2 (OBn C-2 FA )), 73.37 73.51 (m, CH (C-5 Glc ) and CH 2 (OBn C-3 LCB )), 74.78 (CH 2 (OBn C-2 Glc ), 74.97 (br, CH 2 (OBn C-4 Glc )), 75.62 and 75.64 (2 s, CH 2 (OBn C-3 Glc )), 77.21 (CH (C-4 Glc )), 79.22 and 79.35 and 79.98 (br) and 80.18 (br) (3 CH (C-2 FA, C-3 LCB, C-4 LCB ), 81.91 (br, CH (C-2 Glc )), 84.32 (br, CH (C-3 Glc )), 103.77 (CH (C-1 Glc )), 117 (HMBC, C N (C-3 HG )), 127.54 128.53 (35 CH (6 5 CH-OBn, 5 CH-Cbz)), 137.43 138.48 (7 C (6 1 C-OBn, 1 C-Cbz)), 156 (HMBC, C=O (Cbz HG )), 173 (HMBC, C=O (C-1 FA )). 31 P NMR (243 MHz, CDCl 3 ): (ppm) = 1.45, 1.69. ESI-MS: m/z (50 V) = 912.9 [M (sugar moiety+hg)+h] +, 1669.0 [M+H] +, 1691.1 [M+Na] +. Elemental analysis: C 101 H 142 N 3 O 15 P. Calcd.: C: 72.67%; H: 8.57%; N: 2.52%, Found: C: 72.53%; H: 8.90%; N: 3.04%. 14

mmpegc-c22 (1) ((R)-2-(Hydroxy)-N-((2S,3S,4R)-3,4-dihydroxy-1-((O 6 -(O-(2-(methylamino)ethyl)- phosphato)-β-d-glucopyranosyl)oxy)-15-methylhexadecan-2-yl)docosanamide) Dimethylamine (5.6 M in EtOH, 2.2 ml, 12.32 mmol) was added at room temperature to a solution of phosphoglucosylceramide 12 (51.2 mg, 31 µmol) in CH 2 Cl 2 and the mixture was stirred for 30 min. The mixture was partitioned between CH 2 Cl 2 and H 2 O. The aqueous layer was extracted with CH 2 Cl 2 two times and the combined organic layers were dried over MgSO 4. The solvent was evaporated and the residue was dried in a 100 ml round-bottom flask in vacuo overnight. Pd(OH) 2 /C (H 2 O ~50%, 63.1 mg), CH 2 Cl 2 (10 ml), and isopropanol (10 ml) were added to this residue. The mixture was set under H 2 -atmosphere (balloon) and HCO 2 H (6 µl, 159 µmol) was added. The mixture was vigorously stirred for 23 h at room temperature. The catalyst was removed by filtration over a short pad of Celite (CH 2 Cl 2 /isopropanol 1:1) and the solvent was evaporated. Due to incomplete conversion (ESI-MS), Pd(OH) 2 /C (H 2 O ~50%, 60.2 mg), CH 2 Cl 2 (10 ml), and isopropanol (10 ml) were added. The mixture was set under H 2 -atmosphere (balloon) and HCO 2 H (7 µl, 186 µmol) was added. The mixture was vigorously stirred for 28 h at room temperature. The catalyst was removed by filtration over a short pad of Celite (CH 2 Cl 2 /isopropanol 1:1) and the solvent was evaporated. Due to incomplete conversion (ESI-MS), Pd(OH) 2 /C (H 2 O ~50%, 61.3 mg), CH 2 Cl 2 (10 ml), and isopropanol (10 ml) were added. The mixture was set under H 2 -atmosphere (balloon) and HCO 2 H (7 µl, 186 µmol) was added. The mixture was vigorously stirred for 16 h at room temperature. The catalyst was removed by filtration over a short pad of Celite (CH 2 Cl 2 /isopropanol 1:1) and the solvent was evaporated. Due to incomplete conversion (ESI-MS), Pd(OH) 2 /C (H 2 O ~50%, 58.6 mg), CH 2 Cl 2 (10 ml), and isopropanol (10 ml) were added. The mixture was set under H 2 -atmosphere (balloon) and HCO 2 H (10 µl, 265 µmol) was added. The mixture was vigorously stirred for 26 h at room temperature. The catalyst was removed by filtration over a short pad of Celite (CH 2 Cl 2 /isopropanol 1:1) and the solvent was evaporated. As ESI-MS indicated complete conversion, the residue was purified via preparative HPLC (Grace-Vydac 208TP1050 RP C8 50 250 mm, 55 ml/min, MeOH/H 2 O 70:30 to 95:5, 27 min) to yield mmpegc- C22 (1) (8.5 mg, 9.0 mmol, 29%). 15

1 H NMR (600 MHz, CD 3 OD (3.38)): (ppm) = 0.95 (d, J = 6.7 Hz, 6 H (H-16 LCB )), 0.97 (t, J = 6.9 Hz, 3 H (H-22 FA )), 1.23 1-27 (m, 2 H (H-14 LCB )), 1.28 1.52 (m, 52 H (H 2 -[4-21] FA, H 2 -[7-13] LCB, H 2-6 LCB )), 1.60 (non, J = 6.7 Hz, 1 H (H-15 LCB )), 1.57 1.71 (m, 4 H (H 2-3 FA, H 2-5 LCB )), 2.82 (s, 3 H (H 3-3 HG )), 3.23 3.26 (m, 1 H (H- 2 Glc )), 3.32 (t, J = 4.9 Hz, 2 H (H 2-2 HG )), 3.44 3.46 (m, 3 H (H-2 FA, H-3 Glc, H-5 Glc )), 3.58 3.61 (m, 1 H (H-4 LCB )), 3.71 (t, J = 6.1 Hz, 1 H (H-3 LCB )), 3.81 (dd, J AB = 10.3 Hz; J = 3.8 Hz, 1 H (H A -1 LCB )), 4.09 4.14 (m, 3 H (H B -1 LCB, H-4 Glc, H A -6 Glc )), 4.16 4.20 (m, 2 H (H 2-1 HG )), 4.22 (dd, J AB = 10.7 Hz, J = 5.2 Hz, 1 H (H B -6 Glc )), 4.28 4.31 (m, 1 H (H-2 LCB )), 4.37 (d, J = 7.8 Hz, 1 H (H-1 Glc )). 13 C NMR (HSQC-projection) (150 MHz, CD 3 OD (47.96)): (ppm) = 13.10 (CH 3 (C-22 FA )), 21.72 (2 CH 3 (C-16 LCB )), 22.41 (CH 2 (C-21 FA )), 24.78 (CH 2 ), 27.28 (CH 2 ), 27.79 (CH (C-15 LCB )), 28.0 30.5 (24 CH 2 ), 31.73 (CH 2 ), 32.22 (CH 3 (C-3 HG )), 38.97 (CH 2 (C-14 LCB )), 49.67 (CH 2 (C-2 HG )), 50.22 (CH (C-2 LCB )), 60.33 (CH 2 (C-1 HG )), 64.57 (CH 2 (C-6 Glc )), 68.84 (CH 2 (C- 1 LCB )), 69.69 (CH), 71.62 (2 CH (C-4 Glc, C-4 LCB )), 74.05 (br, 2 CH (C-2 Glc, C-3 LCB )), 75.43 (CH), 76.33 (CH), 103.48 (CH (C-1 Glc ). 31 P NMR (HMBC-projection) (243 MHz, CD 3 OD): (ppm) = 0.69. ESI-MS: m/z (10 V) = 942.1 [M+H] +, 1882.3 [2M+H] +. [ ] 20 D = +2 (c = 0.04, MeOH). HRMS: C 48 H 97 N 2 O 13 P [M H] : calcd.: 939.6650, found: 939.6605. 16

2.97 2.03 0.54 0.45 1.51 0.57 0.47 0.61 1.29 4.72 37.91 3.45 NMR-Spectra: in CDCl 3 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm 17

4.84 0.98 0.99 1.00 2.43 2.72 35.37 3.84 in CDCl 3 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm 18

6.05 2.98 6.09 2.00 2.75 1.03 4.04 1.95 14.00 5.86 in CDCl 3 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm 19

2.00 0.97 0.96 0.96 2.06 20.05 18.70 2.43 7.04 in CDCl 3 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm 20

10.01 2.95 1.00 1.00 3.05 0.93 4.82 17.00 2.35 16.87 6.40 in CDCl 3 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm 21

9.85 1.98 1.06 1.02 2.03 2.00 0.94 1.09 3.07 15.95 2.08 6.00 raw material in CDCl 3 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm 22

18.15 4.19 1.06 1.03 1.00 1.96 1.00 1.02 0.98 0.82 2.65 6.17 2.43 54.93 2.59 9.56 in CDCl 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm 23

35.52 0.85 1.00 3.99 1.08 1.14 1.96 1.21 2.08 3.01 0.92 0.97 1.00 2.08 1.92 1.06 2.27 14.11 56.12 23.50 3.11 9.95 in CDCl 3 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm 24

37.77 0.95 1.91 1.96 1.93 3.94 1.00 1.95 2.88 7.04 2.92 2.01 2.80 1.03 1.00 2.82 1.97 8.26 2.79 57.62 9.71 in CDCl 3 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm 25

21.48 0.69 6.73 1.03 1.00 3.11 0.88 23.49 in CDCl 3 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 26

0.44 0.98 1.00 2.00 0.99 1.00 1.02 0.99 0.94 1.79 0.45 4.75 2.35 0.97 0.65 1.18 5.55 3.88 56.98 2.91 10.20 in CD 3 OD 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm 27

d17iso-glccer (11) COSY 28

d17iso-glccer (11) HSQC 29

d17iso-glccer (11) NOESY 30

d17iso-glccer (11) HMBC 31

3.10 1.99 1.21 1.11 1.00 1.22 1.32 1.16 2.93 1.01 1.92 2.89 7.35 83.11 3.96 13.47 in CD 3 OD 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm 32

mmpegc-c22 (1) COSY 33

mmpegc-c22 (1) HSQC 34

mmpegc-c22 (1) NOESY 35

mmpegc-c22 (1) HMBC 36

mmpegc-c22 (1) 1 H/ 31 P-HMBC mmpegc-c22 (1) 1 H-projection 1 H/ 31 P-HMBC 37

2. Supplementary References 1 Yu, X. M., Han, H. & Blagg, B. S. J. Synthesis of mono- and dihydroxylated furanoses, pyranoses, and an oxepanose for the preparation of natural product analogue libraries. J. Org. Chem. 70, 5599 5605 (2005). 2 Livi, S., Gérard, J.-F. & Duchet-Rumeau, J. Ionic liquids: structuration agents in a fluorinated matrix. Chem. Commun. (Camb.) 47, 3589 3591 (2011). 3 Becker, C. F. W., Seeberger, P. H. et al. Semisynthesis of a Glycosylphosphatidylinositol- Anchored Prion Protein. Angew. Chem. Int. Ed. 47, 8215 8219 (2008). 4 Murakata, C. & Ogawa, T. Stereoselective total synthesis of the glycosyl phosphatidylinositol (GPI) anchor of Trypanosoma brucei. Carbohyd. Res. 235, 95 114 (1992). 38