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1 S1 The Synthesis and Biological Characterization of a Ceramide Library Young-Tae Chang*, Jaehwa Choi, Sheng Ding, Eva E. Prieschl, Thomas Baumruker, Jae-Mok Lee, Sung-Kee Chung*, Peter G. Schultz* Department of Chemistry, The Scripps Research Institute, 155 N. Torrey Pines Rd., San Diego, CA 9237 Department of Immunology, Novartis Research Institute, Vienna, Austria Department of Chemistry, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang , Korea Supporting Information Full names for the abbreviations used for compounds. [Cores] DES: D-erythro-sphingosine, LES: L-erythro-sphingosine, DTS: D-threosphingosine, LTS: L-threo-sphingosine, DED: D-erythro-dihydro-sphingosine, ED:erythro-dihydro-sphingosine (racemic), TD: threo-dihydro-sphingosine (racemic), PS: phytosphingosine, PC: psychosine, 1ES: 1-carbon-sphingosine, 12ES: 12-carbonsphingosine, 15ES: 15-carbon-sphingosine, 2ES: 2-carbon-sphingosine, NE: (+)- norephedrine, AP: 2-(aminomethyl)pyridine, DT: ditetradecylamine. [Tails] 2: acetyl, 3: propionyl, 4: butyryl, 5: valeryl, 6: hexanoyl, 7: heptanoyl, 8: octanoyl, 9: nonanoyl, 1: decanoyl, 12: lauroyl, 14: myristoyl, 16: palmitoyl, 18: stearoyl, 31: isobutyryl, 32: t-butylacetyl, 33: isovaleryl, 34: cyclobutane carbonyl, 35: cyclopentane carbonyl, 36: cyclohexane carbonyl, 37: cyclopentylpropionyl, 38: 2- methylvaleryl, 39: 3-methylvaleryl, 4: 4-methylvaleryl, 41: vinylacetyl, 42: pentenoyl, 43: heptenoyl, 44: trans-3-hexenoyl, 45: 2,2,-dimethyl-4-pentenoyl, 46: myristoleyl (cis- 9-tetradecenoyl), 47: myristoleyl (cis-9-hexadecenoyl), 48: oleyl (cis-9-octadecenoyl), 49: all-trans-retinoyl, 5: benzoyl. General Synthetic Methods Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. N,N'-dimethylformamide (DMF) was purchased as the anhydrous grade and stored over 4 Å molecular sieves. Unless otherwise reported all reactions were performed under a nitrogen atmosphere. For moisture sensitive reactions the reaction flasks were flame dried under vacuum (ca. 1mm Hg) prior to use. Removal of solvent in vacuo refers to distillation using a Buchi rotary evaporator attached to a vacuum pump (approx. 3 mmhg). Products obtained as solids or high boiling-point oils were dried under vacuum (ca. 1 mmhg). Department of Immunology, Novartis Research Instiute, Viena, Austria Department of Chemistry, Pohang University of Science and Technology

2 S2 Analytical thin-layer chromatography was performed on precoated silica plates (Merck 6-F 254,.25 mm thickness); compounds were visualized by UV light (254 nm) or by staining with phosphomolybdic acid or ninhydrin. 1 H NMR spectra were recorded on an AMX-4 (3 MHz), DRX-5 (5 MHz) or DRX-6 (6 MHz) at the Scripps Research Institute NMR facility. The high resolution mass spectra were recorded on an IonSpec Ultima FTMS at the Scripps Research Institute MS facility. Core structure of sphingosines Four stereoisomers of sphingosine, DES, DTS, LES and LTS were synthesized by a slightly modified literature procedure. 1 1ES, 12ES, 15ES and 2ES were synthesized using Ru catalyzed olefin metathesis. 2 The other core structures were purchased from Sigma-Aldrich. General procedure for activated ester synthesis using acyl chloride (for 2, 3, 4, 5, 6, 7, 8, 1, 12, 14, 16, 18, 31-37, 48, 5) Nitrophenol resin (1g,.87 mmol), which was prepared by a slightly modified literature procedure 3 was suspended in 1-methyl-2-pyrrolidinone (1mL). After 1 min, pyridine (1mL, 12.4 mmol) and the appropriate acyl chloride (4 mmol) were added to the reaction mixture and stirred overnight at room temperature. The slurry was filtered through a 7 µm frit and washed with 1-methyl-2-pyrrolidinone (3 x 2 ml), methanol-dmf (1:1, 3 x 2 ml) and methylene chloride (5 x 2 ml) and dried under a stream of nitrogen. General procedure for activated ester synthesis using acid (for 38-47, 49) Diisopropylcarbodiimide (DIC, 1mL, 6.4 mmol) was added to a mixture of nitrophenol resin (1g,.87 mmol), the appropriate acid (4. mmol) and 4-(dimethylamino)pyridine (5 mg) in 1-methyl-2-pyrrolidinone (1mL), and stirred overnight at room temperature. The resin was filtered, washed and dried as before. General procedure for ceramide synthesis An activated ester resin (5 mg, 75 umol) was added to a core amine (3 umol) in THF (1 ml) and agitated on a rocker overnight at room temperature. The reaction mixture was filtered and washed with a portion of THF (1 ml) and the filtrate was combined and dried. Completion of the reaction was confirmed by TLC and negative ninhydrin staining. The purity of each compound was checked by TLC and phosphomolybdic acid staining; each gave a single spot (except the DT compounds which stained poorly, and the 38 compounds which showed 2 diastereomers (table 1)). The R f value of the synthetic molecules are listed in Table 1. About 1 synthetic compounds among the 544 in the library were selected randomly and characterized by high resolution mass spectrometry (MALDI-FTMS) and H-NMR to confirm their identity and purity. Cytotoxicity assay in U937 cells Human U937 leukemic cells were obtained from the American Type Culture Collection (ATCC). Cells were grown in RPMI 164 medium supplemented with 1% fetal bovine 1 Chung, S. K.; Lee, J. M. Tetrahedron: Asymmetry 1999, 1, Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett. 1999, 1, Cohen, B. J.; Karoly-Hafeli, H.; Patchornik, A. J. J. Org. Chem. 1984, 49,

3 S3 serum (FBS) and kept at 37 o C in 5% CO 2 /95% air. To a 1 ul cell suspension (2, cells/well) in a 96-well plate, library compounds were added to give final concentrations of 1, 5, 25, 12.5, 6.2, 3.1, 1.6,.8 um and the cells were incubated at 37 o C in 5% CO 2 /95% air. After 24 h, MTS (2 ul, Promega, CellTiter 96 Aqueous One solution reagent) was added to the cells and incubated for an additional 2 h. The absorbance of the blue formazan product was measured at 492 nm with Multiskan Ascent plate reader (Labsystems). The IC 5 values for all library compounds are listed in Table 2. NF-κB transcriptional assay The NF-κB luciferase reporter construct was a generous gift from Dr. Ghosh. 4 C6 glioma (ATCC) were electroporated with 5 µg each of NF-κB-luciferase and CMV-βgalactosidase reporter vector. The cells were plated in 1 % FBS/DMEM, and after overnight incubation the medium was changed to DMEM. After 1 day, library compounds (1 µm) were added to the cells (Figure 1). The activation assay did not require further treatment; for the inhibition assay, TNF-α (5 ng/ml) or IL-1β (1 ng/ml) were added to the wells 3 min after of the library compound addition. The cells were then incubated for an additional 8 h and were harvested and assayed for luciferase and β- galactosidase activity as previously described. 5 Mast cell activation assay The assay used a genetically modified subline of the murine mast cell line CPII (clone 12), which contains a TNFα promoter-luciferase-tnfα 3 region reporter gene construct (NF-AT and AP1 dependent) stably integrated into the genome. 4 x 1 4 cells were seeded in 5 µl Tyrode-BSA buffer per well and stimulated for 4 h with the compounds in the presence or absence of 1 ng/ml ionomycin (Sigma, St. Louis, MO). As a positive control 2 µg / ml anti DNP/TNP-IgE (Pharmingen, La Jolla, CA) and 1 ng/ml DNP- BSA (Calbiochem, La Jolla, CA) were applied as stimulants. Cells were lysed by the addition of 13 µl 5 x lysis buffer (Promega, Madison, WI) and one freeze / thaw cycle before adding the luciferase substrate (Promega) and measuring the readout. Selected data are summarized in Figure 2 and 3. 4 Kopp, E.; Ghosh, S. Science 1994, 265, Crossin, K. L.; Tai, M. H.; Krushel, L.; Mauro, V. P.; Edelman, G. M. Proc. Natl. Acad. Sci. U. S. A. 1997, 94,

4 S NF-kB activity (%) DMSO PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC1 PC12 PC14 PC16 PC18 PC31 PC32 PC33 PC34 PC35 PC36 PC37 PC38 PC39 PC4 PC41 PC42 PC43 PC44 PC45 PC46 PC47 PC48 PC49 PC5 Figure 1. NF-κB activation by PCs in C6 glyoma cells (at 1 µm).

5 S5 7 luciferase values (TNF α ) nst PC-1 PC-3 PC-4 PC-41 PC-42 PC-44 7 luciferase values (TNF α ) nst IgE / Ag Iono. PC-1 PC-3 PC-4 PC-41 PC-42 PC-44 Iono. Figure 2. Mast cell activation by PC-ceramides. Values represent mean of quadruplicate experiments +/- SD. Nst means non-stimulated, iono. means ionomycin.

6 S6 7 luciferase values (TNF α ) nst GP-1 GP-5 GP-7 GP-9 GP-12 GP-32 nst IgE / Ag Iono luciferase values (TNF α ) GP-1 GP-5 GP-7 GP-9 GP-12 GP-32 Iono. Figure 3. Mast cell activation by GP-ceramides. Values represent mean of quadruplicate experiments +/- SD. Nst means non-stimulated, iono. means ionomycin.

7 S7 Table1. Rf values on TLC 1ES 12ES 15ES 2ES DES/LES DTS/LTS ED/DE D TD NE PS PC AP condition A A A A A A B B B B C B ,.4.3,.4.4,.5.4,.5.4,.5.5,.6.5,.7.6, , , , A: Ethyl Acetate:Hexane (3:1), phosphomolybdic acid staining B: Ethyl Acetate, phosphomolybdic acid staining C: Methanol:Methylene Chloride (1:1), phosphomolybdic acid staining

8 S8 Table2. IC5 values of cytotoxicity in U937 cells 1ES 12ES 15ES DES 2ES LES DTS LTS ED TD DED NE PS PC AP DT > >1 >1 2 >1 >1 8 6 > >1 >1 >1 > >1 >1 3 >1 > > >1 7 >1 > >1 >1 4 > >1 >1 >1 > >1 >1 5 > >1 >1 >1 > >1 > >1 >1 >1 > >1 > >1 >1 >1 > >1 > > >1 > >1 > > >1 >1 >1 > >1 > > >1 >1 > >1 > >1 >1 >1 >1 >1 >1 >1 >1 >1 8 6 >1 >1 > >1 >1 >1 >1 >1 >1 >1 >1 >1 >1 9 >1 >1 > >1 >1 >1 >1 >1 >1 1 >1 >1 >1 55 >1 >1 >1 18 >1 7 >1 8 >1 >1 >1 >1 >1 >1 >1 >1 25 >1 >1 >1 31 > > > >1 >1 32 > > >1 2 3 >1 >1 33 > >1 >1 >1 >1 2 2 >1 >1 34 > >1 >1 >1 >1 4 2 >1 >1 35 > > >1 >1 > >1 >1 36 > >1 >1 >1 > >1 > > >1 >1 >1 > >1 > >1 8 >1 >1 2 2 >1 > >1 75 >1 > >1 >1 4 > > >1 75 >1 >1 2 3 >1 >1 41 > > >1 >1 >1 > >1 >1 42 > > >1 >1 >1 > >1 >1 43 > > >1 >1 >1 >1 1 3 >1 > > >1 >1 9 > >1 >1 45 > > > >1 > >1 9 >1 >1 >1 >1 > > >1 96 >1 >1 >1 >1 > > >1 75 >1 >1 >1 >1 >1 >1 > >1 > > > >1 >1 5 > > >1 >1 6 > >1 Average of at least two independent experiments