Paper
Compound 18d: In a manner similar to the synthesis of
Organic & Biomolecular Chemistry
1d: ESI-MS (negative) m/z = 1133.88 [M − H]−; HR-MS (m/z):
18a, the azido group of 17d (9.1 mg, 0.00615 mmol) was Calcd for C60H115N2O15P [M
reduced to an acetylamino group to give 18d (9.2 mg, 100%) as 1133.7961.
−
H]−, 1133.7962: Found,
a white syrup. ESI-MS (positive) m/z = 1496.02 [M + H]+;
1e: ESI-MS (negative) m/z = 1161.87 [M − H]−; HR-MS (m/z):
HR-MS (m/z): Calcd for C88H139N2O15NaP [M
1517.9811: Found, 1517.9814.
+
Na]+, Calcd for C62H119N2O15P [M
1161.8274.
−
H]−, 1161.8275: Found,
Compound 18e: In a manner similar to the synthesis of
Cytokine assay. The cytokine inducing activities of synthetic
18a, the azido group of 17e (23.1 mg, 0.0153 mmol) was phosphoglycolipids and LPS (E. coli O111:B4, Sigma-Aldrich
reduced to an acetylamino group to give 18e (19.5 mg, 84%) as Chemicals Co., as a positive control) were tested in hepari-
a white syrup. ESI-MS (positive) m/z = 1524.03 [M + H]+; nized human peripheral whole blood (HPWB) cells, collected
HR-MS (m/z): Calcd for C90H143N2O15NaP [M
+
Na]+, from a blood of an adult volunteer. The synthesized samples
1546.0124: Found, 1546.0129.
(dissolved in 25 μL of 5% DMSO in saline) and HPWB (25 μL)
Phosphoglycolipid 1a. To a solution of compound 18a in an RPMI1640 medium (75 μL), including 3% meylon, were
(15.8 mg, 0.0103 mmol) in distilled THF (1.5 mL) was added incubated in triplicate in a 96-well plastic plate at 37 °C in 5%
Pd/C (44 mg). The mixture was stirred under 7.0 kg cm−2 of H2 CO2. To maintain the solubility of synthetic compounds, the
at room temperature for 4 days. The catalyst was filtered off by culture contained 1% DMSO.20 After 20 h of incubation and
a membrane filter and the filtrate was extracted with MeOH. subsequent centrifugal separation (at 300g for 2 min), the
The solution was concentrated in vacuo to give 1a (9.7 mg, supernatants were collected and then the induced quantities
75%) as a white solid.
ESI-MS (negative) m/z = 1175.92 [M − H]−; HR-MS (m/z): and TNF-α: ELISA Ready-SET-Go! (eBioscience)).
Calcd for C63H120N2O15P [M
H]−, 1175.8432: Found,
of cytokines were measured by ELISA assay (for human IL-6,
−
1
1175.8434; H-NMR (600 MHz, CDCl3–CD3OD = 3/1) δ (ppm) =
5.25–5.22 (m, 1H, glycerol H-2), 4.78–4.75 (m, 1H, glyceroyl
H-2), 4.77 (d, J = 3.3 Hz, 1H, Glc H-1), 4.38 (dd, J = 12.1 Hz, 3.3
Hz, 1H, glycerol H-1a), 4.16 (dd, J = 12.1 Hz, 6.8 Hz, 1H, gly-
Acknowledgements
This work was supported in part by Grants-in-Aid for Scientific
Research from the Japan Society for the Promotion of Science,
by grants from Suntory Institute for Bioorganic Research
(SUNBOR Grant), the Houansha Foundation, Takeda Science
Foundation, the Naito Foundation, Osaka University Global
COE program (Frontier Biomedical Science Underlying Orga-
nelle Network Biology), ERATO, Murata Lipid Active Structure
Project, and a funding program for Next Generation World-
Leading Researchers (NEXT Program; LR025) from JSPS and
CSTP.
3
cerol H-1b), 3.99 (t, J = 6.6 Hz, J (P,H) = 6.6 Hz, 2H, glycerol
H-3), 3.91 (dd, J = 10.4 Hz, 3.3 Hz, 1H, Glc H-2), 3.80–3.78 (2H,
glyceroyl H-2)a, 3.80–3.74 (4H, Glc, H-3 and H-5 and H-6)a,
3.37–3.34 (ddb, 1H, Glc H-4), 3.27–3.19 (m, 2H, –CONH–CH2–),
2.34–2.30 (m, 4H, –OCOCH2– × 2), 2.04 (s, 3H, –NHCO–CH3),
1.63–1.58 (m, 4H, –OCOCH2–CH2– × 2), 1.55–1.47 (m, 5H, –CH-
(CH3)2 × 3 and –CO–NH–CH2–CH2–), 1.30–1.26 (m, 58H, –
(CH2)– × 29), 1.17–1.14 (m, 6H, –CH2–CH(CH3)2 × 3), 0.86 (d, J =
6.6 Hz, 18H, –CH(CH3)2 × 3); 13C-NMR (150 MHz, CDCl3–
CD3OD = 3/1) δ (ppm) = 174.1 (s), 174.0 (s), 173.1 (s), 170.0 (d),
97.4 (s), 75.6 (d), 72.2 (s × 2), 71.5 (s), 70.7 (d), 68.3 (s),
64.0 (d), 62.8 (s), 62.0 (s), 53.9 (s), 39.6 (s), 39.3 (s × 3), 34.4 (s),
34.3 (s), 30.2–27.2 (30C), 28.2 (s × 3), 25.1 (s × 2), 22.8 (s × 6),
22.7 (s).
Notes and references
1 O. Takeuchi and S. Akira, Cell, 2010, 140, 805.
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7, e35067.
The diastereomers 1b, 1c, 1d and 1e were synthesized from
18b in a manner similar to that for 1a.
1b: ESI-MS (negative) m/z = 1175.32 [M − H]−; 1H-NMR
(500 MHz, CDCl3–CD3OD = 3/1) δ (ppm) = 5.17 (m, 1H, glycer-
oyl H-2), 4.74 (d, J = 3.0 Hz, 1H, Glc H-1), 4.72 (m, 1H, –CH-
(OP)–CONHR), 4.33 and 4.12 (m, 2H, glycerol 3-CHaHbOCOR),
3.98 (m, 1H, glycerol 1-CH2OP), 3.87 (dd, J = 10.7, 3.0 Hz, 1H,
H-2), 3.81–3.74 (m, 3H, Glc–O–CH2–CH(OP)– and Glc H-6a),
3.68–3.65 (m, 2H, Glc H-3 and Glc H-6b), 3.63–3.61 (m, 1H, Glc
H-5), 3.32–3.30 (m, 1H, Glc H-4), 3.21–3.13 (m, 2H, –CONH–
CH2–), 2.29–2.23 (m, 4H, OCOCH2– × 2), 2.00–1.97 (m, 3H,
NHCOCH3), 1.57–1.52 (m, 4H, OCOCH2–CH2– × 2), 1.50–1.43
(m, 5H, –CONH–CH2–CH2– and –CH(CH3)2– × 2), 1.26–1.11 (m,
64H, –(CH2)– × 32), 0.86 (d, J = 4.8, 18H, –CH(CH3)2 × 3).
1c: ESI-MS (negative) m/z = 1203.95 [M − H]−; HR-MS (m/z):
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M. Parrilli, R. Lanzetta and O. Holst, Glycobiology, 2006, 16,
766.
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Calcd for C65H124N2O15P [M
−
H]−, 1203.8745: Found, 10 T. Tanaka, M. Noguchi, A. Kobayashi and S.-I. Shoda,
1203.8749.
Chem. Commun., 2008, 2016.
5040 | Org. Biomol. Chem., 2013, 11, 5034–5041
This journal is © The Royal Society of Chemistry 2013