Synthesis and Proinflammatory Responses of H. pylori LPS Structures
FULL PAPER
2-Deoxy-6-O-{2-deoxy-2-[(R)-3-(octadecanoyloxy)octadecanoylamino]-b-
d-glucopyranosyl}-2-[(R)-3-hydroxyoctadecanoylamino]-a-d-glucopyra-
nose 1-phosphate (1): Pd-black (10.0 mg) was added to a solution of 15
(11.0 mg, 5.63 mmol) in distilled THF (1.0 mL). The mixture was stirred
under hydrogen (2.0 MPa) at room temperature for 18 h and then neu-
tralized with Et3N in THF (10%, v/v, 20.0 mL), and the Pd catalyst was
removed by filtration. After removal of the solvent in vacuo, the residue
was lyophilized from tBuOH to afford 1 as a triethylammonium salt
(white solid, 7.0 mg, quant). 1H NMR (500 MHz, CDCl3/CD3OD): d=
5.47 (d, J=4.4 Hz, 1H; H-1), 5.27 (brs, 1H; b-CH of 2’-N-acyl), 4.62 (d,
J=8.8 Hz, 1H; H-1’), 4.08 (t, J=9.8 Hz, 1H; H-5), 4.02 (d, J=12.1 Hz,
1H; b-CH of 2-N-acyl), 4.02 (d, J=8.6 Hz, 1H; H-2), 3.89 (d, J=11.8 Hz,
1H; H-6a), 3.83 (dd, J=11.8, 6.3 Hz, 1H; H-6b), 3.78–3.65 (m, 2H; H-3,
H-2’), 3.45–3.24 (m, 4H; H-3’, H-4’, H-6’a, H-6’b), 3.23–3.16 (m, 2H; H-
4, H-5’), 2.58 (t, J=6.3 Hz, 2H; a-CH2 of 2’-N-acyl main chain), 2.39–
2.27 (m, 4H; a-CH2 of 2’-N-acyl side chain and 2-N-acyl), 1.76–1.15 (m,
84H; CH2 of 2-N-acyl and 2’-N-acyl), 0.89 ppm (t, J=6.2 Hz, 9H; -CH2-
CH3 of 2-N-acyl and 2’-N-acyl); HRMS (ESI-Q-TOF, negative): m/z:
calcd. for C66H127N2O17P [MꢀH]ꢀ: 1249.8794; found: 1249.8794.
hexane and water and then was lyophilized from tBuOH to afford 4 as a
white solid (110 mg, 15%). H NMR is shown in the Supporting Informa-
1
tion. HRMS (ESI-Q-TOF, negative): m/z: calcd for C76H144N3O24P
[MꢀH]ꢀ: 1512.9799; found: 1512.9792.
Cytokine assay: The cytokine-inducing activities of the synthetic lipid A
compounds and LPS (E. coli O111:B4, Sigma–Aldrich as a positive con-
trol) were tested in heparinized human peripheral whole blood (HPWB)
cells collected from the blood of an adult volunteer. The synthesized
samples (dissolved in 25 mL of 5% DMSO in saline) and HPWB (25 mL)
in RPMI1640 medium (75 mL) including meylon (3%), were incubated in
triplicate in a 96-well plastic plate at 378C under CO2 (5%). To maintain
the solubility of the synthetic compounds this culture included DMSO
(1%).[54] After 20 h of incubation and subsequent centrifugal separation
(at 300g for 2 min), the supernatant proteins were collected and the in-
duced quantities of cytokines were then measured by ELISA assay
(ELISA Ready-SET-Go! (eBioscience) for human IL-1b, IL-6, IL-12p70,
IL-8, and TNF-a; Human IL-18 ELISA Kit (MBL) for human IL-18).
For the inhibitory activities of the synthetic lipid A compounds and Kdo-
lipid A compounds against E. coli LPS, the synthetic compounds and ad-
ditional E. coli LPS (500 pgmLꢀ1) in RPMI 1640 medium including
meylon (3%) and a buffer solution were incubated and measured by a
method similar to that used for the inducing activities. Data represent
averages of three repeated assays with standard deviations from individu-
al experiments.
2-Deoxy-6-O-{2-deoxy-2-[(R)-3-(octadecanoyloxy)octadecanoylamino]-b-
d-glucopyranosyl}-2-[(R)-3-hydroxyoctadecanoylamino]-a-d-glucopyra-
nose 1-(aminoethyl)phosphate (2): Pd(OH)2/C (20 wt% on carbon,
20.0 mg) was added to a solution of 17 (20.0 mg, 9.79 mmol) in distilled
THF (1.0 mL), water (100 mL), and AcOH (50 mL). The mixture was
stirred under hydrogen (2.0 MPa) at room temperature for 2 d and then
neutralized with Et3N in THF (10%, v/v, 20.0 mL), and Pd catalyst was
removed by filtration. After removal of the solvent in vacuo, the residue
was lyophilized from tBuOH to afford 2 as a colorless solid (11.2 mg,
89%). 1H NMR (500 MHz, CDCl3/CD3OD): d=5.47 (d, J=8.3 Hz, 1H;
H-1), 5.26–5.22 (m, 1H; b-CH of 2’-N-acyl), 4.62 (d, J=8.5 Hz, 1H; H-
1’), 4.21 (dd, J=18.2, 8.5 Hz, 1H; H-5), 4.04 (d, J=12.1 Hz, 1H; H-6a),
3.95–3.90 (m, 2H; H-2, b-CH of 2-N-acyl), 3.88 (d, J=11.8 Hz, 1H;
-OPO-CH2-CH2-), 3.81 (t, J=10.9 Hz, 1H; H-6b), 3.72–3.65 (m, 2H; H-3,
-OPO-CH2-CH2-), 3.60–3.55 (m, 1H; H-2’), 3.48–3.20 (m, 6H; H-3’, H-4’,
H-6’a, H-6’b, -OPO-CH2-CH2-), 3.13–3.10 (m, 1H; H-5’), 2.95 (brs, 1H;
H-4), 2.56–2.42 (m, 2H; a-CH2 of 2’-N-acyl main chain), 2.41–2.22 (m,
4H; a-CH2 of 2’-N-acyl side chain and 2-N-acyl), 1.62–1.15 (m, 84H;
CH2 of 2-N-acyl and 2’-N-acyl), 0.88 ppm (brs, 9H; -CH2-CH3 of 2-N-
acyl and 2’-N-acyl); HRMS (ESI-Q-TOF, negative): m/z: calcd for
C68H132N3O17P [MꢀH]ꢀ: 1292.9216; found: 1292.9211.
Acknowledgements
This work was supported in part by Grants-in Aid for Scientific Research
(Nos. 22310136, 20241053, 19310144, 17310128, and 20–870) and the
NEXT program from the Japan Society for the Promotion of Science, as
well as by grants from Osaka University Global COE program (Frontier
Biomedical Science Underlying Organelle Network Biology), the Naito
Foundation, and the Takeda Science Foundation. The authors would also
like to acknowledge Prof. Yasuhiro Kajihara, Seiji Adachi, and Naoya In-
azumi of Osaka University for the NMR measurements and fruitful dis-
cussions.
2-Deoxy-6-O-{2-deoxy-6-O-(3-deoxy-a-d-manno-oct-2-ulopyranosid)onic
acid]-2-[(R)-3-(octadecanoyloxy)octadecanoylamino]-b-d-glucopyrano-
[4] S. O. Hynes, J. A. Ferris, B. Szponar, T. Wadstrom, J. G. Fox, J. OꢀR-
ourke, L. Larsson, E. Yaquian, A. Ljungh, M. Clyne, L. P. Andersen,
[6] G. I. Perez-Perez, V. L. Shepherd, J. D. Morrow, M. J. Blaser, Infect.
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J. E. Conlon, K. Fukase, S. Kusumoto, C. Sweet, K. Miyake, S.
syl}-2-[(R)-3-benzyloxyoctadecanoylamino]-a-d-glucopyranose
1-phos-
phate (3): Pd-black (6.0 mg) was added to a solution of 23 (6.3 mg,
2.70 mmol) in distilled THF (500 mL). The mixture was stirred under hy-
drogen (2.0 MPa) at room temperature for 5 d and then neutralized with
Et3N in THF (10%, v/v, 20.0 mL), and Pd catalyst was removed by filtra-
tion. After removal of the solvent in vacuo, the residue was lyophilized
from tBuOH to afford 3 as a triethylammonium salt (white solid, 4.3 mg,
quant). 1H NMR (600 MHz, CDCl3/CD3OD): d=5.48 (brs, 1H; H-1),
5.26–5.18 (m, 1H; b-CH of 2’-N-acyl), 4.46–4.40 (m, 1H; H-1’), 4.08–3.92
(m, 5H; H-2, H-5, H-4’’, H-7’’, b-CH of 2-N-acyl), 3.84–3.56 (m, 9H; H-3,
H-6a, H-6b, H-2’, H-5’, H-5’’, H-6’’, H-8’’a, H-8’’b), 3.51–3.42 (m, 3H; H-
3’, H-6’a, H-6’b), 3.38–3.26 (m, 2H; H-4, H-4’), 2.56–2.50 (m, 2H; a-CH2
of 2’-N-acyl main chain), 2.42–2.26 (m, 4H; a-CH2 of 2’-N-acyl side chain
and 2-N-acyl), 2.10–2.04 (m, 1H; H-3’’a), 1.92–1.84 (m, 1H; H-3’’b), 1.78–
1.14 (m, 84H; CH2 of 2-N-acyl and 2’-N-acyl), 0.89 ppm (t, J=7.2 Hz,
9H; -CH2-CH3 of 2-N-acyl and 2’-N-acyl); HRMS (ESI-Q-TOF, nega-
tive): m/z: calcd for C74H139N2O24P [MꢀH]ꢀ: 1469.9377; found: 1469.9371.
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2-Deoxy-6-O-[2-deoxy-6-O-(3-deoxy-a-d-manno-oct-2-ulopyranosid)onic
acid]-2-{[(R)-3-(octadecanoyloxy)octadecanoylamino]-b-d-glucopyrano-
syl}-2-[(R)-3-benzyloxyoctadecanoylamino]-a-d-glucopyranose 1-(amino-
ethyl)phosphate (4): Pd(OH)2/C (20 wt% on carbon, 1.0 mg) was added
to a solution of 25 (1.3 mg, 0.495 mmol) in distilled THF (435 mL), water
(43 mL), and AcOH (22 mL). The mixture was stirred under hydrogen
(2.0 MPa) at room temperature for 2 d and then neutralized with Et3N in
THF (10%, v/v, 20.0 mL), and Pd catalyst was removed by filtration.
After removal of the solvent in vacuo, the residue was washed with n-
[11] J. Danesh, Y. Wong, M. Ward, J. Muir, Heart 1999, 81, 245–247.
[12] C. C. Kuo, J. T. Grayston, L. A. Campbell, Y. A. Goo, R. W. Wissler,
[14] A. Schumacher, I. Seljeflot, A. B. Lerkerod, L. Sommervoll, J. E.
[15] G. Hajishengallis, A. Sharma, M. W. Russell, R. J. Genco, Ann. Pe-
Chem. Eur. J. 2011, 17, 14464 – 14474
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