6846
For a-selective introduction of the dihydrogen phosphate group at the glycosidic position of
the disaccharide backbone of lipid A, we have already established an ecient method by using
tetrabenzyl diphosphate.8 In the case of an asymmetric phosphodiester-type structure, like the
present 2-aminoethyl hydrogen phosphate linked at the same position, no promising route to its
stereoselective formation was known. The typical phosphoramidite procedure was, therefore, ®rst
examined and it turned out to give the desired a-anomer exclusively, though the yield was not
very high. Thus, successive phosphitylation of 17 with the phosphoramidite 189 in the presence of
1H-tetrazole followed by oxidation with m-CPBA gave the fully protected lipid A 19 as a
diastereomeric mixture at the phosphorus atom. No trace of the corresponding b-phosphate
isomer was detected after the next hydrogenolytic deprotection though the starting 17 was used as
an anomeric mixture. This exclusive stereoselectivity would be due to the instability of the
b-phosphate moiety that might readily isomerize to the a-con®guration or be cleaved spontaneously.
The ®nal one-step hydrogenolytic deprotection was performed by 9 kg cm^2 of H2 over Pd(OH)2/
C to provide the desired lipid A 1 as a white powder in 25% yield after silica gel chromatography.
The synthetic lipid A proved to be identical with the natural counterpart1 in chromatographic
1
aspects. This was also con®rmed by comparison of their NMR (1H, H±1H COSY) and ESI-MS
(negative mode) spectra. By the use of the p-(tri¯uoromethyl)benzyl group for protection of the
3-hydroxy fatty acid, oxidative formation of the corresponding benzoyl derivative was completely
avoided, which certainly made the ®nal puri®cation easier.
An analogue 2 that lacks the ethanolamine residue of 1 was also synthesized by treating 17 with
8,10
tetrabenzyl diphosphate and LiN(TMS)2
followed by hydrogenolytic deprotection (8 kg cm^2
of H2, Pd black) of 20. The crude material was puri®ed by liquid±liquid partition chromatography5
on Sephadex1 LH-20 gel furnishing the analogue 2.
The synthetic lipid A was found to show identical biological activities (mitogenic eect and
TNF-a production) with the natural product whose activity is known to be weaker than that of
Escherichia coli lipid A.2 The amounts of interleukin-1b, induced in human peripheral blood
mononuclear cells by 0.1 ng/well of 1 and 2, were determined to be 2125 pg/mL and 982 pg/mL,
respectively. The analogue 2 thus showed lower activity in interleukin-1b production at this
concentration than H. pylori lipid A 1, suggesting the positive eect of the ethanolamine residue
in the cytokine-inducing activity. Further studies on the bioactivity and other physical properties
of 1 and 2 are currently under way.
Acknowledgements
The authors thank Mr. Seiji Adachi of Osaka University for his skillful measurement of NMR
spectra.
References
1. Suda, Y.; Ogawa, T.; Kashihara, W.; Oikawa, M.; Shimoyama, T.; Hayashi, T.; Tamura, T.; Kusumoto, S.
J. Biochem. 1997, 121, 1129±1133.
2. Ogawa, T.; Suda, Y.; Kashihara, W.; Hayashi, T.; Shimoyama, T.; Kusumoto, S.; Tamura, T. Vaccine 1997, 15,
1598±1605.
3. Yoshizaki, H.; Wada, A.; Oikawa, M.; Suda, Y.; Fukase, K.; Kusumoto, S. 72th National Meeting of the Chemical
Society of Japan, March 1997, Abstract No. 3G312.