Synthesis of Helicobacter pylori lipid A and its analogue using p-(trifluoromethyl)benzyl protecting group

Article abstract of DOI:10.1016/S0040-4039(00)01158-8

Synthesis of lipid A 1 isolated from Helicobacter pylori strain-206-1 has been achieved in 2.2% total yield through 14 steps from D-glucosamine by employing a p-(trifluoromethyl)benzyl group for protection of the hydroxy group on the 3-hydroxy fatty acid residue. The synthetic specimen was identical with the natural counterpart in chromatographic, spectroscopic, and biological aspects. A structural analogue 2 which lacks the ethanolamine residue of 1 was also synthesized, and 2 was found to exhibit less potent IL-1β-inducing activity than 1. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01158-8

Tetrahedron Letters 41 (2000) 6843±6847
Synthesis of Helicobacter pylori lipid A and its analogue using
p-(tri¯uoromethyl)benzyl protecting group
Yasuhiro Sakai,a Masato Oikawa,b,* Hiroaki Yoshizaki,b Tomohiko Ogawa,c
Yasuo Suda,b Koichi Fukaseb and Shoichi Kusumotob,*
aNutrition Research Institute, Otsuka Pharmaceutical Factory, Inc., Muya-Cho, Naruto,
Tokushima 772-8601, Japan
bDepartment of Chemistry, Graduate School of Science, Osaka University, Machikaneyama-cho 1-1, Toyonaka,
Osaka 560-0043, Japan
cDepartment of Oral Microbiology, Asahi University School of Dentistry, 1851-1 Hozumi, Hozumi-cho, Motosu-gun,
Gifu 501-0296, Japan
Received 12 June 2000; revised 3 July 2000; accepted 7 July 2000
Abstract
Synthesis of lipid A 1 isolated from Helicobacter pylori strain 206-1 has been achieved in 2.2% total yield
through 14 steps from d-glucosamine by employing a p-(tri¯uoromethyl)benzyl group for protection of the
hydroxy group on the 3-hydroxy fatty acid residue. The synthetic specimen was identical with the natural
counterpart in chromatographic, spectroscopic, and biological aspects. A structural analogue 2 which lacks
the ethanolamine residue of 1 was also synthesized, and 2 was found to exhibit less potent IL-1b-inducing
activity than 1. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: lipopolysaccharides; lipid A; Helicobacter pylori; IL-1b induction.
Helicobacter pylori is Gram-negative bacteria often observed in the stomach of patients with
chronic gastritis. The bacteria are also considered to be a possible cause of gastric and duodenal
ulcers. We have studied the chemical components of H. pylori strain 206-1 and succeeded in the
isolation and structural determination of its characteristic lipid A 1 (Fig. 1),1 which is the
proximal partial structure of lipopolysaccharide constituting the outermost lea¯et of the outer
membrane bilayer. The chemical structure of H. pylori lipid A 1 is di€erent from that of
Escherichia coli as follows: (1) the presence of a smaller number of fatty acid residues but of
longer chain lengths; (2) the absence of the phosphate group at the C40-position; and (3) the
presence of an ethanolamine group linked to the glycosyl phosphate functionality. This particular
lipid A 1 was found to exhibit lower mitogenic activity than E. coli lipid A, whereas 1 induces the
production of a comparable amount of interleukin-6 (IL-6) from human peripheral blood cells.2
* Corresponding authors. Tel: 81-6-6850-5391; fax: 81-6-6850-5419; e-mail: moik@chem.sci.osaka-u.ac.jp
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01158-8

6844
Figure 1.
In this letter, we report the synthesis of 1 and its analogue 2, which lacks the ethanolamine group,
by using novel methods for hydroxy group protection and phosphono group introduction. The
IL-1b-inducing activity of the synthetic preparations is also described.
The synthesis was achieved using the benzyl-type protecting groups for all active functionalities
to be protected until the ®nal step. The parent benzyl (Bn) group used for the protection of the
hydroxy groups on the 3-hydroxyacyl residue was, however, found in our previous study to be
susceptible to oxidation: it is partly converted into a benzoyl group even by air oxidation during
storage. To solve this problem, the p-(tri¯uoromethyl)benzyl group was newly developed in the
present study.3,4 Comparison of reactivities of unsubstituted and p-tri¯uoromethylated benzyl
ethers is summarized in Table 1. Toward DDQ oxidation, the p-(tri¯uoromethyl)benzyl group
was found to be stable under the conditions where the Bn group was nearly completely cleaved.
For the reductive cleavage of the former, hydrogenolysis under higher hydrogen pressure was
required than for the Bn group, but the cleavage yield was quantitative.
Table 1
Removal of Bn and p-(tri¯uoromethyl)benzyl groups under oxidative or reductive conditions
The known protected glucosamine 6,5 prepared from d-glucosamine hydrochloride in three
steps, was benzylated to give 7 in 89% yield (Scheme 1). Reductive opening of the benzylidene
6
.
.
group in 7 was then e€ected by our protocol (BH3 Me2NH, BF3 OEt2, CH2Cl2) in 86% yield,
providing exclusively the 4-O-Bn ether 8 which is a common intermediate for the synthesis of
both glucosamine components (10, 13) for the subsequent disaccharide formation. The glycosyl

6845
acceptor 10 was prepared by the reductive deprotection of the N-Troc group in 8 followed by
acylation of the generated amino group with (R)-3-(p-(tri¯uoromethyl)benzyloxy)octadecanoic
acid (RCOOH, 9).y
Scheme 1.
Benzyloxymethylation of the free hydroxy group of 8 gave a fully protected glucosamine
derivative 11 quantitatively. After cleavage of the allyl group in 11 by successive treatments with
an activated cationic iridium complex and aqueous iodine,7 the product 12 was treated with
CCl3CN in the presence of Cs2CO3 as a catalyst to give the glycosyl trichloroacetimidate 13.
Coupling of the two glucosamine derivatives (10, 13) proceeded smoothly by the action of a
catalytic amount of TMSOTf to a€ord the b(1,6)-disaccharide 14 in a stereoselective manner by
neighboring carbamate group participation (Scheme 2). N-acylation with (R)-3-(octadecanoyloxy)-
octadecanoic acid (R0COOH, 15) followed by allyl group removal was then carried out by
standard reaction procedures to furnish 17 ready for the next phosphorylation.
Scheme 2.
y
The carboxylic acids 9 and 15 were synthesized according to our published procedure.10

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 ecient 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 e€ect 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 e€ect 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
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6847
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