Synthesis of immunoregulatory Helicobacter pylori lipopolysaccharide partial structures

Article abstract of DOI:10.1016/j.tetlet.2007.07.036

The synthesis of immunoregulatory glycoconjugates, namely the active entity of lipopolysaccharide (LPS) from Helicobacter pylori was achieved. The results of biological activities of the LPS partial structures provide the structural basis for the immunobi

Full text of DOI:10.1016/j.tetlet.2007.07.036

Tetrahedron Letters 48 (2007) 6577–6581
Synthesis of immunoregulatory Helicobacter
pylori lipopolysaccharide partial structures
Yukari Fujimoto,a Masato Iwata,a Noriko Imakita,a Atsushi Shimoyama,a Yasuo Suda,b
Shoichi Kusumotoc and Koichi Fukasea,*
aDepartment of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
bDepartment of Nanostructure and Advanced Materials, Graduate School of Science and Engineering Kagoshima University,
Kagoshima 890-0065, Japan
cSuntory Institute for Bioorganic Research, Shimamoto, Osaka 618-8503, Japan
Received 8 June 2007; revised 30 June 2007; accepted 3 July 2007
Available online 2 August 2007
This Letter is dedicated to the memory of the late Professor Yoshihiko Ito
Abstract—The synthesis of immunoregulatory glycoconjugates, namely the active entity of lipopolysaccharide (LPS) from Helico-
bacter pylori was achieved. The results of biological activities of the LPS partial structures provide the structural basis for the immu-
nobiological activity, especially for the immune inhibitory activity of H. pylori LPS.
Ó 2007 Elsevier Ltd. All rights reserved.
Helicobacter pylori is a Gram-negative bacterium and an
etiological agent of gastroduodenal diseases such as
chronic gastritis, gastroduodenal ulcers and gastric can-
cer.1,2 Gram-negative bacteria are distinguished by the
presence of lipopolysaccharide (LPS) in their outer
membrane. LPS, also called an endotoxin, is a potent
immunostimulator, often causing sepsis in severe infec-
tions. However, H. pylori lipopolysaccharide (LPS)
shows very low endotoxic activity compared to other
enterobacterial preparations such as Escherichia coli
LPS.3–6 The H. pylori LPS has the characteristic lipid
A component, the structure of which is distinct from
those of the enterobacterial ones as in the following
ways: (1) the presence of fewer, but longer fatty acid res-
idues, (2) the absence of the 40-phosphate group and (3)
the occasional presence of an ethanolamine group linked
to the glycosyl phosphate functionality.7,8 We have pre-
viously synthesized H. pylori strain 206-1 lipid A, which
has tri-acyl groups and ethanolamine at the anomeric
phosphate (1b). This lipid A induced lower levels of
cytokines such as IL-18 and TNF-a upon activation of
the LPS receptor, a Toll-like receptor 4 (TLR4)/MD-2
complex,9,10 as it was known that the activation leads
to induction of proinflammatory cytokines such as IL-
6, IL-18 and TNF-a. On the other hand, it has been
recently reported that H. pylori LPS from other strains
has inhibitory effects on TLR4 activation.11 This dis-
crepancy in biological activities may come from the
structural heterogeneity of LPS, especially in the acyl
group distribution and in the phosphate part in lipid
A (Fig. 1, 1a–d). Synthetic study is therefore required
for elucidation of biological activity of H. pylori LPS,
since chemical synthesis can provide homogeneous
preparations.
It has been reported that the number of acyl groups is
crucial for the biological activity of lipid A.12,13 E. coli
type lipid A having hexaacyl groups has strong endo-
toxic activity, whereas a biosynthetic precursor Ia hav-
ing tetraacyl groups shows antagonistic activity in
humans.12 Triacyl lipid As also showed antagonistic
activity though the potency was low. There have been
known some bacteria, LPS of which have significant
inhibitory activity against E. coli LPS, such as Rhodo-
bacter sphaeroides,14,15 and Rhodobacter capsulatus,16
which have an unsaturated acyl group, and also Rhizo-
bium sin-117,18 which has pentaacyl groups of relatively
longer fatty acids and lactone structure at the anomeric
position. However, in the case of H. pylori, the LPS
shows weaker antagonistic activity, and at the same
time, shows weak proinflammatory activity. Chain
*
Corresponding author. Tel.: +81 6 6850 5388; fax: +81 6 6850
5419; e-mail: koichi@chem.sci.osaka-u.ac.jp
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.07.036

6578
Y. Fujimoto et al. / Tetrahedron Letters 48 (2007) 6577–6581
OH
contamination of immunoreactive compounds in prepa-
OH
Kdo
HO
rations from natural sources. We thus planned to syn-
thesize the series of the lipid A and LPS partial
structures to clarify the structural basis of the immuno-
regulatory activities.
O
CO2H
HO
OH
O
O
O
O
HO
HO
O
HO
HO
O
O
HO
HO
R2O
HO
O
P
O
P
HN
HN
OH
HN
HN
O
O
O
OH
OH
OR1
O
O
O
O
Lipid A generally connects to the polysaccharide
domain via an acidic sugar, 3-deoxy-D-manno-2-octuro-
sonic acid (Kdo). Because H. pylori lipid A lacks a 40-
phosphate group, the acidic group of Kdo might alter
the anionic charge and affect recognition by the LPS
receptor. In the present study, we thus synthesized the
H. pylori LPS partial structures, lipid A 1a and Kdo-
lipid A 2, which do not have ethanolamine at the
anomeric phosphate to observe the effect of Kdo and
ethanolamine in comparison with our previously synthe-
sized ethanolamine-type lipid A (Fig. 1).9 Scheme 1
shows the basic strategy for lipid A synthesis. Glycosyl
donors 4a/4b and glycosyl acceptors 5a/5b were pre-
pared from properly protected glucosamine 3a/3b,
respectively. The acyl group of the glycosyl acceptors
5a/5b was introduced before glycosylation. Glycosyl
donor, trichloroacetimidate 5a/5b, which possessed a
2-N-Troc group, was used for b-selective glycosylation
to construct the lipid A backbone. H. pylori lipid A 1a
was synthesized from disaccharide 6a with 1-O-phos-
phorylation and deprotection. Kdo-lipid A 2 was syn-
thesized by glycosylation of 6b with Kdo donor 7 and
O
HO
HO
O
O
(C18)
(C18)
(C18)
(C18)
(C18)
(C18)
R1= -H,
R1= -CH2CH2-NH2,
R = -H,
R)-hydroxy-octadecyl-
: 1a
: 1b
: 1c
R2= -H
R2= -H
2
R2= 3(
R1= -CH2CH2-NH2,
R2= 3( )-hydroxy-octadecyl-
1
R
: 1d
Figure 1. Chemical structures of Helicobacter pylori lipid A and Kdo-
lipid A.
length of fatty acids in lipid A also influences the biolog-
ical activity. A biosynthetic precursor Ia analog having
shorter lipids (two C14 and two C10 fatty acids) showed
much weaker antagonistic activity, whereas that having
four C10 fatty acids did not show the activity.19 On the
other hand, an E. coli lipid A analogue having shorter
lipids (two C14 and four C12 fatty acids) were ca. 100
times more active than E. coli lipid A.20
HO
O
BnO
3a (R3=MPM)
3b (R3=Bn)
R3O
TrocNH
OAllyl
HO
BnO
R4O
AllocO
BnO
O
O
NH
OCCl3
The acidic functional groups of lipid A are also crucial
for the biological activity.12,13 Monophosphoryl lipid
A analogues show much weaker activity than the
corresponding diphosphates and the analogues lacking
phosphoryl group did not show the activity. The phos-
phate groups in lipid A can be replaced with carboxy-
methyl groups without loss of the activity in the case
of E. coli lipid A and the tetraacyl biosynthetic precur-
sor.21–24 However, in our recent studies with lipid A
analogues containing acidic amino acid residues,
immunostimulating or antagonistic activity was ob-
served depending on the anionic charges (phosphoric
acid vs carboxylic acid).25,26
+
R3O
NH
TrocNH
O
OAllyl
R1
4a (R3=MPM)
5a (R1=(R)-3-(4-trifluorobenzyl-
4b (R3=Bn)
oxy)octadecanoic acid, R4=H)
5b (R1=(R)-3-(benzyloxy)-
octadecanoic acid, R4=Bn)
R5O
O
BnO
6a
O
O
BnO
R3O
R4O
H. pylori lipid A 1a
NH
OAllyl
NH
R2
O
O
R1
6a (R3= MPM, R4= H, R5= Alloc)
6b (R3= Bn, R4= Bn, R5= Alloc)
6c (R3= Bn, R4= Bn, R5= TES)
OBn
O
BnO
TBSO
As for another aspect, it has been reported that H. pylori
LPS activates TLR2.11,27,28 Some of them also indicated
that H. pylori LPS antagonizes TLR4.11,28 Another par-
asitic bacteria, Porphyromonas gingivalis, which has sim-
ilar acylated patterns of lipid A to the one of H. pylori
though the acyl groups have branched terminus,29,30
also showed TLR2 stimulatory and TLR4 antagonizing
activities.28 These biological activities are considered to
be associated with atherosclerosis, which is a chronic
inflammatory vascular disease and leads to cerebral
stroke and cardiac affection—most major causes of
death in industrial countries. The heterogeneity of the
lipid A structures in H. pylori LPS, however, complex-
ifies understanding the molecular basis of these biologi-
cal activities. There is also a potential issue on the
OBn
O
BnO
TBSO
CO2Bn
TBSO
F
7
CO2Bn
O
TBSO
O
O
O
BnO
R3O
BnO
R4O
NH
R1
NH
R2
OAllyl
BnO
O
O
R1= CH3(CH2)14
8
O
2
CH (CH )
R =
3
2 16
O
CH3(CH2)14
H. pylori Kdo-lipid A 2
Scheme 1. Outline for the synthesis of Helicobacter pylori lipid A and
Kdo-lipid A. Alloc = allyloxycarbonyl, Bn = benzyl, MPM = p-meth-
oxybenzyl, TBS = t-butyldimethylsilyl, Troc = 2,2,2-trichloroethoxy-
carbonyl, TMS = trimethylsilyl.

Y. Fujimoto et al. / Tetrahedron Letters 48 (2007) 6577–6581
6579
1-O-phosphorylation. Kdo fluoride 7 protected with
TBS groups at the 4- and 5-positions was used as a do-
nor for a-selective glycosylation, because we previously
found in our E. coli Re-LPS synthesis that a-selectivity
increased when large protective groups such as TBS
were introduced at the 4- and 5-positions.31 Benzyl-type
protecting groups were used for hydroxyl, carboxy and
phosphate functions, and the final deprotection of all
the benzyl-type protecting groups was carried out by
catalytic hydrogenolysis.
5b in subsequent procedures. The glycosyl donor 4b
and the acceptor 5b were synthesized in a manner simi-
lar to compounds 4a and 5a, respectively.31 The glycos-
ylation was carried out in the presence of a catalytic
amount of TMSOTf in THF solution at ꢀ78 °C to give
the disaccharide 12 in 70% yield. The Troc group at the
20-position of 12 was cleaved with Zn–Cu couple in ace-
tic acid, and (R)-3-octadecanoyloctadecanoic acid was
introduced with WSCDÆHCl at 40 °C to give 6b in
58% yield from compound 12. The Alloc group at the
60-position was cleaved with Pd(PPh3)4 in the presence
of PPh3 and ammonium formate in THF, and then the
TES group was introduced at this position for subse-
quent glycosylation to give compound 6c.
Scheme 2 shows the synthesis of lipid A 1a. Glycosyl
donor 4a and the acceptor 5a were synthesized in a
manner similar to previously published methods.31 The
glycosylation was carried out in the presence of a cata-
lytic amount of TMSOTf in THF solution at 0 °C to
give disaccharide 9 in 85% yield. The Troc group at
the 20-position of 9 was cleaved with Zn–Cu couple in
acetic acid, and (R)-3-octadecanoyloxyoctadecanoic
acid was introduced with WSCDÆHCl at 40 °C to give
6a from compound 9 in 56% yield. The Alloc group at
the 60-position was cleaved with Pd(PPh3)4, PPh3 and
ammonium formate in THF selectively in the presence
of an allyl group at 1-position, and then the MPM
group was introduced at this liberated 60-position. The
allyl group at the 1-position was cleaved via isomeriza-
tion to a vinyl group with an iridium complex and then
with iodine and water in 87% yield, and the anomeric
position was selectively phosphorylated with tetrabenzyl
pyrophosphate to give 11. Cleavage of all the benzyl-
type protecting groups in 11 by hydrogenolysis gave
the desired lipid A 1a.
Scheme 4 shows the preparation of Kdo fluoride 7, the
glycosyl donor for the next glycosylation, from com-
pound 13.31 In the previous study, we used (diethyl-
amino)sulphur trifluoride (DAST) for fluorination.
However, the reaction was difficult due to the high reac-
tivity of DAST and a considerable amount of glycal was
formed as a by-product. Hence, we used a new, mild
fluorination reagent, N,N-diethyl-a,a-difluoro-(m-meth-
ylbenzyl)amine (DFMBA).32 The anomeric stereoselec-
tivity for fluorination was different for these two
compounds: 3/1 (a/b) for DAST and 1/10 (a/b) for
DFMBA. The formation of glycal 14 was suppressed
when DFMBA was used at a lower temperature.
Scheme 5 shows the glycosylation between Kdo-fluoride
7 and lipid A backbone 6c. The reaction conditions were
examined using a variety of Lewis acids, solvents, and
molecular sieves. The molecular sieve 5A (MS5A) was
found to be effective for this reaction compared to
MS4A. Because MS5A contains calcium ions, MS5A
should trap fluoride anions to promote glycosylation.
Thus, we successfully obtained trisaccharide 8 with the
a-glycoside linkage between Kdo and lipid A, using
BF3/Et2O and MS5A in CH2Cl2 at ꢀ20 °C with a high
For the synthesis of Kdo-lipid A, the lipid A backbone
was first prepared as shown in Scheme 3. Although we
initially tried to synthesize Kdo-lipid A from 6a, the
30-O-MPM group was cleaved under the glycosylation
conditions using Kdo fluoride 7 in the preliminary
experiments. We, therefore, used 3-O-benzyl acceptor
O
AllocO
a
b, c
O
O
BnO
BnO
HO
5a
4a
+
MPMO
NH
R1
TrocNH
OAllyl
O
9
O
O
MPMO
BnO
MPMO
AllocO
BnO
MPMO
O
O
d, e
OAllyl
O
O
BnO
HO
BnO
HO
NH
R1
NH
R2
NH
R1
OAllyl
NH
R2
O
O
O
O
6a
10
O
MPMO
BnO
MPMO
f, g
h
O
O
BnO
HO
1a
O
NH
R1
NH
R2
OP(OBn)2
O
O
11
Scheme 2. Synthesis of Helicobacter pylori lipid A. (a) TMSOTf (0.1 equiv), MS4A, THF, 0 °C, 1 h, 85%; (b) Zn–Cu, AcOH, rt; (c) (R)-3-
octadecanoyloxyoctadecanoic acid, WSCDÆHCl, HOAt, CHCl3, 40 °C, 56% (two steps); (d) Pd(PPh3)4 (1.2 equiv), PPh3 (3 equiv), HCOONH4
(5 equiv), THF, rt, 2 h, 94%; (e) MPM imidate Sn(OTf), MS4A, THF, 88%; (f) [Ir(H)(cod)(MePh2P)2]PF6, then I2, H2O, 87%; (g) tetrabenzyl
pytrophosphate, LiN(TMS)2, THF, ꢀ78 °C, 47%; (h) H2 (15 kg cmꢀ2), Pd (black), THF. R1: (R)-3-(4-trifluorobenzyloxy)octadecanoyl, R2: (R)-3-
octadecanoyloxyoctadecanoyl, HOAt = 1-hydroxy-7-azabenzotriazole, TMSOTf = trimethylsilyl trifluoromethanesulfonate, WSCD = 1-(3-dimeth-
ylaminopropyl)-3-ethylcarbodiimide hydrochloride.

6580
Y. Fujimoto et al. / Tetrahedron Letters 48 (2007) 6577–6581
O
removed with HF, and the allyl group at the 1-position
AllocO
BnO
BnO
O
O
a
BnO
b, c
was cleaved via isomerization to a vinyl group with
iridium complex to give 15 in 78% yield. After selective
phosphorylation at the anomeric position with tetra-
benzyl pyrophosphate, all the benzyl-type protecting
groups were removed by hydrogenolysis to give the
desired compound 2.
4b
5b
+
BnO
NH
R1
TrocNH
OAllyl
O
12
O
O
TESO
BnO
BnO
AllocO
BnO
BnO
O
O
O
O
d, e
BnO
BnO
BnO
BnO
NH
R1
NH
NH
R1
NH
R2
OAllyl
OAllyl
O
O
O
O
R2
The immunostimulating activities of the synthetic
H. pylori lipid A 1a and Kdo-lipid A 2 were examined
by measuring IL-6 production from human whole blood
cells (HWBC)33 (Fig. 2). Both 1a and 2 did not induce
IL-6, but showed competitive inhibition of IL-6 induc-
tion by E. coli LPS (0.5 ng/mL, O111:B4). Kdo-lipid A
2 showed more potent inhibitory activity than lipid A 1a.
6b
6c
Scheme 3. Synthesis of lipid A backbone disaccharide for Helicobacter
pylori Kdo-lipid A. (a) TMSOTf (0.2 equiv), MS4A, THF, ꢀ78 °C,
1 h, 80%; (b) Zn–Cu, AcOH, rt; (c) (R)-3-octadecanoyloxyoctadeca-
noic acid, WSCDÆHCl, HOAt, CHCl3, 40 °C, 58% (two steps); (d)
Pd(PPh3)4, PPh3, HCOONH4, THF, 94%; (e) TESCl, imidazole,
CHCl3, 88%. R1: (R)-3-benzyloxyoctadecanoyl, R2: (R)-3-octadeca-
noyloxyoctadecanoyl, TES = triethyl.
These results demonstrated that the H-form tri-acylated
lipid A 1a and Kdo-lipid A 2 of H. pylori competitively
inhibit E. coli LPS immunostimulation. These results
indicated that 1a and 2 are TLR4 antagonist but not
TLR2 or TLR4 agonist. We also observed that intro-
ducing an acidic Kdo residue to lipid A enhanced this
inhibitory activity by 10-fold. The present study pro-
vides the molecular basis for antagonistic activity of
H. pylori LPS from certain strains.
OBn
O
OBn
O
OBn
O
BnO
TBSO
BnO
TBSO
BnO
TBSO
Reagent
CH2Cl2
CO2Bn
CO2Bn
OH
CO2Bn
TBSO
TBSO
TBSO
F
13
7
14
Entry Reagent
Conditions
Time
3 h
Yield[a]
88% (α:β= 3:1), 14
1
2
3
DAST (3 equiv)
-78°C
60°C
(7%)
It should be noted that the ethanolamine-form of lipid A
1b showed weak immunostimulating activity in our pre-
vious study.10 These results indicate that the number of
anionic charges influences the biological activity of lipid
A and LPS. Similar results were obtained from our stud-
ies with lipid A analogues containing acidic amino acid
residues as mentioned above. Depending on the anionic
charges (phosphoric acid vs carboxylic acid) in the lipid
A analogues, immunostimulating or antagonistic activ-
ity was observed.26 The present study also explains
why some strains of H. pylori LPS show weak immuno-
stimulating activity while others show antagonistic
activity.
10 min
68% (α:β= 1:10),
14 (12%)
DFMBA (2 equiv)
(microwave 50 W)
88% (α:β= 1:10),
14 (ND)
DFMBA (4 equiv) -14°C 1.5 d
[a] The ratio of the α, β-glycoside and glycal 14 was determined by the area
ratio of the PhCH2OCO signal in 1H-NMR.
Scheme 4. Fluorination of Kdo.
OBn
BnO
HO
O
CO2Bn
HO
b ,c
a
+
6c
7
8
O
O
O
In conclusion, we synthesized tri-acylated lipid A 1a and
LPS partial structure 2 of H. pylori and found that both
compounds antagonize E. coli LPS immunostimulation.
BnO
O
BnO
BnO
BnO
OH
NH
R2
NH
R1
O
O
OBn
O
BnO
HO
15
1.4
CO2Bn
d
HO
e
2
1.2
50 ng/mL
O
O
500 ng/mL
1
O
BnO
O
5000 ng/mL
BnO
BnO
BnO
O
NH
0.8
0.6
0.4
0.2
0
NH
R1
O
OP(OBn)2
R2
O
16
Scheme 5. Synthesis of Helicobacter pylori Kdo-lipid A. (a) BF3ÆEt2O
(10 equiv), MS5A, CHCl3, ꢀ20 °C, 1.5 h, 85%; (b) 47%
HF:CH2Cl2:CH3CN = 1:5:5, 81%; (c) [Ir(H)(cod)(MePh2P)2]PF6, then
I2, H2O, 78%; (d) tetrabenzyl pyrophosphate, LiN(TMS)2, THF,
ꢀ78 °C, 47%; (e) H2 (20 kg cmꢀ2), Pd (black), THF, 91%. R1: (R)-3-
benzyloxyoctadecanoyl, R2: (R)-3-octadecanoyloxyoctadecanoyl.
2
2a
2
2a
LPS (E.coli)
1a
1a
LPS (E.coli)
+ LPS (E.coli)
+ LPS (E.coli)
Figure 2. Helicobacter pylori Kdo-lipid A and lipid A competitively
inhibit IL-6 induction by E. coli LPS. Noted concentrations of Kdo-
lipid A 2a and lipid A 1a and 0.5 ng/mL of E. coli (O111:B4) LPS were
added to human whole blood cells (HWBC). Amount of IL-6
produced by HWBC was determined by ELISA. Data represent
averages of three repeated assays with standard deviations from
individual experiments.
yield. The stereochemistry at the anomeric position was
determined with the chemical shift of the protons at the
30-position from 1H NMR in comparison with the
previously reported data.31 Then, the TBS groups were

Y. Fujimoto et al. / Tetrahedron Letters 48 (2007) 6577–6581
6581
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Yoshizaki, H.; Sato, K.; Oikawa, M.; Suda, Y. J.
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B.; Haslov, K.; Qureshi, N.; Takayama, K. Infect. Immun.
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In future studies, we will use these H. pylori LPS com-
pounds to investigate the biological activities of LPS
and the factors that mediate host receptor LPS
recognition.
15. Kirikae, T.; Schade, F. U.; Kirikae, F.; Qureshi, N.;
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Acknowledgements
This work was supported in part by Grants-in Aid for
Scientific research (Nos.17035050, 17310128, 18032046)
from Japan Society for the Promotion of Science, grants
from Suntory Institute for Bioorganic Research (SUN-
BOR Grant), the Houansha Foundation, and Hayashi
Memorial Foundation for Female Natural Scientists
(to Y.F.). DFMBA was a generous gift of Mitsubishi
Gas Chemical Company, Inc.
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