Synthesis of the trisaccharide repeating unit of the atypical O-antigen polysaccharide from Danish Helicobacter pylori strains employing the 2′-carboxybenzyl glycoside

Article abstract of DOI:10.1021/ol048648u

(Chemical Equation Presented) Synthesis of the unique trisaccharide repeating unit of the O-polysaccharide of the lipopolysaccharide from Danish Helicobacter pylori strains has been accomplished. Key steps include the coupling of three monosaccharide moie

Full text of DOI:10.1021/ol048648u

ORGANIC
LETTERS
2004
Vol. 6, No. 22
3901-3904
Synthesis of the Trisaccharide
Repeating Unit of the Atypical
O-Antigen Polysaccharide from Danish
Helicobacter pylori Strains Employing
the 2′-Carboxybenzyl Glycoside
Yong Tae Kwon, Yong Joo Lee, Kyunghoon Lee, and Kwan Soo Kim*
Center for BioactiVe Molecular Hybrids and Department of Chemistry, Yonsei
UniVersity, Seoul 120-749, Korea
kwan@yonsei.ac.kr
Received July 15, 2004
ABSTRACT
Synthesis of the unique trisaccharide repeating unit of the O-polysaccharide of the lipopolysaccharide from Danish Helicobacter pylori strains
has been accomplished. Key steps include the coupling of three monosaccharide moieties by glycosylations employing the 2 -carboxybenzyl
-mannose, which is one of three
′
glycoside method. Also presented is a method for the synthesis of the novel branched sugar, 3-C-methyl-
D
monosaccharide components.
Since the first isolation of Helicobacter pylori,1 extensive
studies have led to recognition of this bacterium as the major
cause of chronic gastritis and gastric and duodenal ulcers.2
Moreover, persistent infection with H. pylori is strongly
associated with the risk of development of gastric cancer.2
H. pylori is estimated to infect over one-half of the world’s
population and thus has been classified as a category 1
(definite) human carcinogen.3 Like the cell envelope of other
gram-negative bacteria, that of H. pylori contains lipopolysac-
charides (LPS). The O-antigen polysaccharide is a part of
the LPS, which interacts with the bacterial microenvironment
and the infected host. Structural studies on the LPS of a
number of H. pylori strains have shown that the O-antigen
polysaccharide exhibits mimicry of Lewis blood group
antigens by expression of the corresponding determinants
in the O-chain or at the nonreducing end of the O-chain
polysaccharide.4 Consequently, a number of studies have
been performed to account for the pathogenic relevance of
Lewis antigen mimicry by H. pylori.5 There have also been
immunization studies with inactivated H. pylori whole-
cell vaccines having homologous and heterologous LPS
(1) (a) Warren, J. R.; Mashall, B. J. Lancet 1983, 321, 1273-1275. (b)
Mashall, B. J.; Warren, J. R. Lancet 1984, 323, 1311-1315.
(2) (a) Dunn, B. E.; Cohen, H.; Blaser, M. J. Clin. Microbiol. ReV. 1997,
10, 720-741. (b) Blaser, M. J. Sci. Am. 1996, 274, 104-109. (c) Dubois,
A. Emerg. Infect. Dis. 1995, 1, 79-85.
(3) (a) Covacci, A.; Telford, J. L.; Giudice, G. D.; Parsonnet, J.;
Rappuole, R. Science, 1999, 284, 1328-1333. (b) Logan, R. P. H. Lancet
1994, 344, 1078-1079.
(4) (a) Monteiro, M. A.; Chan, K. H. N.; Rasko, D. A.; Taylor, D. E.;
Zheng, P. Y.; Appelmelk, B. J.; Wirth, H. P.; Yang, M. Q.; Blaser, M. J.;
Hynes, S. O.; Moran, A. P.; Perry, M. B. J. Biol. Chem. 1998, 273, 11533-
11543. (b) Aspinall, G. O.; Monteiro, M. A. Biochemistry 1996, 35, 2498-
2504. (c) Aspinall, G. O.; Monteiro, M. A.; Pang, H.; Walsh, E. J.; Moran,
A. P. Biochemistry 1996, 35, 2489-2497.
10.1021/ol048648u CCC: $27.50
© 2004 American Chemical Society
Published on Web 09/29/2004

O-antigens expressing different Lewis antigens6 and with H.
pylori outer membrane vesicles, which are enriched with
LPS.7 Recently, however, an atypical O-antigen polysaccha-
ride of LPS was isolated from Danish H. Pylori strains D1,
D3, and D6, and the following repeat trisaccharide structure
was established: f3)-R-L-Rhap-(1f3)-R-D-Rhap-(1f2)-R-
D-Manp3CMe-(1f (A).8 An unusual feature of this O-
polysaccharide is the occurrence of the novel branched sugar,
3-C-methyl-D-mannose, which has not been found in Nature
before. The simultaneous occurrence of L- and D-rhamnose
is also unusual.
Scheme 1. Retrosynthesis of Trisaccharide 1
Due to its structural uniqueness and biomedical potential,
the repeat unit A is an attractive synthetic target. Although
a number of methods for the synthesis of Lewis antigens
have been reported,9 the synthesis of the repeating unit A or
compounds with the similar structure has not been reported.
Herein we report the synthesis of trisaccharide 1 in a suitably
protected form of the repeating unit A.
protective groups in the target trisaccharide 1 were chosen
after consideration of the future synthesis of a hexasaccharide
or a nonasaccharide by dimerization or trimerization of 1.
Thus, the latent 2′-(benzyloxycarbonyl)benzyl (BCB) group10a
at C-1 in the reducing end of trisaccharide 1 would be readily
converted into the active CB group to give the trisaccharide
donor, while the p-methoxybenzyl (PMB) group at C-3 in
the nonreducing end of 1 would be selectively cleaved to
provide the trisaccharide acceptor. Retrosynthesis of 1 leads
to disaccharide donor 2 and acceptor 3, and further analysis
of 2 provides L-rhamnosyl donor 4 and D-rhamnosyl acceptor
5 as shown in Scheme 1.
Key problems needing to be addressed in the synthesis of
1 are the synthesis of the unique 3-C-methyl-D-mannose
moiety and the coupling of three monosaccharide moieties
by efficient glycosylation methods. We have recently de-
veloped a glycosylation method that employs a new type of
glycosyl donor, 2′-carboxybenzyl (CB) glycosides, and have
used these extensively for the stereoselective â-mannopy-
ranosylation10 and 2-deoxyglycosylation of various acceptors.
We have also used this method in the synthesis of a
tetrasaccharide.11
Before commencing the synthesis of the BCB R-D-
mannopyranoside 3, we carried out a model study on
elaboration of the 3-C-methyl group in the mannoside with
simpler methyl R-D-mannopyranoside as shown in Scheme
2. The model study began with a selective protection of the
C-2 axial hydroxyl group of methyl 4,6-O-benzylidene-D-
mannoside 6 with PMBCl and oxidation of the resulting
PMB ether 7 with PDC to obtain ketosugar 8. Axial attack
of dimethylsulfonium methylide12 to the C-3 carbonyl carbon
of 8 afforded only the spiroepoxide 9, which was reduced
with LiAlH4 to desired 3-C-methylmannoside 10. The
stereochemistry at C-3 of 10 was confirmed by NOE
experiments and by comparison with the corresponding 3-C-
methylaltroside, the C-3 epimer of 10, which was obtained
by direct addition of methyllithium to the ketosugar 8.
The sequence developed in the model study with methyl
4,6-O-benzylidenemannoside 6 was then applied to BCB 4,6-
The CB glycosylation method would be here used for the
coupling of all three monosaccharide components of 1. The
(5) Raghavan, S.; Hjulsrtom, M.; Holgren, J.; Svennerholm, A.-M. Infect.
Immun. 2002, 70, 6383-6388.
(6) (a) Lozniewski, A.; Haristoy, X.; Rasko, D. A.; Hatier, R.; Plenat,
F.; Taylor, D. E.; Angioi-Duprez, K. Infect. Immun. 2003, 71, 2902-2906.
(b) Appelmelk, B. J.; Simmons-Smit, I.; Negrini, R.; Moran, A. P.; Aspinall,
G. O.; Forte, J. G.; De Vries, T.; Quan, H.; Verboom, T.; Maaskant, J. J.;
Ghiara, P.; Kuipers, E. J.; Bloemena, E.; Tadema, T. M.; Townsend, R. R.;
Tyagarajan, K.; Crothers, J. M.; Monteiro, M. A.; Savio, A.; Graaff, J. D.
Infect. Immun. 1996, 64, 2031-2040.
(7) (a) Keenan, J. I.; Rijpkema, S. G.; Durrani, Z.; Roake, J. A. FEMS
Immun. Med. Microbiol. 2003, 36, 199-205. (b) Keenan, J.; Oliaro, J.;
Domigan, N.; Potter, H.; Aitken, G.; Allardyce, R.; Roake, J. Infec. Immun.
2000, 68, 3337-3343.
(8) Kocharova, N. A.; Knirel, Y. A.; Widmalm, G.; Jansson, P.-E.;
Moran, A. P. Biochemistry 2000, 39, 4755-4760.
(9) (a) Vankar, Y. D.; Schmidt, R. R. Chem. Soc. ReV. 2000, 29, 201-
216. (b) Brocke, C.; Kunz, H. Bioorg. Med. Chem. 2002, 10, 3085-3112.
(10) (a) Kim, K. S.; Kim, J. H.; Lee, Y. Joo; Lee, Y. Jun; Park, J. J. Am.
Chem. Soc. 2001, 123, 8477-8481. (b) Kim, K. S.; Park, J.; Lee, Y. J.;
Seo, Y. S. Angew. Chem., Int. Ed. 2003, 42, 459-462.
(11) Kim, K. S.; Kang, S. S.; Seo, Y. S.; Kim, H. J.; Lee, Y. J.; Jeong,
K.-S. Synlett 2003, 1311-1314.
(12) Corey, E. J.; Chaykovsky, M. J. J. Am. Chem. Soc. 1965, 87, 1353-
1364.
3902
Org. Lett., Vol. 6, No. 22, 2004

Scheme 2. Model Study for the Synthesis of
3-C-Methylmannoside
Scheme 4. Synthesis of CB L-Rhamnoside 4
O-benzylidenemannoside 1110a (Scheme 3). Treatment of 11
with Bu2SnO in refluxing methanol followed by reaction of
the resulting crude O-stannylene acetal13 with PMB chloride
in the presence of Bu4NI in DMF afforded the C-3 PMB
ether 12 in 78% yield for the two steps with complete
regioselectivity. Reaction of 12 with TBS chloride gave the
fully protected sugar 13, and deprotection of the PMB group
of 13 with DDQ provided the C-3 hydroxyl sugar 14 in high
yield. Oxidation of 14 with Dess-Martin periodinane14 gave
ketosugar 15 in 93% yield.
OBCB group. Reaction of the same sulfur ylide with other
ketosugars having different C-2 protective groups such as
PMB and acetyl groups did not occur. Ketosugar 15,
therefore, was converted into methylene sugar 16 in 92%
yield by Wittig reaction with triphenylphosphonium meth-
ylide. The TBS group of 16 was removed by Bu4NF, and
epoxidation of the resulting hydroxy methylene sugar 17
afforded spiroepoxide 18 in 80% yield with complete
stereoselectivity. Chemoselective reduction of spiroepoxy
sugar 18 with Bu3SnH in the presence of NaI and AIBN
under reflux in DME15 afforded the desired BCB 3-C-
methylmannoside 3. The stereochemistry at C-3 of compound
3 was confirmed on the basis of NOE experiments. Thus,
NOE was observed between the methyl protons at C-3 and
the proton at C-5 in compound 3.
However, unlike the model study with the ketosugar 8,
reaction of the keto sugar 15 with dimethylsulfonium
methylide did not proceed, possibly due to steric congestion
between the incoming sulfur ylide and the bulky C-1 axial
Scheme 3. Synthesis of BCB 3-C-Methylmannoside 3
Synthesis of the L-rhamnose moiety 4 started with conver-
sion of triacetylrhamnosyl bromide 19 into BCB triacetyl-
rhamnoside 20 by coupling of 19 with benzyl 2-(hydroxy-
methyl)benzoate10a in the presence of Hg(II) salts (Scheme
4). Sodium methoxide converted triacetate 20 into triol 21
in 91% yield without affecting the BCB moiety. Compound
21 was treated with Bu2SnO in methanol at 60 °C and
subsequently reacted with PMB chloride in the presence of
Bu4NI in DMF to afford exclusively the C-3 PMB ether 22
in 83% yield. Pivaloylation of the diol 22 and hydrogenolysis
of resultant BCB glycoside 23 in the presence of ammonium
acetate gave the desired CB L-rhamnoside 4 in 85% yield.
The D-rhamnose unit 5 was prepared starting from the
BCB mannoside 12 (Scheme 5). Pivaloylation of 12 followed
by reductive cleavage of the benzylidene group in the
resulting pivaloate 24 with borane in the presence of Bu2-
BOTf16 afforded the C-4 benzyl ether 25 having a free
(13) For a review on stannylene acetals of carbohydrates, see: Grindley,
T. B. AdV. Carbohydr. Chem. Biochem. 1998, 53, 17-142.
(14) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155-4156.
(15) Bonini, C.; Fablo, R. D. Tetrahedron Lett. 1988, 29, 819-822.
(16) Jiang, L.; Chan, T.-H. Tetrahedron Lett. 1998, 39, 355-358.
Org. Lett., Vol. 6, No. 22, 2004
3903

Scheme 5. Synthesis of BCB D-Rhamnoside 5
Scheme 6. Completion of the Synthesis of Trisaccharide 1
disaccharides 28 and 2 and trisaccharide 1 were unequivo-
1
cally determined on the basis of their H and 13C NMR
spectral data, especially one-bond C1′-H1′ coupling con-
1
stants: JC1′-H1′ ) 171.1 Hz in compound 2 and 1JC1′-H1′
)
hydroxyl group at C-6 in high yield. Iodination of the primary
alcohol 25 with I2, Ph3P, and imidazole provided the iodide
26 without difficulty. Reduction of the iodide 26 with Bu3-
SnH in the presence of AIBN and subsequent removal of
PMB group of the rhamnoside 27 with DDQ gave the desired
BCB D-rhamnoside 5 in high yield.
170.8 Hz17 in compound 1.
In summary, we have described the synthesis of the
suitably protected trisaccharide repeating unit 1 of an unusual
O-antigen polysaccharide of the lipopolysaccharide from
Danish H. pylori strains. The 2′-carboxybenzyl glycosides
method proved to be effective in the coupling of three
monosaccharide components of the trisaccharide, 3-5. We
also presented the first synthesis of a novel branched sugar,
3-C-methyl-D-mannose.
Coupling of CB L-rhamnoside 4 and BCB D-rhamnoside
5 was achieved by the addition of Tf2O to the mixture of 4
and 5 in the presence of DTBMP (2,6-di-tert-butyl-4-
methylpyridine) and 4 Å MS in CH2Cl2 at -78 °C followed
by warming to 0 °C; the desired R-disaccharide 28 was
obtained in 88% yield (Scheme 6). Selective hydrogenolysis
of the BCB disaccharide 28 in the presence of NH4OAc in
methanol afforded the CB disaccharide 2 in 95% yield.
Finally, glycosylation with the CB disaccharide donor 2 and
BCB 3-C-methylmannoside acceptor 3 was carried out by
the addition of Tf2O to a solution of 2 and 3 in CH2Cl2 in
the presence of DTBMP at -78 °C. Warming the reaction
mixture to 0 °C, quenching with NaHCO3, and purification
by chromatography afforded the target R-trisaccharide 1
along with its â-anomer in a 7:1 ratio in 80% yield. The
stereochemistries at newly generated anomeric centers of
Acknowledgment. This work was supported by a grant
form the Korea Science and Engineering Foundation through
Center for Bioactive Molecular Hybrids (CBMH).
Supporting Information Available: Synthetic procedures
and spectral/analytical data of all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL048648U
(17) For C-H coupling constants in pyranoses, see: Bock, K.; Pedersen,
C. J. Chem. Soc., Perkin Trans. 2 1974, 293-297.
3904
Org. Lett., Vol. 6, No. 22, 2004