Design and Synthesis of C3-Symmetric LewisX Antigen

Article abstract of DOI:10.1021/ol0351293

(Matrix presented) C₃-Symmetric glycoconjugates carrying three equivalent Lewis^X antigens or β-lactosides were synthesized from p-nitrophenyl glycosides and trimesic acid via regio- and stereocontrolled glycosylation reactions. An

Full text of DOI:10.1021/ol0351293

ORGANIC
LETTERS
2003
Vol. 5, No. 21
3775-3778
Design and Synthesis of C -Symmetric
3
LewisX Antigen
Yoshihiro Nishida,* Tomoaki Tsurumi, Kenji Sasaki, Kenta Watanabe,
Hirofumi Dohi, and Kazukiyo Kobayashi*
Department of Molecular Design and Engineering, Graduate School of Engineering,
Nagoya UniVersity, Chikusa-ku, Nagoya 464-8603, Japan
nishida@mol.nagoya-u.ac.jp; kobayash@mol.nagoya-u.ac.jp
Received June 19, 2003 (Revised Manuscript Received August 28, 2003)
ABSTRACT
C -Symmetric glycoconjugates carrying three equivalent LewisX antigens or â-lactosides were synthesized from p-nitrophenyl glycosides and
3
1
trimesic acid via regio- and stereocontrolled glycosylation reactions. An H NMR study has shown that the C -symmetric glycoconjugates
3
soluble in water provide useful probes to investigate the Ca2+-dependent LewisX−LewisX association.
Intense interest is being directed to the design and application
of multivalent glycoconjugates1,2 carrying human oligo-
saccharide antigens.3,4 Several multivalent models have
already been proposed for sialyl LewisX and globotriaosyl
antigens, in which polymers,3,4a dendrimers,4c and starfish
models4d are widely examined. In this paper, we describe
the synthesis and potential use of C3-symmetric LewisX
trisaccharide and â-lactoside.
Many studies have been directed toward this intriguing
phenomenon,6 in which LewisX-based dimers,6c liposomes,6b
giant-liposomes,6g gold nanoparticles,6e and self-assembling
monolayers6f have been proposed as multivalent probes.
The C3-symmetric LewisX 3 (Figure 1) was designed as
an alternative probe with some expectations as described
below. (1) The trivalent structure will induce enhanced self-
association due to multivalent effects, (2) allow a simpler
analysis than nonsymmetric or dendric models due to the
C3-symmetry, and (3) show higher water solubility than usual
glycolipid models because three hydrophilic LewisX are
directed outside of the hydrophobic core. Regarding the
multivalent effect, Rao, Whitesides, and co-workers7 have
evidenced in their pioneering study that a vancomycin trimer
Hakomori et al.5 had previously disclosed that LewisX
oligosaccharides bound to cell membrane lipids cause
homophilic association in the presence of a calcium ion.
(1) Lee, Y. C.; Lee, R. T. Neoglycoconjugates: Preparation and
Applications; Academic Press: San Diego, 1994.
(2) (a) Bovin, N. V.; Gabius, H. J. Chem. Soc. ReV. 1995, 24, 413-422.
(b) Roy, R. Curr. Opin. Struct. Biol. 1996, 6, 679. (c) Rye, P. D.
Biotechnology 1997, 14, 155.
(3) Miyauchi, H.; Tanaka, M.; Koike, H.; Kawamura, N.; Hayashi, M.
Bioorg. Med. Chem. Lett. 1997, 7, 985. (b) Gordon, E. J.; Sanders, W. J.;
Kiessling, L. L. Nature 1998, 392, 30.
(4) (a) Nishida, Y.; Dohi, H.; Kobayashi, K. Tetrahedron Lett. 1998,
39, 8681. (b) Matsuoka, K.; Terabatake, M.; Esumi, Y.; Terunuma, D.
Kuzuhara, H. Tetrahedron Lett. 1999, 40, 7839. (c) Kitov, P. I.; Sadowska,
J. M.; Mulvey, G.; Armstrong, G. D.; Ling, H.; Pannu, N. S.; Read, R. J.;
Bundle, D. R. Nature 2000, 403, 669.
(6) (a) Siuadak, G.; Ichikawa, Y.; Caulfield, T. J.; Munoz, B.; Wong,
C.-H. Nicolaou, K. C. J. Am. Chem. Soc. 1993, 115, 2877. (b) Geyer, A.;
Gege, C.; Schmidt, R. R. Angew. Chem., Int. Ed. 1999, 38, 1446. (c) Geyer,
A.; Gege, C.; Schmidt, R. R. Angew. Chem., Int. Ed. 2000, 39, 3246. (d)
Zhang, Y.; Dausse, B.; Sinay, P.; Afsahi, M.; Berthault, P.; Desvaux, H.
Carbohydr. Res. 2000, 324, 231. (e) Fuente, J. M.; Barrientos, A. G.; Rojas,
T. C.; Rojo, J.; Canada, J.; Fernandez, A.; Penades, S. Angew. Chem., Int.
Ed. 2001, 40, 2258. (f) Tromas, C.; Rojo, J.; Fuente, J. M.; Barrientos, A.
G.; Garcia, R.; A.; Penades, S. Angew. Chem., Int. Ed. 2001, 40, 2258. (g)
Pincet, F.; Bouar, T. L.; Zhang, Y.; Esnault, J.; Mallet, J.-M.; Perez, E.;
Sinay, P. Biophys. J. 2001, 80, 1354.
(5) (a) Hakomori, S. Pure Appl. Chem. 1991, 63, 473. (b) Hakomori, S.
Cancer Res. 1996, 56, 5309.
10.1021/ol0351293 CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/17/2003

Scheme 1. Syntheses of Compounds 7 and 10a
a Reagents: (a) (i) tetrachlorophthalic anhydride, Et3N/MeOH,
(ii) Ac2O/Py 80%; (b) HBr-AcOH/CH2Cl2 58%; (c) AgOTf,
p-nitrophenol/CH2Cl2 46%; (d) NaOMe/MeOH quant; (e) p-
toluenesulfonic acid, benzaldehyde dimethyl acetal/DMF, 77%; (f)
triethylsilane, trifluoroacetic acid, trifluoroacetic anhydride/CH2Cl2,
75%; (g) per-O-acetyl-R-D-galactopyranosyl trichloroacetiimidate,
TMSOTf/CH2Cl2, 83%.
Figure 1. Conversion of an asymmetric LewisX trisaccharide 1 to
a C3-symmetric conjugate 3 with trimesic acid 2. (The linker moiety
shown with a red bar was required for successful conversion as
cited in the text).
The tetrachlorophthalimide (TCP) group12 in 7 was chosen
as an N-protecting group expecting that the TCP will be
removed under basic conditions without affecting the pNP
O-glycoside linkage. Moreover, the bulky group seemed to
work effectively in regioselective glycosylation reactions.9
Compound 7 was converted to 6-O-benzyl derivative 9 in a
conventional way. â-Galactosylation was performed using
a trichloroacetiimidate method established by Schmidt et al.13
The glycosylation of 9 was regio- and â-specific to afford
pNP â-lactosaminide 10.
For R-L-fucosylation, we applied our thio-glycosylation
method using o-methoxycarbonyl phenyl (o-MCP) 1-thio-
glycosides.14 A 1-thiofucosyl donor 14 (mp ) 125 °C) was
prepared via an SN2 reaction between fucosyl bromide 11
and thiosalicylate (Scheme 2). No unpleasant thiol odor was
composed of trimesic acid 2 can induce a remarkable
enhancement in the antibiotic activity. They have provided
also theoretical bases to rationalize this enhancement. Thus,
it can be reasonably assumed that the trimeric LewisX can
provide a useful probe to investigate the weak LewisX-
LewisX interaction.
Though there are many synthetic studies targeting the
LewisX skeleton 18 and its multivalent models,9 there is no
established way to assemble the Cn-symmetric structure.
Here, choice of an appropriate aglycon (R in 1) convertible
to the C3-symmetric structure provides a key investigative
issue. In our continuous studies on the synthesis of artificial
glycopolymers,10 we have employed p-nitrophenyl (pNP)
glycosides. In addition to the UV absorption useful for TLC
detection, reduction of the pNP group affords an arylamine
useful for multivalent assembly.4a,11 Also in this work, we
undertook the same strategy using pNP D-glucosaminide 7
and 9 as key intermediates (Scheme 1).
Scheme 2 o-MCP 1-Thio-L-fucosyl Donor 14a
(7) (a) Rao, J.; Lahiri, J.; Lyle, I.; Weis, R. M.; Whitesides, G. M. Science
1998, 280, 708. (b) Rao, J.; Lahiri, J.; Weis, R. M.; Whitesides, G. M. J.
Am. Chem. Soc. 2000, 122, 2698.
(8) (a) Jacquinet, J. C.; Sinay, P. J. Chem. Soc., Perkin Trans 1 1979,
314. (b) Hindsgaul, O.; Norberg, T.; Pendu, J. Le; Lemieux, R. U.
Carbohydr. Res. 1982, 109, 109. (c) Sato, S.; Itoh, Y.; Nukada, T.; Nakahara,
Y.: Ogawa, T. Carbohydr. Res. 1987, 167, 197. (d) Nicolaou, K. C.;
Hummel, C. W.; Iwabuchi, Y. J. Am. Chem. Soc. 1992, 114, 3126. (e)
Hasegawa, A.; Ando, T.; Kameyama, A.; Kiso, M. J. Carbohydr. Chem.
1992, 11, 645. (f) Jain, R. K.; Matta, K. L. Carbohydr. Res. 1992, 226, 91.
(j) Windmueller, R.; Schmidt, R. R. Tetrahedron Lett. 1994, 35, 7927. (h)
Ehara, T.; Kameyama, A.; Yamada, Y.; Ishida, H.; A.; Kiso, M.; Hasegawa,
A. Carbohydr. Res. 1996, 281, 237. (i) Ellervik, U.; Magnusson, G. J. Org.
Chem. 1998, 63, 9314.
(9) (a) Esnault, J.; Mallet, J.-M.; Zhang, Y.; Sinay, P.; Bouar, T. L.;
Pincet, F.; Perez, E.; Eur. J. Org. Chem. 2001, 253. (b) Fuente, J. M.;
Penades, S. Tetrahedron: Asymmetry 2002, 13, 1879.
(10) Sasaki, K.; Nishida, Y.; Tsuruimi, T.; Uzawa, H.; Kondo, H.;
Kobayashi, K. Angew. Chem., Int. Ed. 2002, 41, 4463. (b) Dohi, H.; Nishida,
Y.; Furuta, Y.; Uzawa, H.; Yokoyama, S.-I.; Ito, S.; Mori, H.; Kobayashi,
K. Org. Lett. 2002, 4, 355.
a Reagents: (a) methyl thiosalicylate, K2CO3/DMF, 80%; (b)
NaOMe/MoOH, 77%; (c) NaH, BnBr/DMF, 0 °C, 90%.
evolved during the preparation of 14 to allow a practical
thioglycosylation methodology.
Prior to the fucosylation, the reactivity of 14 was examined
with cetyl alcohol (A) and 1,2;5,6-di-O-isopropylidene-R-
(11) Page, D.; Roy, R. Glycoconjugate J. 1997, 14, 345.
3776
Org. Lett., Vol. 5, No. 21, 2003

Scheme 3. Synthesis of LewisX Trisaccharides 17 and 18a
Table 1. Glycosylation of Cetyl Alcohol (A) and
1,2:5,6-di-O-isopropylidene-R-D-glucofuranose (B) with 14
entry
acceptors
solvents
CH2Cl2
CH2Cl2
CH2Cl2 + Et2O
CH2Cl2
T (°C)
timea
R/âb
1
2
3
4
5
6
Ac
Ac
Ac
Bc
Bc
Bd
0
-40
-40
0
-40
-40
2
2
24
1
2
20
3:7
3:7
3:7
>9:1
8:2
7:3
CH2Cl2
CH2Cl2 + Et2O
a Reagents: (a) (i) ethylenediamine/EtOH, 50 °C, (ii) MeOH,
H2O, Ac2O, 62%; (b) 14, NIS, TfOH/CH2Cl2, 57%; (c) NaOMe/
MeOH, 91%; (d) H2, Pd(OH)2/C, MeOH; (e) succinic anhydride,
DIPEA/MeOH, quant.
a Reaction time (h) until the donor was consumed out in the reaction.
b Determined by 1H NMR analysis (accuracy within 5%). The yield based
on the amount of 14 is quantitative in every case. c 1.5 molar equiv of
acceptor was required for the donor 14 to complete the reaction in the
presence of 2 molar equiv. N-Iodosucciimide (NIS) and a catalytic amount
of triflic acid. d 6 molar equiv of the acceptor (A or B) was used.
diaminopropane 19 to give 20 carrying three amino groups
(Scheme 4). Condensation between 18 and 20 was completed
nearly quantitatively with 1-hydroxybenzotriazole (HOBT)
and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC)
in the presence of DIPEA. Product 3 was identified with
MALDI-TOF-MS [(M + Na)+ ) 2603] and 1H NMR
spectroscopy.
The above processes generated a hydrophobic amide linker
with some flexibility in the conformation. However, the
nature of this linker, in terms of conformation, length, and
hydrophobicity, was not evaluated or optimized in the present
study.
D-glucofuranose (B) as the acceptor substrates (Table 1). The
reaction of 14 with the primary alcohol (A) gave â-glycoside
predominantly, while it gave R-glycoside from the secondary
alcohol (B). A similar tendency was observed for o-MCP
1-thio-D-galactosyl donors, and this was explained in terms
of the participation of the o-carboxylate group.14 Though the
mechanism remains tentative, glycosylation reactions ap-
plying o-MCP 1-thio glycosides show a remarkable change
in the R- and â-selectivity depending on the reactivity of
the acceptors.
When the fucosyl donor 14 was applied to 10, no product
was obtained. On the other hand, an R-L-fucosyl product 16
was obtained in the reaction with 15 having an N-acetyl
group in the vicinity of OH-3. No â-isomer was detected by
the 1H NMR analysis of the product, and every acceptor was
consumed in the reaction [the yield (57%) of 16 after
purification on silica gel column is not optimized]. After the
removal of O-Ac and O-benzyl groups followed by the
reduction of a pNP group, pAP LewisX 17 was obtained
(Scheme 3).
Scheme 4 C3-Symmetric LewisX 3 and â-lactoside 21a
In our initial design, we had assumed direct condensation
between 2 and 17 to construct a rigid linker for a LewisX
trimer. This is because the rigidity is thought to minimize
the loss of entropy during a binding event and induce strong
bindings.7b The direct condensation, however, did not proceed
satisfactorily. This seemed ascribable to the low nucleo-
philicity of the arylamine. Thus, compound 17 was treated
with succinic anhydride and diisopropylethylamine (DIPEA)
and converted to 18 carrying a carboxyl group at the terminal.
The core 2 was modified with 1-N-tert-butoxycarbonyl-1,4-
(12) Debenham, J. S.; Madsen, R.; Roberts, C.; Fraiser-Reid, B. J. Am.
Chem. Soc. 1995, 117, 3302.
(13) Schmidt, R. R. AdV. Carbohydr. Chem. Biochem. 1994, 50, 21.
(14) (a) Dohi, H.; Nishida, Y.; Tanaka, H.; Kobayashi, K. Synth. Lett.
2001, 1446. (b) Dohi, H.; Nishida, Y.; Takeda, T, H.; Kobayashi, K.
Carbohydr. Res. 2002, 337, 983.
a Reagents: (a) 2, HOBT, EDC/DMF; (b) 4 N HCl in dioxane,
quant.; (c) 18, HOBT, EDC, DIPEA/DMF, 94%.
Org. Lett., Vol. 5, No. 21, 2003
3777

As a reference compound, a C3-symmetric â-lactoside 21
was also prepared from pNP â-lactoside4a,11 in the same way
as described above. Though the glycoconjugates 3 and 21
provide an apparent feature like amphiphilic molecules, they
show higher water solubility than glycero-glycolipids.15 Thus,
their 1H NMR spectra measured in D2O gave relatively sharp
signals at room temperature (Figure 2). The spectra have
1
Figure 3. Effect of Ca2+ ion (100 mM) on H NMR signals of
LewisX 3 (a) and â-lactoside 21 (b) trimers (10 mM, D2O, 500
MHz, room temperature).
association as Hakomori et al.5 proposed. The broadening is
considered to reflect the self-association that increases the
apparent molecular weight of 3 in aqueous solution and
1
accelerates H-spin-spin relaxation.16 These preliminary
results17 support that the C3-symmetric conjugates can
provide useful probes to investigate the LewisX-LewisX
interaction. More precise analyses presently in progress will
be reported elsewhere.
Figure 2. 1H NMR spectra of 3 (500 MHz, D2O, room temper-
ature).
Acknowledgment. This study was supported in part by
the 21st Century COE program “Nature-Guided Materials
Processing” of the Ministry of Education, Culture, Sports,
Science and Technology of the Japanese government.
also indicated that both of the glycoconjugates hold the C3-
symmetry in the aqueous solution displaying the three sugars
equivalently.
When the aqueous solution was treated with CaCl2, the
two conjugates turned out to behave in a different way from
each other. The 1H NMR signals of 3 became broader with
the addition of Ca2+ ion between 0 and 100 mM, while those
of 21 showed little broadening even at 100 mM Ca2+
concentration (Figure 3). The selective broadening for 3 is
in agreement with the Ca2+-dependent LewisX-LewisX
Supporting Information Available: Experiments and 1H
NMR data of the main products in Schemes 1-4. 1H NMR
and DLS profiles of compounds 3 and 21. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL0351293
(16) Bovey, F. A. High-Resolution NMR of Macromolecules; Academic
Press: New York, 1972.
(15) (a) Nishida, Y.; Ohrui, H. Meguro, H.; Ishizawa, M.; Matsuda, K.;
Taki, T.; Handa, S.; Yamamoto, N. Tetrahedron Lett. 1994, 35, 5465. (b)
Nishida, Y.; Takamori, Y.; Ohrui, H.; Ishizuka, I.; Matsuda, K.; Kobayashi,
K. Tetrahedron Lett. 1999, 40, 2371.
(17) A dynamic light-scattering (DLS) analysis also indicated a clear
difference. In the presence of Ca2+ ion (5 mM), the LewisX trimer 3 turns
to take two types of self-assembly forms with an averaged size of ca. 0.5
× 102 nm and larger than 2.0 × 102 nm, respectively.
3778
Org. Lett., Vol. 5, No. 21, 2003