Sialyl α(2 → 3) lactose clusters using carbosilane dendrimer core scaffolds as influenza hemagglutinin blockers

Article abstract of DOI:10.1016/j.bmcl.2008.06.101

An efficient synthesis of a series of carbosilane dendrimers uniformly functionalized with sialyl α(2 → 3) lactose (Neu5Acα(2 → 3)Galβ(1 → 4)Glcβ1→) moieties was accomplished. The results of a preliminary study on biological responses against influenza virus hemagglutinin, using the sialyl lactose clusters showed unique biological activities on the basis of the structure-activity relationship according to the carbosilane scaffolds.

Full text of DOI:10.1016/j.bmcl.2008.06.101

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4405–4408
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Sialyl
a
(2 ? 3) lactose clusters using carbosilane dendrimer core scaffolds
as influenza hemagglutinin blockers q
Hiroyuki Oka a, Tomotsune Onaga a, Tetsuo Koyama a, Chao-Tan Guo b,c,d, Yasuo Suzuki b,c
,
Yasuaki Esumi e, Ken Hatano a, Daiyo Terunuma a, Koji Matsuoka a,
*
a Division of Material Science, Graduate School of Science and Engineering, Saitama University, Sakura, Saitama 338-8570, Japan
b Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Aichi 487-8501, Japan
c Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
d Zhejiang Academy of Medical Science, 182 Tianmushan Road, Hangzhou 310016, China
e The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient synthesis of a series of carbosilane dendrimers uniformly functionalized with sialyl
a(2 ? 3)
Received 7 April 2008
Revised 16 June 2008
Accepted 17 June 2008
Available online 3 July 2008
lactose (Neu5Ac (2 ? 3)Galb(1 ? 4)Glcb1?) moieties was accomplished. The results of a preliminary
a
study on biological responses against influenza virus hemagglutinin, using the sialyl lactose clusters
showed unique biological activities on the basis of the structure–activity relationship according to the
carbosilane scaffolds.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Sialyl lactose
Hemagglutinin
Sialic acid
Influenza virus
Dendrimers
Carbosilanes
Glycoclusters
Carbosilanes are hybrid materials and show unique characteris-
tics in view of the interdisciplinary region of organic chemistry and
inorganic chemistry.1 We have therefore selected carbosilanes as
supporting materials for constructing glycodendrimers including
bioactive carbohydrate moieties.2 The chemical and biological sta-
bilities of carbosilane dendrimers uniformly functionalized with
carbohydrate moieties have been verified using Shiga toxin–carbo-
hydrate interactions,3 Dengue virus–carbohydrate interactions,4
and influenza virus neuraminidase–thiosialoside cluster-type
inhibitor interactions.5
Influenza viruses are unique because each virus has two glyco-
proteins with different roles on the surfaces of viral particles.6
Hemagglutinin (HA) is one of the proteins and it shows lectin-like
activity against sialyl oligosaccharides as specific receptors on host
cells.7 The other protein is a glycosidase, which is referred to as
either sialidase or neuraminidase (NA), and it selectively creaves
sialic acid residues from sialoglycoproteins as well as gangliosides
on the surfaces of host cells.8 Therefore, the virus is significantly
attractive and the proteins on the surfaces of influenza viruses
have completely different roles, such as adhesion to the host cell
and secession from the host cell.6 Many efforts have been made
to prepare multivalent-type glycomaterials as HA blockers for
inhibiting adhesion of the virus to a host cell.9 We have also estab-
lished a procedure for coupling between the carbosilane dendri-
mers and sialic acid derivatives including a sugar moiety of GM3
to afford corresponding glycodendrimers as multivalent-type HA
blockers. In this paper, we report a synthetic assembly of sialyl
a(2 ? 3) lactosyl (Neu5Aca(2 ? 3)Galb(1 ? 4)Glcb1?) moieties
using a series of carbosilane dendrimer scaffolds and the results
of biological evaluations of the glycodendrimers as potential candi-
dates for influenza virus hemagglutinin blockers.
Our synthetic target compounds having unique shapes, genera-
tions, and different numbers of saccharide residues are shown in
Figure 1. A compound 2 having zero generation and three sugar
moieties and 4 having first generation and six sugar moieties were
prepared by the method previously reported.10 A monomeric com-
pound 1 having a 4-pentenyl moiety as the aglycon was quantita-
tively prepared from a known compound 8 by a combination of
transesterification and saponification to remove all protections
q
Glyco-Silicon Functional Materials. Part 10. For Part 9, see Ref. 5.
* Corresponding author. Tel./fax: +81 48 858 3099.
E-mail address: koji@fms.saitama-u.ac.jp (K. Matsuoka).
All new compounds with specific rotation data gave satisfactory results of
elemental analyses or high-resolution mass spectra.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.101

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H. Oka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4405–4408
OH
OH
COOH
OH
OSLac
OSLac
OH
O
S
HO
AcHN
O
Ph Si
S
O
O
O
SLacO :
O
SLacO
S
HO
OSLac
OH
HO
HO
OH
1
2
OSLac
SLacO
S
S
S
Si
SLacO
OSLac
S
OSLac
O
SLacO
O
S
S
S
S
N
H
H
N
3
N
H
H
N
Si
6
O
OSLac
OSLac
O
SLacO
Me
Me
S
S
S
S
SLacO
OSLac
Si
Si
4
SLacO
SLacO
S
Si
S
OSLac
OSLac
SLacO
OSLac
S
S
S
O
N
O
O
N
N
H
O
Me
Me
O
H
O
H
Si
S
OSLac
Si
7
Si
SLacO
S
N
H
Ph Si
S
OSLac
N
H
N
H
H
N
H
H
N
N
O
O
O
S
S
S
OSLac
5
SLacO
OSLac
Figure 1. A series of multivalent-type synthetic substrates having sialyl
a
(2 ? 3) lactose.
18
(Scheme 1), ½aꢀD ꢁ 3:4ꢂ (c 0.96, H2O), 1H NMR (D2O) d 4.40 (d, 1 H,
methoxide in methanol–DMF solvent systems, followed by the
usual reacetylation to provide fully protected carbosilane dendri-
mers having sialyl lactose moieties at each terminal, Fan(0)3-type
16,10 Ball(0)4-type 17, integral ratio of the H atoms by 1H NMR:
SiCH2:H-1:H-10 = 8:4:4, Dumbbell(1)6-type 18,10 Fan(0)3-amide-
J1 ;2 ¼ 7:7 Hz, H-10), 4.35 (d, 1 H, J1,2 = 8.1 Hz, H-1), 2.63 (dd, 1 H,
0
0
¼ 4:0 Hz, H-300eq) 1.77 (t,
1
H,
00ax;300eq
00eq;400
J3
J3
¼ 12:5 Hz, J3
¼ 12:1 Hz, H-300ax). The other transformation of 8 was also
00ax;400
performed at the terminal C@C double bond by a radical addition
of thioacetic acid in the presence of AIBN as the radical initiator to
furnish thioacetate 9 in high yield.
type 19, integral ratio of the
SiCH2:CH2SCH2:Ph:H-300eq = 6:12:5:3, Ball(0)4-amide-type 20,
integral ratio of the
atoms by 1H NMR: SiCH2:NCH2:H-
H
atoms by 1H NMR:
A series of alkyl halide-type carbosilane compounds as support-
ing materials for carbohydrates are shown in Figure 2. Dumb-
bell(1)6-type 15 was additionally synthesized by the method
previously reported.11 In brief, a known carbosilane having six bro-
mine atoms at their terminal ends3e was converted into the azide,
which was then transformed into the amine in the presence of
Ph3P and water. The amine was further treated with 6-bromohexa-
noyl chloride to yield an amide 15 after chromatographic
purification.
A synthetic scheme for construction of a series of glycodendri-
mers is illustrated in Scheme 2. Coupling reaction between the car-
bosilane dendrimer scaffolds 10–15 and sialyl lactose derivative 9
was performed in a one-pot reaction in the presence of sodium
H
10 = 8:8:4, Dumbbell(1)6-amide-type 21, integral ratio of the H
atoms by 1H NMR not determined due to intramolecular lactoniza-
tion. Removal of all protective groups in the dendrimers was car-
ried out by the same two-step procedure as that described for
the preparation of 1 to give white powdery compounds after chro-
matographic purification, followed by lyophilization, Fan(0)3-type
2,10 Ball(0)4-type 3, integral ratio of the H atoms by 1H NMR:
SiCH2:H-1:H-10:H-300eq = 8:4:4:4, MALDI-TOF MS calcd for
[M+Na]+: 3158.4; found m/z: 3159.37, Dumbbell(1)6-type 4,10
Fan(0)3-amide-type 5, integral ratio of the H atoms by 1H NMR:
SiCH2:CH2SCH2:Ph:H-1:H-10:H-300eq = 6:12:5:3:3:3,
MS calcd for [M+Na]+: 2798.1; found m/z: 2799.26, Ball(0)4-
amide-type 6, integral ratio of the
atoms by 1H NMR:
MALDI-TOF
H
SiCH2:NCH2:H-1:H-10:H-300eq = 8:8:4:4:4, MALDI-TOF MS calcd
for [M+Na]+: 3611.0; found m/z: 3610.44, Dumbbell(1)6-amide-
type 7, integral ratio of the H atoms by 1H NMR: CH3Si:H-1:H-
10:H-300eq = 6:6:6:6, MALDI-TOF MS calcd for [M+Na]+: 5561.4;
found m/z: 5562.36.
OAc
OAc
OAc
COOMe
OAc
AcO
AcHN
(i)
O
O
O
O
1
OR
O
AcO
OAC
AcO
AcO
OAc
Since the systematic synthesis of glycoclusters having sialyl
a
(2 ? 3) lactose (Neu5Aca(2 ? 3)Galb(1 ? 4)Glcb1?) moieties
8 : R = (CH2)3CH=CH2
9 : R = (CH2)5SAc
(ii)
1–7 was efficiently accomplished, our attention was directed to-
ward the structure–activity relationship (SAR) of the sialyl com-
pounds as HA blockers against influenza virus. Thus, the
hemagglutinin inhibition (HAI) assay12 was preliminarily per-
Scheme 1. Reagents and conditions: (i) NaOMe, MeOH, rt, overnight, then 0.05 M
aq. NaOH, rt, 2 h, 84%; (ii) AIBN, HSAc, 1,4-dioxane, 50 ? 80 °C, 3 h, 99%.

H. Oka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4405–4408
4407
O
Br
Ph Si
Br
Ph Si
N
H
3
3
10
Fan(0)3-Br
13
Fan(0)3-amide-Br
O
Si
Br
Br
Si
N
H
4
4
11
14
Ball(0)4-Br
Ball(0)4-amide-Br
O
N
Me
Me
Me
Me
Br
Si
Si
Br
Si
Si
H
3
3
2
2
15
Dumbbell(1)6-amide-Br
12
Dumbbell(1)6-Br
Figure 2. A series of alkyl halide-type carbosilane dendrimers uniformly functionalized with a bromo atom at each terminal end.
OAc
OAc
OAc
COOMe
OAc
(i)
AcO
AcHN
(ii)
O
Br
O
O
O
9
2 ~ 7
OR
+
O AcO
10: Fan(0)3-Br
11: Ball(0)4-Br
12: Dumbbell(1)6-Br
OAC
AcO
AcO
OAc
16: R = Fan(0)3
17: R = Ball(0)4
18: R = Dumbbell(1)6
19: R = Fan(0)3-amide
20: R = Ball(0)4-amide
13: Fan(0)3-amide-Br
14: Ball(0)4-amide-Br
15: Dumbbell(1)6-amide-Br
21: R = Dumbbell(1)6-amide
Scheme 2. Reagents and conditions: (i) NaOMe, MeOH–DMF, rt, then Ac2O–Pyr, rt, then CH2N2, Et2O, rt, for 16 (Ref. 10), for 17 (33%), for 18 (Ref. 10), for 19 (66%), for 20 (15%),
for 21 (35%); (ii) NaOMe, MeOH, rt, overnight, then 0.05 M aqueous NaOH, rt, for 2 (Ref. 10), for 3 (94%), for 4 (Ref. 10), for 5 (quant.), for 6 (64%), for 7 (77%).
formed using various virus strains, and the results are summarized
in Figure 3. The virus strains used in this study were human influ-
enza viruses A/PR/8/34 (H1N1), A/Aich/2/68 (H3N2), and A/Mem-
phis/1/71 (H3N2). The strain A/PR/8/34 recognizes
Neu5Ac (2 ? 3)Gal residues in glycoconjugates and binds to
erythrocytes. A/Aich/2/68-type virus recognizes both Neu5A-
(2 ? 3)Gal and Neu5Ac (2 ? 6)Gal residues and A/Memphis/
1/71-type virus recognizes Neu5Ac (2 ? 6)Gal residues. Inhibitory
a
1000
A/PR/8/34
(H1N1)
A/Aichi/2/68 A/Memphis/1/71
(H3N2) (H3N2)
ca
a
a
activity of the series of glycodendrimers against hemagglutination
of various human influenza viruses to erythrocytes was clearly ob-
served when A/PR/8/34 and A/Aich/2/68 were used. Inhibitory po-
tency of the glycodendrimers against A/Memphis/1/71 was weak
because of an unsuitable carbohydrate structure for the HA on
the virus strain. These results strongly suggested that oligosaccha-
ride chains of the glycoclusters were closely recognized by the cor-
responding HA on the virus surface. In addition, we found by using
SARs methodology that Dumbbell(1)6-amide 7 showed the highest
level of inhibitory activity in glycolibrary. These carbosilane den-
100
10
1
drimers uniformly functionalized with sialyl a(2 ? 3) lactose moi-
eties have unique characteristics, such as different numbers of
sugar moieties depending on the core structure, different degree
of freedom of the sugar moieties depending on the spacer length
and core shape, and different 3-D structures. This phenomenon is
useful for consideration of synthetic construction of other glyco-
dendrimers as well as expansion of the glycolibrary. Furthermore,
1
2
3
4
5
6
7
Glycodendrimers
Figure 3. Inhibitory activity of a series of glycodendrimers against hemagglutina-
tion of various human influenza viruses. Concentrations of glycodendrimers were
these glycodendrimers having Neu5Aca(2 ? 3)Gal residues at
calculated on the basis of a sialyl a(2 ? 3) lactose unit.
their terminal ends are promising agents for prevention of

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H. Oka et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4405–4408
7669; (b) Nishikawa, K.; Matsuoka, K.; Watanabe, M.; Igai, K.; Hino, K.; Hatano,
K.; Yamada, A.; Abe, N.; Terunuma, D.; Kuzuhara, H.; Natori, Y. J. Infect. Dis.
2005, 191, 2097; (c) Matsuoka, K.; Terabatake, M.; Esumi, Y.; Terunuma, D.;
Kuzuhara, H. Tetrahedron Lett. 1999, 40, 7839; (d) Matsuoka, K.; Terabatake, M.;
Umino, A.; Esumi, Y.; Hatano, K.; Terunuma, D.; Kuzuhara, H.
Biomacromolecules 2006, 7, 2274; (e) Matsuoka, K.; Terabatake, M.; Esumi, Y.;
Hatano, K.; Terunuma, D.; Kuzuhara, H. Biomacromolecules 2006, 7, 2284.
4. (a) Aoki, C.; Hidari, K. I. P. J.; Itonori, S.; Yamada, A.; Takahashi, N.; Kasama, T.;
Hasebe, F.; Islam, M. A.; Hatano, K.; Matsuoka, K.; Taki, T.; Guo, C.-T.; Takahashi,
T.; Sakano, Y.; Suzuki, T.; Miyamoto, D.; Sugita, M.; Terunuma, D.; Morita, K.;
Suzuki, Y. J. Biochem. 2006, 139, 607; (b) Yamada, A.; Hatano, K.; Koyama, T.;
Matsuoka, K.; Esumi, Y.; Terunuma, D. Carbohydr. Res. 2006, 341, 467.
5. Sakamoto, J.-I.; Koyama, T.; Miyamoto, D.; Yingsakmongkon, S.; Hidari, K. I. P. J.;
Jampangern, W.; Suzuki, T.; Suzuki, Y.; Esumi, Y.; Hatano, K.; Terunuma, D.;
Matsuoka, K. Bioorg. Med. Chem. Lett. 2007, 17, 717.
infection with avian influenza (H5N1), since avian influenza virus
recognizes Neu5Ac
(2 ? 3)Gal residues on host cells.13
In conclusion, an efficient synthesis of a series of glycoclusters
having sialyl
(2 ? 3) lactose moieties 1 ꢃ 7 was systematically
a
a
accomplished using various carbosilane dendrimers as supporters
for the oligosaccharides. Biological evaluations of these glycoden-
drimers against influenza virus were preliminarily carried out,
and the results showed that a glycodendrimer having longer
spacer-arms and most carbohydrate epitopes has the highest activ-
ity. The results suggested SARs in this biological response using the
glycolibrary was observed. Experimental details for the syntheses
of the series of glycodendrimers in this study and other biological
activities will be reported elsewhere in the near future.
6. Webster, R. G.; Bean, W. J.; Gorman, O. T.; Chambers, T. M.; Kawaoka, Y.
Microbiol. Rev. 1992, 56, 152. and references cited therein.
7. For example, see Wiely, D. C.; Skehel, J. J. Ann. Rev. Biochem. 1987, 56, 365. and
references cited therein.
8. For example, see Hayden, F. G.; Calfee, D. P. Drugs 1998, 56, 537. and references
cited therein.
Acknowledgments
9. (a) Spaltenstein, A.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 686; (b)
Tsuchida, A.; Kobayashi, K.; Matsubara, N.; Muramatsu, T.; Suzuki, T.; Suzuki, Y.
Glycoconjugate J. 1998, 15, 1047; (c) Kamitakahara, H.; Suzuki, T.; Nishigori, N.;
Suzuki, Y.; Kanie, O.; Wong, C.-H. Angew. Chem. Int. Ed. 1998, 37, 1524; (d)
Fruike, T.; Aiba, S.; Suzuki, T.; Takahashi, T.; Suzuki, Y.; Yamada, K.; Nishimura,
S.-I. J. Chem. Soc., Perkin Trans. 1 2000, 3000; (e) Makimura, Y.; Zhonghong, G.;
Roy, R. Int. Congress Series 2001, 1223, 45; (f) Ohta, T.; Miura, N.; Fujitani, N.;
Nakajima, F.; Niikura, K.; Sadamoto, R.; Guo, C.-T.; Suzuki, T.; Suzuki, Y.;
Monde, K.; Nishimura, S.-I. Angew. Chem. Int. Ed. 2003, 42, 5186; (g) Ogata, M.;
Murata, T.; Murakami, K.; Suzuki, T.; Hidari, K. I. P. J.; Suzuki, Y.; Usui, T. Bioorg.
Med. Chem. 2007, 15, 1383.
10. Matsuoka, K.; Oka, H.; Koyama, T.; Esumi, Y.; Terunuma, D. Tetrahedron Lett.
2001, 42, 3327.
11. Matsuoka, K.; Terabatake, M.; Saito, Y.; Hagihara, C.; Esumi, Y.; Terunuma, D.;
Kuzuhara, H. Bull. Chem. Soc. Jpn. 1998, 71, 2709.
12. Suzuki, Y.; Suzuki, T.; Matsumoto, M. J. Biochem. (Tokyo) 1983, 93, 1621.
13. (a) Rogers, G. N.; Paulson, J. C. Virology 1983, 127, 361; (b) Shinya, K.; Kawaoka,
Y. Uirusu 2006, 56, 85.
We are grateful to Snow Brand Milk Products Co., Ltd. for pro-
viding the sialic acid used in this study. This work was partly sup-
ported by a Grant-in-Aid for Encouragement of Young Scientists
(12750754) from the Ministry of Education, Science, and Culture
of Japan.
References and notes
1. For example, see Majoral, J.-P.; Caminade, A.-M. Chem. Rev. 1999, 99, 845. and
references cited therein.
2. Yuasa, H. Ed. Nanotechnology in carbohydrate chemistry; Transworld
Research Network: Kerala, 2006, Matsuoka, K.; Hatano, K.; Terunuma, D.
pp. 89–102.
3. (a) Nishikawa, K.; Matsuoka, K.; Kita, E.; Okabe, N.; Mizuguchi, M.; Hino, K.;
Miyazawa, S.; Yamasaki, C.; Aoki, J.; Takashima, S.; Yamakawa, Y.; Nishijima,
M.; Terunuma, D.; Kuzuhara, H.; Natori, Y. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,