Syntheses and biological evaluations of carbosilane dendrimers uniformly functionalized with sialyl α(2→3) lactose moieties as inhibitors for human influenza viruses

Article abstract of DOI:10.1016/j.bmc.2009.06.035

A series of carbosilane dendrimers uniformly functionalized with sialyl lactose moieties (Neu5Acα2→3Galβ1→4Glc) was systematically synthesized, and biological evaluations for anti-influenza virus activity using the glycodendrimers were performed. The resu

Full text of DOI:10.1016/j.bmc.2009.06.035

Bioorganic & Medicinal Chemistry 17 (2009) 5465–5475
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Syntheses and biological evaluations of carbosilane dendrimers uniformly
functionalized with sialyl
a(2?3) lactose moieties as inhibitors for human
influenza viruses 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:
A series of carbosilane dendrimers uniformly functionalized with sialyl lactose moieties (Neu5Aca2?3-
Received 5 June 2009
Revised 17 June 2009
Accepted 18 June 2009
Available online 21 June 2009
Galb1?4Glc) was systematically synthesized, and biological evaluations for anti-influenza virus activity
using the glycodendrimers were performed. The results suggested that the glycodendrimers had unique
biological activities depending on the form of their core frame, and Dumbbell(1)6-amide type glycoden-
drimer 7 showed particularly strong inhibitory activities against human influenza viruses [A/PR/8/34
(H1N1) and A/Aichi/2/68 (H3N2)]. The results suggested that the structure–activity relationship (SAR)
on the glycolibrary against various influenza viruses was observed, and dumbbell-shaped dendrimers
as supporting carbohydrate moieties were found to be the most suitable core scaffolds in this study.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Sialyl lactose
Hemagglutinin
Sialic acid
Influenza virus
Dendrimers
Carbosilanes
Glycoclusters
1. Introduction
Sialyl lactose (Neu5Aca2?3Galb1?4Glc), a sugar chain of gan-
glioside GM3 on the cell surface, is known to a specific receptor of
HAs of influenza A viruses.6 HA has an important role on the basis
of carbohydrate–protein interaction in order to adhere the surface
glycosyl receptor on the host cell. Three HAs aggregate to form a
trimeric structure by hydrophobic interaction on the viral particle,
and HAs therefore have three sugar-binding sites on each unit.6
These three sugar-binding sites locate on a each end of ca. 45 Å tri-
angle.6 Cluster-type carbohydrate derivatives7 are expected to be
effective artificial receptors to the binding sites. Synthetic assem-
bly of sialyl lactose against the trimeric binding sites of HAs was
valuable approach for the prevention from infection of influenza
A viruses. Similar strategies to inhibit receptor-ligand interaction
have recently been reported.8 For example, linear-type polystyrene
bearing sialyl lactose moieties as a polymeric HA receptor has been
synthesized.9 Linear-type polyacylamide as backbone has been
also synthesized.10 Both of them were showed high biological
activities against viral HAs. In order to construct clustered sialyl
lactose moieties, carbosilane dendrimers were conveniently
selected as core scaffolds. The advantages of using carbosilane den-
drimers are easy control of the number of functional groups at ter-
minal ends, easy accessibility to a macromolecule having suitable
Influenza virus is a highly pathogenic virus, and an average of
about 226,000 patients with influenza virus have been hospitalized
every year in the USA.1 Influenza A viruses have two unique glyco-
proteins, hemagglutinin (HA) and neuraminidase (NA), on the
surfaces of viral particles for infection and replication.2 Neuramin-
idase inhibitors, including zanamivir3 and oseltamivir,4 have been
synthesized and widely used worldwide for clinical treatment of
influenza A virus. Although these agents have strong inhibitory
activities against influenza virus, several strains of resistant viruses
were emerged and their crystal structures of neuraminidase mu-
tants have been studied.5 In view of the outbreak of neuramini-
dase-resistant viruses as well as the possibility of appearance of
further mutants related to influenza virus, alternative types of
therapeutic agents are needed. One of the approaches to prevent
first contact of the virus and a host cell has focused on HAs, which
participate in infection of influenza virus to host cells.
q
Glyco-silicon functional materials, Part 12. For Part 11, see Ref. 25b.
* Corresponding author. Tel./fax: +81 48 858 3099.
E-mail address: koji@fms.saitama-u.ac.jp (K. Matsuoka).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.035

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H. Oka et al. / Bioorg. Med. Chem. 17 (2009) 5465–5475
molecular size, and low toxicity for living organisms.11 In our pre-
vious studies, some functionalized carbosilane dendrimers uni-
formly functionalized with cyclodextrins for host–guest
interactions,12 with globotriaoses for Shiga toxin–carbohydrate
interactions,13 and with lacto-N-neotetraoses for Dengue virus–
carbohydrate interactions,14 were efficiently synthesized and
showed attractive biological activities.
In this paper, we report the systematic synthesis of various car-
bosilane dendrimers having sialyl lactose moieties at each terminal
end on the surface of dendritic structures, as shown in Figure 1,
and the results of biological evaluations of the synthesized glyco-
dendrimers for human influenza viruses.
gar aglycon, such as a butenyl glycoside, was progressed in good
yield.13 Although the acetyl-thio functional group at the end of su-
gar aglycon is readily de-S-acetylated to give the corresponding
thiolate anion, which is a superior nucleophile for SN2 reaction
with alkyl halide, a radical addition of thioacetic acid to the termi-
nal double bond, especially sugar aglycon, had not been reported.
Thus, radical additions of thioacetic acid into allyl glycosides,
either acetate 8 or benzylate 10, were successfully performed to
give corresponding thioacetate 9 (67%) or 11 (56%) and recovered
starting materials 8 (31%) or 10 (23%). In the addition reaction,
0.5 equiv molar use of AIBN for the double bond was required to
achieve the above yields. When a small amount of AIBN was use
for the same reaction, prolonged reaction time was needed. Addi-
tion of more AIBN (1 equiv molar or more) to the reaction mixture
did not improve the reaction yields. However, when pentenyl gly-
cosides, either acetate 12 or benzylate 14, were used for the reac-
tion, addition of 0.5 equiv molar of AIBN gave the corresponding
thioacetate 13 or 15 in quantitative yield. The results suggest that
it is important to choose appropriate chain length for a radical
addition of thioacetic acid to the terminal C@C double bond of su-
gar aglycon.16
Scheme 2 summarizes synthetic assembly of lactose moieties
using dendritic core scaffolds as shown in Figure 2. A one-pot pro-
cedure involving de-S-acetylation of 13 with NaOMe and subse-
quent coupling reaction to a known carbosilane dendrimer 1613a
in MeOH–DMF was carried out, and the reaction proceeded
smoothly to afford 22 in 71% yield. Introduction of lactosyl deriva-
tives 13 into a dumbbell-type carbosilane dendrimer 1813b was
then carried out in the same manner as that described for fan-type
carbosilane dendrimers to provide carbosilane dendrimer 24 hav-
ing six lactose moieties in 62% yield. A disulfide-linked lactoside di-
mer 23 was also obtained as by-product in the reaction.
2. Results and discussion
Sialyl lactose is chemically labile under strong basic conditions
resulting to form intra-molecular lactam.15 In our previous study,
introduction of sugar moieties into carbosilane dendrimers in li-
quid ammonia in the presence of Na metal was reported.12,13 This
method could not be used for introduction of sialyl acid-containing
sugar moieties because of the strong basic conditions. Thus, we
have established an alternative route for the introduction of sialyl
lactose moieties into carbosilane dendrimers using lactose as a
model sugar.16 Application of the methodology for assembly of sia-
looligosaccharide using carbosilane scaffolds was tested to provide
a library of carbosilane dendrimers carrying sialyl lactose moieties
as the epitope for influenza HAs.
2.1. Synthesis of carbosilane dendrimers having lactose
moieties as model introduction for unstable sialyl lactose
moieties
Deprotection of fan-type dendrimer 22 was performed with a
combination of Zemplén’s transesterification and subsequent
saponification reaction to yield the corresponding water-soluble
carbosilane dendrimer 25 having three lactose moieties quantita-
tively. In the course of this reaction, partially deprotected products
Synthetic introduction of a thioacetate moiety into unsaturated
aglycons of lactosides is demonstrated in Scheme 1. A pentenyl lac-
toside 12 was readily synthesized from b-lactose per acetate and 4-
penten-1-ol in the presence of BF3–OEt2 as Lewis acid.13a Radical
addition of alkyl thiol to a C@C double bond at the terminal of su-
OH
OH
O
COOH
O
OH
OH
OH
O
HO
S
:
O
O
S
S
S
Ph Si
S
AcHN
HO
Si
OH
S
HO
S
OH
3
2
1
O
O
S
S
Si
6
N
O
N
Me
Me
S
H
H
O
S
S
Si
S
S
S
Si
Si
N
N
H
H
S
S
4
H
N
O
H
N
S
S
S
N
Me
Me
O
H
N
H
H
O
O
H
S
Ph Si
Si
7
Si
N
H
N
Si
N
S
S
O
H
O
O
N
S
N
H
S
5
O
S
O
Figure 1. A series of carbosilane dendrimers having sialyl a(2?3) lactose.

H. Oka et al. / Bioorg. Med. Chem. 17 (2009) 5465–5475
5467
OR
O
OR
OR
O
OR
O
O
RO
O
SAc
(i)
RO
O
O
RO
OR
O
OR
RO
OR
n
OR
OR
OR
n
8 : R = Ac, n = 1
10 : R = Bn, n = 1
12 : R = Ac, n = 3
14 : R = Bn, n = 3
9 : R = Ac, n = 1
11 : R = Bn, n = 1
13 : R = Ac, n = 3
15 : R = Bn, n = 3
Scheme 1. Reagents and conditions: (i) AIBN, HSAc, 1,4-dioxane, 50 °C?80 °C, 3 h, for 9 (67%), for 11 (56%), for 13 (quant), for 15 (quant).
OR
O
OR
O
OAc
O
OAc
(i)
O
O
RO
OR
S
O
O
AcO
S
RO
+
Si Ph
13 + 16
O
AcO
OR
OR
3
OAc
OAc
2
OAc
22 : R = Ac
25 : R = H
(ii)
23
OR
O
OR
O
Me
Me
(i)
O
O
RO
OR
+ 23
S
Si
Si
13 + 18
RO
OR
2
3
OR
24 : R = Ac
26 : R = H
(iii)
Scheme 2. Reagents and conditions: (i) NaOMe, MeOH–DMF, rt, overnight, then Ac2O–Pyr, rt, overnight, for 22 (71%), for 24 (62%); (ii) NaOMe, MeOH, rt, overnight, then
0.1 M aq NaOH, rt, overnight, quant.; (iii) NaOMe, MeOH, rt, overnight, then 0.07 M aq NaOH, rt, overnight, quant.
H
Br
N
S
H
O
Br
Ph Si
N
S
S
S
Br
Br
S
S
Br
Br
Ph Si
H
N
O
O
16
19
Br
Br
H
Br
Br
O
S
S
N
Br
Br
Br
N
S
S
S
O
H
N
Si
Si
O
H
S
Br
S
N
S
O
H
20
17
Br
Br
O
H
S
Br
Br
S
Br
Br
Br
Br
N
N
Me
S
Me
O
Me
Me
S
S
H
O
O
H
S
Si
Si
N
H
Si
Si
Si
S
S
Si
N
S
S
O
H
N
S
S
Br
N
Br
Br
Br
H
O
18
21
Figure 2. A series of carbosilane dendrimers uniformly functionalized with a bromo atom at each terminal end.
were observed on TLC and the reaction mixture became a suspen-
sion due to precipitation of the partially deprotected compounds.
Therefore, two-step reaction, such as ester exchange reaction and
subsequent saponification, was needed for complete deacetylation.
In the same manner, deprotection of dumbbell-type dendrimer 24
gave the corresponding water-soluble carbosilane dendrimer 26
having six sialyl lactose moieties in quantitative yield.
2.2. Synthesis of sialyl lactoside having a thioacetylated pentyl
group as the aglycon
Construction and chemical modification of a sialyl lactose deriv-
ative are shown in Scheme 3. Sialyl lactose moiety of 31 was pre-
pared by coupling reaction between lauryl thioglycoside of Neu5Ac
2817 and a known lactosyl diol 3018 with good
a-selectivity. An

5468
H. Oka et al. / Bioorg. Med. Chem. 17 (2009) 5465–5475
OBn
O
OBn
HO
O
OSE
O
BnO
OBn
OBn
OH
30
ii)
OAc
1
OAc
OAc
O
OAc
O
R
OAc
COOMe
AcO
AcHN
2
i)
AcO
AcHN
R
OAc
OAc
27
1
2
28 : R = COOMe, R = SLauryl
1
2
29 : R = SLauryl, R = COOMe
OAc
OAc
OAc
OAc
COOMe
OBn
iii)
OBn
O
COOMe
OAc
O
iv)
OAc
O
AcO
AcHN
AcO
AcHN
O
O
NH
CCl
O
O
OSE
O
O
O
BnO
OBn
AcO
OAc
OAc
OBn
OAc
OH
AcO
OAc
O
3
31
32
OAc
OAc
OAc
OAc
COOMe OAc
OAc
O
COOMe OAc
OAc
O
v)
AcO
AcHN
AcO
AcHN
SAc
O
AcO
O
O
O
O
O
O
O
O
O
AcO
OAc
OAc
OAc
OAc
OAc
OAc
OAc
33
OAc
34
vi)
1
Scheme 3. Reagents and conditions: (i) 1-dodecanethiol, BF3–OEt2, CH2Cl2, 0 °C?rt, 3 h, a-laurylate 28 (26%) and b-laurylate 29 (59%); ii) 28 (2 equiv molar), 30 (1 equiv.
molar), NIS, TfOH, 3 Å MS, CH3CN, 55%; (iii) Ref. 14; (iv) 4-penten-1-ol, BF3–OEt2, 4 Å MS, CH2Cl2, ꢀ25 °C?-5 °C, 4 h, 84%; (v) AIBN, HSAc, 1,4-dioxane, 50 °C?80 °C, 3 h, 99%;
(vi) NaOMe, MeOH, rt, overnight, then 0.05 M aq NaOH, rt, 2 h, 84%.
anomeric mixture of lauryl thiosialosides 28 and 29 was found to
be an efficient glycosyl donor for sialylation, and the thioglycoside
was easily prepared from a known acetate 27.19 Trichloroacetoim-
idate 32 was derived from 31 by applying the method reported by
Tietze et al. in four steps in good total yield.20
Glycosidation reaction between imidate 32 and 4-penten-1-ol
(n-pentenyl alcohol) was carried out in the presence of a Lewis acid
as a promoter to give b-pentenyl sialyl lactoside 33 with high ano-
meric b-selectivity in high yield. Radical addition of thioacetic acid
into the terminal C@C double bond of 33 required one more equiv-
alent molar of AIBN per double bond for completion of the reaction.
When 0.5 equiv molar of AIBN was used for the reaction, unreacted
33 remained. NMR spectra of 34 suggested that the radical addition
reaction of thioacetic acid into the alkenyl moiety was accom-
plished with antiMarkovnikov’s orientation.
the carboxyl group of sialic acid were carried out for temporal puri-
fication of the desired dendrimers. A combined purification by sil-
ica gel column chromatography and gel permeation
chromatography gave pure 35 in 80.2% yield including 100,20-lac-
tone form, which was formed by intramolecular ester exchange
reaction. In the same manner, coupling reactions into ball-type
dendrimer 17,13b dumbbell-type dendrimer 18 and amide-type
dendrimers 19, 20, 2112 were performed and gave corresponding
sugar-modified dendrimers 36, 37, 39, 40, and 41 in adequate
yields. A disulfide-linked sialyl lactoside dimer 38 was also ob-
tained as by-product in the reaction. Ball-type dendrimers 36
and 40 showed unfortunately lower yields than those of other
dendrimers.
As a model reaction for complete deprotection of sialyl lactose
moieties in Scheme 3, removal of all protection of 33 was achieved
with two-step reaction, such as ester exchange reaction and subse-
quent saponification reaction, to provide corresponding sialyl lac-
toside 1 having a pentenyl moiety as the aglycon in 84.4% yield.
Saponification of sialoside 33 was carried out using 0.05 M aq
NaOH in order to avoid lactam formation under strong basic condi-
tions. The analogues of GM3 were known to show an equilibrium
between normal sialic acid formation and it is lactone formation
in aqueous solution.21 Pentenyl sialyl lactoside 1 also formed an
equilibrium between free sialic acid and it’s lactone in aq solution
in a few days.
2.3. Synthesis of carbosilane dendrimers uniformly
functionalized with sialyl lactosyl moieties as multivalent-type
epitopes
We have established a model reaction for introduction of lac-
tose derivatives 13 into carbosilane dendrimers 16 and 18 by mak-
ing sulfide linkage in DMF–MeOH as a solvent system in the
presence of NaOMe. This reaction involves removal of the S-acetyl
group to generate thiolate anion and subsequent replacement of
the bromine atom in alkyl halide-type dendrimers as shown in Fig-
ure 2. The one-pot reaction, de-S-acetylation, followed by coupling
reaction, was applied for sialyl lactosyl derivatives 34. Under these
conditions, partial de-O-acetylation and demethylation of the
methyl ester occurred, and the purification of the reaction mixture
was impossible. Thus, re-O-acetylation and methyl esterification of
Deprotection of a series of dendrimers was demonstrated as
shown in Scheme 4. Thus, deprotection of fan-type dendrimer 35
was carried out with ester exchange reaction and subsequent
saponification reaction to yield the corresponding water-soluble
carbosilane dendrimer having three sialyl lactose moieties 2 quan-
titatively. In the same manner, removal of all protections of 36, 37,

H. Oka et al. / Bioorg. Med. Chem. 17 (2009) 5465–5475
5469
OAc
OAc
COOMe
OAc
OAc
COOMe
OAc
i)
OAc
O
OAc
OAc
O
AcO
AcHN
O
AcO
OR
O
AcO
AcHN
Br
O
S
34 +
O
+
O
OAc
O
AcO
O
O
O
OAc
OAc
OAc
2
OAc
OAc
OAc
OAc
16 : Fan(0)3-Br
17 : Ball(0)4-Br
18 : Dumbbell(1)6-Br
19 : Fan(0)3-amide-Br
20 : Ball(0)4-amide-Br
35 : R = Fan(0)3
36 : R = Ball(0)4
37 : R = Dumbbell(1)6
39 : R = Fan(0)3-amide
40 : R = Ball(0)4-amide
38
21 : Dumbbell(1)6-amide-Br 41 : R = Dumbbell(1)6-amide
ii)
35, 36, 37, 39, 40, 41
2
7
Scheme 4. Reagents and conditions: (i) NaOMe, MeOH–DMF, rt, then Ac2O–Pyr, rt, then CH2N2, Et2O, rt, for 35 (80%), for 36 (33%), for 37 (77%), for 39 (66%), for 40 (16%), for
41 (35%); (ii) NaOMe, MeOH, rt, then 0.05 M aq NaOH, rt, for 2 (quant), for 3 (94%), for 4 (quant), for 5 (quant), for 6 (64%), for 7 (77%).
39, 40, and 41 provided corresponding water-soluble dendrimers
3, 4, 5, 6, 7, respectively, in good yields. Structural elucidation
was performed by a combination of 1H NMR spectroscopic analysis
and mass spectroscopy. The results of the NMR showed near-dou-
blet peaks at around 2.6–2.7 ppm, which were assignable to H-300eq
of sialyl acid residue in dendrimers, while H-300eq of lactone
showed doublet–doublet peaks at around 2.2–2.3 ppm.
Table 1
Inhibitory activities of
human influenza viruses (type-A) to erythrocytes
a series of glycodendrimers against hemagglutination of
Compounds
Inhibition activities (
lM)
A/PR/
A/Aichi/
A/Memphis/
8/34 (H1N1)
2/68 (H3N2)
1/71 (H3N2)
1 [Pent-SLac]
2 [Fan(0)3-SLac]
3 [Ball(0)4-SLac]
4 [Dumbbell(1)6-SLac]
5 [Fan(0)3-amide-SLac]
6 [Ball(0)4-amide-SLac]
7 [Dummbell(1)6-amide-Slac]
125
64
64
32
16
16
8
250
125
32
32
8
500
250
250
250
250
250
250
2.4. Biological evaluations of the activities of carbosilane
dendrimers having sialyl lactose moieties against human
influenza virus
4
4
Since systematic synthesis of a glycolibrary of sialyl lactose
using carbosilane dendrimers was successfully accomplished, our
attention was directed toward the biological activity of the glycol-
ibrary against influenza viruses. The inhibitory activities of these
dendrimers against three types of human influenza A virus, (A/
PR/8/34, H1N1), (A/Aichi/2/68, H3N2) and (A/Memphis/1/71,
H3N2), were initially evaluated by means of hemagglutination
inhibition (HAI) assays. The hemagglutinin on viral particles of
Values are IC50 values based on a sialyl lactose unit.
inhibition assay because the virus strain of A/PR/8/34 strongly rec-
ognizes the Neu5Ac (2?3)Gal structure, which was appropriate
a
for our synthesized glycodendrimers. The inhibitory activities of
glycodendrimers in the hemolysis inhibition assay of influenza
virus to erythrocytes are shown in Figure 3, where concentrations
of glycodendrimers are represented by [SLac], which was corrected
the viral strain A/PR/8/34 recognizes Neu5Aca(2?3)Gal residues
in glycoconjugates, and the virus adheres to erythrocytes through
on the basis of a Neu5Aca(2?3)Galb(1?4)Glc unit. According to
protein—carbohydrate interactions. The hemagglutinin of A/Aichi/
Figure 3, both Dumbbell(1)6-SLac 4 and Dumbbell(1)6-amide-SLac
7 showed strong inhibitory activity. The results suggested that the
shape of the core dendrimer has a remarkable influence on the
inhibitory activities in the hemolysis inhibition assay. The half
maximal inhibitory concentration (IC50) values of the inhibitors
from Figure 3 were estimated and are shown in Table 2. Other gly-
codendrimers also have stronger inhibitory activity than Pent-SLac
1, and the order of the inhibitory effects of the glycolibrary in the
hemolysis inhibition assay was 4 [Dumbbell(1)6-SLac] = 7 [Dumb-
2/68 strain recognizes both Neu5Ac
Gal sequences, and the hemagglutinin of A/Memphis/1/71 strain
recognizes Neu5Ac (2?6)Gal saccharidic unit. Inhibitory activity
a(2?3)Gal and Neu5Aca(2?6)-
a
of the series of glycodendrimers on hemagglutination was clearly
observed when A/PR/8/34 and A/Aichi/2/68 were used as viruses.22
A/Memphis/1/71 was used as the viral strain for HAI assay using
the glycolibrary, and the inhibitory potencies of all of the glycoden-
drimers were extremely weak. The results of HAI assays using the
glycodendrimers are summarized in Table 1, where the inhibitor
concentrations were calculated on the basis of a sialyl lactose unit.
The results suggested that the hemagglutinin of each virus cer-
bell(1)6-amide-SLac] > 5
[Fan(0)3-amide-SLac] = 6
[Ball(0)4-
amide-SLac] > 3 [Ball(0)4-SLac] > 2 [Fan(0)3-SLac] > 1 [Pent-SLac].
Neutralization assays of glycodendrimers to the infection of
influenza virus (A/PR/8/34) using Madin-Darby canine kidney
(MDCK) cells were also performed and the results are shown in Fig-
ure 4. As was found in hemolysis inhibition assays, both Dumb-
bell(1)6-SLac 4 and Dumbbell(1)6-amide-SLac 7 showed strong
inhibitory activity compared to the activities of others. IC50 of the
glycolibrary in the neutralization assay was estimated from the re-
sults shown in Figure 4 and the values are summarized in Table 2.
All glycodendrimers having multivalent sialyl lactose moieties
showed higher levels of inhibitory activities against human influ-
enza virus than that of monomeric-type Pent-SLac 1. Especially,
Dumbbell(1)6-amide-SLac 7 showed 22-times stronger inhibitory
activity than that of 1. The order of the inhibitory effects of the gly-
colibrary in the infection inhibition assay was 7 [Dumbbell(1)6-
amide-SLac] > 4 [Dumbbell(1)6-SLac] > 6 [Ball(0)4-amide-SLac] > 3
tainly recognizes the Neu5Aca(2?3)Gal disaccharidic unit in den-
dritic structures and the results agree well with those in a previous
report.23 A substrate Dumbbell(1)6-amide-SLac 7 showed the
highest inhibitory potency in the HAI assay, and the effective con-
centration of 7 was 16-times lower than that of Pent-SLac 1 against
A/PR/8/34 and 63-times lower than that of 1 against A/Aichi/2/68.
Furthermore, inhibitory activity of glycodendrimers in HAI assays
was significantly related to the shape of core dendrimers and num-
bers of sugar epitopes.
In order to elucidate further the structure–activity relationships
(SARs) of the glycodendrimer against influenza virus, hemolysis
inhibition assay and infection inhibition assay were carried out
using human influenza virus (A/PR/8/34) strain. The strain A/PR/
8/34 was chosen for both hemolysis inhibition assay and infection

5470
H. Oka et al. / Bioorg. Med. Chem. 17 (2009) 5465–5475
1.4
1.2
1
0.8
0.6
0.4
0.2
0
7.8
15.6
31.25
62.5
125
250
500
1000
[SLac] (µM)
Figure 3. Inhibitory activities of glycodendrimers against hemolysis of human influenza virus (A/PR/8/34 (H1N1)) to erythrocytes [-d-: 1 (Pent-SLac),
: 3 (Ball(0)4-SLac), : 4 (Dumbbell(1)6-SLac), : 5 (Fan(0)3-amide-SLac), : 6 (Ball(0)4-amide-SLac), : 7 (Dumbbell(1)6-amide-SLac)]. Concentrations of
2?3Galb1?4Glc unit.
: 2 (Fan(0)3-SLac),
glycodendrimers are represented by [SLac], which was calculated on the basis of a Neu5Ac
a
Table 2
3. Conclusion
Inhibitory activities of a series of glycodendrimers against human influenza virus (A/
PR/8/34 (H1N1) strain)
We have systematically succeeded in the synthesis of a series of
carbosilane dendrimers uniformly functionalized with sialyl lac-
tose moieties (Neu5Aca2?3Galb1?4Glc). Biological evaluations
Compounds
Inhibition activities (
l
M)
Hemagglutinationa
Hemolysis
Infection
(IC50)
(IC50
)
of the glycolibrary against human influenza viruses were also car-
ried out. Inhibitory activities of the glycodendrimers were stronger
than that of monomeric sialyl lactoside. The strongest inhibitor
against human influenza virus in this study was Dumbbell(1)6-
amide-SLac 7. SAR studies of the glycolibrary also suggested that
dumbbell-shaped dendrimers as supporting carbohydrate epitopes
were found to be the most suitable core scaffolds. Furthermore, we
have reported a series of thiosialoside clusters as NA inhibitors25
and both HA inhibitors in this study and NA inhibitors are promis-
ing therapeutic agents for influenza disease.
1 [Pent-SLac]
2 [Fan(0)3-SLac]
3 [Ball(0)4-SLac]
4 [Dumbbell(1)6-SLac]
5 [Fan(0)3-amide-SLac]
6 [Ball(0)4-amide-SLac]
7 [Dummbell(1)6-amide-Slac]
125
64
64
32
16
16
8
500
250
125
32
62.5
62.5
32
125
64
32
7.8
25
16
5.6
Concentrations of glycodendrimers were calculated on the basis of a sialyl lactose
unit.
a
The data are also shown in Table 1.
4. Experimental
[Ball(0)4-SLac] > 5 [Fan(0)3-amide-SLac] > 2 [Fan(0)3-SLac] > 1
[Pent-SLac]. Furthermore, these glycodendrimers having a Neu5A-
4.1. Materials and methods
ca(2?3)Gal residue at each terminal end are promising agents for
prevention of infection with avian influenza virus (H5N1), since
Unless otherwise stated, all commercially available solvents
and reagents were used without further purification. Pyridine
avian influenza virus recognized Neu5Aca(2?3)Gal residues on
host cells.24
0.03
0.025
0.02
0.015
0.01
0.005
0
0.5
1
2
3.9
7.8
15.6
31.25
62.5
[SLac] (µM)
Figure 4. Inhibitory activities of glycodendrimers against infection of human influenza virus (A/PR/8/34 (H1N1)) to MDCK cells [-d-: 1 (Pent-SLac),
: 3 (Ball(0)4-SLac), : 4 (Dumbbell(1)6-SLac), : 5 (Fan(0)3-amide-SLac), : 6 (Ball(0)4-amide-SLac), : 7 (Dumbbell(1)6-amide-SLac)]. Concentrations of
glycodendrimers are represented by [SLac], which was calculated on the basis of a Neu5Ac 2?3Galb1?4Glc unit.
: 2 (Fan(0)3-SLac),
a

H. Oka et al. / Bioorg. Med. Chem. 17 (2009) 5465–5475
5471
(Pyr), N,N-dimethylformamide (DMF), and 1,4-dioxane were stored
over molecular sieves (4 Å MS), and methanol (MeOH) was stored
over 3 Å MS before use. Tetrahydrofuran (THF) was distilled in the
presence of sodium benzophenone ketyl just before use. Melting
points were measured with a Laboratory Devices MELTEMP II
apparatus and were uncorrected. The optical rotations were deter-
mined with a JASCO DIP-1000 digital polarimeter. IR spectra were
obtained using a JASCO FT/IR-300E spectrophotometer. The 1H
NMR spectra were recorded at 400 MHz with a Bruker AM-400
spectrometer or at 300 MHz with a Bruker AC-300P spectrometer
in chloroform-d, deuterium oxide or methyl-d3 alcohol-d. The 13C
NMR spectra were recorded at 100.6 MHz using the same instru-
ments. Tetramethylsilane (TMS), CHCl3 (7.26 ppm for 1H or
77.0 ppm for 13C), and MeOD (3.3 ppm for 1H or 49.0 ppm for
13C) were used as internal standards. Ring-proton assignments in
NMR were made by first-order analysis of the spectra and were
supported by the results of homonuclear decoupling experiments.
Elemental analyses were performed with a Fisons EA1108 on sam-
ples extensively dried at 50–60 °C over phosphorus pentoxide for
4–5 h. Fast atom bombardment mass (FAB-MS) spectra were re-
corded with a JEOL JMS-HX110 spectrometer. Matrix-assisted laser
desorption/ionization time-of-flight mass spectra (MALDI-TOF-
MS) were obtained using a Perseptive Biosystems Voyager Elite
spectrometer. Reactions were monitored by thin-layer chromatog-
raphy (TLC) on a precoated plate of Silica Gel 60F254 (layer thick-
ness, 0.25 mm; E. Merck, Darmstadt, Germany). For detection of
the intermediates, TLC sheets were dipped in (a) a solution of
85:10:5 (v/v/v) MeOH-p-anisaldehyde-concd H2SO4 and heated
for a few minutes (for carbohydrate), (b) an aq solution of 5 wt %
KMnO4 and heated similarly (for C@C double bond), or (c) an eth-
anolic solution of 7% phosphomolybdic acid and heated similarly
(for organic compound). Column chromatography was performed
803. Anal. Calcd for C33H48O19S1: C, 50.76; H, 6.20; N, 0.00. Found:
C, 50.67; H, 6.21; N, 0.00.
4.1.2. Fan(0)3-Lac-Ac (22)
To a suspension of 16 (50.0 mg, 0.107 mmol) in DMF (0.6 mL)
was added compound 13 (500 mg, 0.64 mmol), and the solution
was stirred until it became clear. Then, to the solution was added
MeOH (0.6 mL), and the solution was stirred at room temperature
for 2 h. NaOMe (38.0 mg) was added to the solution, and the solu-
tion was stirred overnight. After addition of AcOH (0.3 mL) to the
solution, the solution was concentrated. The residue was acety-
lated with Ac2O and pyridine and then concentrated. The residue
was extracted with CHCl3, and the organic layer was washed suc-
cessively with cold 1 M aq HCl, cold satd aq NaHCO3 and brine,
dried over anhyd MgSO4, filtered, and evaporated in vacuo. Purifi-
cation of the residue by silica gel column chromatography with 2:1
(v/v) hexane–EtOAc gave pure 22 (184 mg, 70.8%) and a disulfide-
linked lactoside dimer 23 (181 mg), respectively.
4.1.2.1. Data for Fan(0)3-Lac-Ac 22. Rf 0.50 [5:1 (v/v) EtOAc–
hexane]; ½a 2D1
ꢁ
ꢀ14.2 (c 1.40, CHCl3); IR (KBr) 2941 (
mC–H), 1753
(m
C@O), 1234 (
m
CO–C), 1057 (
m
C–OC) cmꢀ1 1H NMR d (CDCl3) 7.41
;
(m, 5H, SiPh), 5.35 (dd, 3H, J3 ,4 = 3.5 Hz, J4 ,5 = 0.9 Hz, H-40), 5.19
0
0
0
0
0
0
(t, 3H, J2,3 = J3,4 = 9.4 Hz, H-3), 5.11 (dd, 3H, J1 ,2 = 7.7 Hz,
J2 ,3 = 10.3 Hz, H-20), 4.95 (dd, 3H, H-30), 4.88 (dd, 3H, J1,2 = 8.1 Hz,
H-2), 4.49 (d, 3H, H-10), 4.47 (m, 3H, H-6b), 4.45 (d, 3H, H-1),
4.11 (m, 9H, H-6a, H-60a, H-60b), 3.83 (m, 9H, H-4, H-50, one of
OCH2–), 3.59 (ddd, 3H, J = 2.2 Hz, J = 5.2 Hz, J = 9.9 Hz, H-5), 3.44
(dt, 3H, Jgem = 9.6 Hz, Jvic = 6.6 Hz, one of OCH2–), 2.49 (t, 6H,
SCH2), 2.43 (t, 6H, SCH2), 2.15, 2.11, 2.06, 2.04, 2.04, 2.03, and
1.97 (each s, 63H, OAc), 1.54 (m, 18H, –CH2), 1.40 (m, 6H, –CH2),
0.90 (m, 6H, –CH2); 13C NMR d (CDCl3) 170.19, 170.16, 169.98,
169.87, 169.63, 169.41, 168.91, 136.13, 133.85, 128.99, 127.74,
100.89, 100.40, 76.14, 72.68, 72.44, 71.54, 70.83, 70.49, 69.74,
68.96, 66.48, 61.90, 60.67, 35.73, 31.83, 29.19, 28.88, 24.97,
23.89, 20.71, 20.65, 20.56, 20.47, 20.34, 11.73. FAB-MS Calcd for
C108H158O54S13Si [M+H]+: 2444.9. Found: m/z 2444.6. Anal. Calcd
for C108H158O54S3Si1: C, 53.06; H, 6.51; N, 0.00. Found: C, 52.94;
H, 6.54; N, 0.00.
0
0
on silica gel (Silica Gel 60; 63–200
chromatography was performed on silica gel (Silica Gel 60, spher-
ical neutral; 40–100 m, E. Merck). All extractions were concen-
trated below 45 °C under diminished pressure.
lm, E. Merck). Flush column
l
4.1.1.
x
-Acetylthiopentyl 4-O-(2,3,4,6-tetra-O-acetyl-b-
D-
galactopyranosyl)-2,3,6-tri-O-acetyl-b-
D
-glucopyranoside (13)
To a solution of 12 (5.00 g, 7.10 mmol) in 1,4-dioxane (25 mL)
was added thioacetic acid (7.45 mL, 106 mmol), and the solution
was heated to 50 °C. To the solution was added AIBN (583 mg,
3.55 mmol) and the solution was heated to 80 °C and stirred for
3 h. The solution was cooled to room temperature. Then, to the
solution was added cyclohexene (10.8 mL, 106 mmol) and the
solution was stirred for a few minutes. The solution was co-evap-
orated with toluene. Purification of the residue by silica gel column
chromatography with 8:7 (v/v) hexane–EtOAc gave 13 (5.52 g,
99.6%).
4.1.2.2. Data for dimer 23. Rf 0.53 [5:1 (v/v) EtOAc–hexane]; 1H
NMR d (CDCl3) 5.35 (dd, 2H, H-40), 5.19 (t, 2H, J2,3 = J3,4 = 9.4 Hz,
H-3), 5.10 (dd, 2H, J1 ,2 = 8.0 Hz, J2 ,3 = 10.2 Hz, H-20), 4.96 (dd,
0
0
0
0
2H, J3 ,4 = 3.5 Hz H-30), 4.88 (dd, 2H, J1,2 = 8.0 Hz, H-2), 4.49 (d,
2H, H-10), 4.49 (m, 2H, H-6b), 4.46 (d, 2H, H-1), 4.11 (m, 6H, H-
6a, H-60a, H-60b), 3.84 (m, 6H, H-4, H-50, one of OCH2–), 3.60
(ddd, 2H, H-5), 3.46 (dt, 2H, Jgem = 9.6 Hz, Jvic = 6.7 Hz, one of
OCH2–), 2.65 (t, 4H, J = 7.2 Hz SCH2), 2.06 (each s, 42H, OAc), 1.61
(m, 8H, OCH2CH2CH2CH2CH2S), 1.41 (m, 4H, OCH2CH2CH2–). FAB-
MS Calcd for C62H90O36S2 [M+H]+: 1475. Found: m/z 1475.
0
0
Rf 0.43 [2:1 (v/v) EtOAc–hexane], 0.69 [5:1 (v/v) EtOAc–hex-
ane]; ½a 2D1
ꢁ
ꢀ15.1 (c 1.77, CHCl3); IR (KBr) 2945 (
m
C–H), 1751 (
mC@O),
1690 (
m
C@O, SAc), 1238 (
m
C–O–C), 1057 (
m
C–O–C) cmꢀ1
;
1H NMR d
4.1.3. Dumbbell(1)6-Lac-Ac (24)
(CDCl3) 5.35 (dd, 1H, J3 ,4 = 3.2 Hz, J4 ,5 = 1.1 Hz, H-40), 5.19 (t, 1H,
Compound 18 (49.7 mg, 0.053 mmol) in DMF (0.6 mL) was cou-
pled with 1316 (500 mg, 0.64 mmol) by the method described for
the preparation of 22. Purification of the reaction mixture by silica
gel column chromatography with 1:5 (v/v) hexane–EtOAc gave
pure 24 (159 mg, 61.5%) and a disulfide-linked lactoside dimer
23, respectively.
0
0
0
0
0
0
0
0
J2,3 = J3,4 = 9.1 Hz, H-3), 5.11 (dd, 1H, J1 ,2 = 7.8 Hz, J2 ,3 = 10.4 Hz,
H-20), 4.95 (dd, 1H, H-30), 4.88 (dd, 1H, J1,2 = 7.8 Hz, H-2), 4.48 (d,
1H, H-10), 4.48 (m, 1H, H-6b), 4.45 (d, 1H, H-1), 4.16–4.06 (m,
3H, H-6a, H-60a, H-60b), 3.89–3.77 (m, 3H, H-4, H-50, one of
OCH2–), 3.59 (ddd, 1H, J = 2.2 Hz, J = 5.2 Hz, J = 9.9 Hz, H-5), 3.48
(dt, 1H, Jgem = 9.6 Hz, Jvic = 6.4 Hz, one of OCH2–), 2.32 (s, 3H, SAc),
2.15, 2.12, 2.06, 2.04, and 1.97 (each s, 21H, OAc), 1.56 (m, 4H,
OCH2CH2CH2CH2CH2SAc), 1.64 (m, 2H, –CH2CH2CH2SAc); 13C
NMR d (CDCl3) 195.34(SC@O), 170.12, 170.07, 169.90, 169.78,
169.52, 169.39, 168.83, 100.80, 100.29, 76.05, 72.57, 72.39, 71.41,
70.75, 70.44, 68.90, 67.95, 66.45, 61.81, 60.64, 30.36, 29.12,
25.35, 25.10, 20.61, 20.55, 20.45, 20.38, 20.26. FAB-MS Calcd for
C33H48O19S1 [M+H]+: 781, [M+Na]+: 803. Found: m/z: 781, m/z:
vRf 0.17 [5:1 (v/v) EtOAc–hexane], 0.39 [10:1 (v/v) CHCl3–
MeOH]; ½a 2D1
ꢁ
ꢀ15.3 (c 0.80, CHCl3); IR (KBr) 2941 (
mC–H), 1753 (mC@O),
1234 (mC–O–C), 1053 (m ;
C–O–C) cmꢀ1 1H NMR d (CDCl3, 7.26 (CHCl3))
5.33 (d, 6H, J = 2.9 Hz, H-40), 5.18 (t, 6H, J2,3 = J3,4 = 9.4 Hz, H-3),
5.09 (dd, 6H, J1 ,2 = 7.9 Hz, J02 = 10.5 Hz, H-20), 4.94 (dd, 6H,
0
0
0
,3
00
J3 = 3.3 Hz, H-30), 4.86 (dd, 6H, J1,2 = 8.1 Hz, H-2), 4.48 (d, 6H, H-
;4
10), 4.46 (m, 6H, H-6b), 4.44 (d, 6H, H-1), 4.09 (m, 18H, H-6a, H-
60a, H-60b), 3.82 (m, 18H, H-4, H-50, one of OCH2–), 3.58 (ddd, 6H,

5472
H. Oka et al. / Bioorg. Med. Chem. 17 (2009) 5465–5475
J = 1.9 Hz, J = 4.9 Hz, J = 9.8 Hz, H-5), 3.44 (dt, 6H, Jgem = 9.5 Hz,
Jvic = 6.7 Hz, one of OCH2–), 2.48 (t, 12H, SCH2), 2.46 (t, 12H,
SCH2), 2.14–1.95 (each s, 126H, OAc), 1.58–1.39 (m, 52H,
OCH2CH2CH2CH2CH2SCH2CH2–, SiCH2CH2CH2Si), 0.55 (m, 20H,
–CH2SiCH2CH2CH2SiMe2), ꢀ0.08 (s, 6H, Si(CH3)2); 13C NMR d (CDCl3)
170.20, 170.19, 169.99, 169.89, 169.64, 169.43, 168.93, 100.92,
100.44, 77.20, 76.18, 72.73, 72.48, 71.59, 70.86, 70.52, 69.77,
69.00, 66.51, 61.93, 60.67, 35.98, 31.98, 29.52, 29.27, 28.96,
25.03, 24.19, 20.74, 20.67, 20.59, 20.49, 20.36, 20.28, 18.22,
16.98, 11.99, ꢀ3.32. FAB-MS Calcd for C212H324O108S6Si3 [M+H]+:
4877.8. Found: m/z 4877.6. Anal. Calcd for C212H324O108S6Si3: C,
52.21; H, 6.70; N, 0.00. Found: C, 51.99; H, 6.65; N, 0.00.
C) cmꢀ1 1H NMR d (CDCl3) 5.36 (ddd, 1H, H-8), 5.33 (dd, 1H, H-
;
7), 5.21 (d, 1H, JNH,5 = 10.0 Hz, NH), 4.86 (ddd, 1H, J3eq,4 = 4.7 Hz, J3-
ax,4 = J4,5 = 10.7 Hz, H-4), 4.32 (dd, 1H, J8,9a = 2.2 Hz, H-9a), 4.12 (dd,
1H, J9a,9b = 12.4 Hz, J8,9b = 4.7 Hz, H-9b), 4.05 (m, 1H, H-5), 3.83 (dd,
1H, H-6), 3.80 (s, 3H, COOCH3), 2.53 (m, 2H, SCH2), 2.16, 2.14, 2.04,
2.02, and 1.88 (each s, 15H, NHAc, 4OAc), 1.49 (m, 2H, SCH2CH2),
1.25 (m, 18H, 9CH2), 0.88 (t, 3H, CH2CH3). Anal. Calcd for
C32H53N1O12S: C, 56.87; H, 7.90; N, 2.07. Found: C, 56.72; H,
7.95; N, 2.01.
4.1.6.2. Data for b-laurylate 29. Rf 0.60 [5:1 (v/v) CHCl3–MeOH];
½
a 2D8
ꢁ
ꢀ67.0 (c 0.91, CHCl3); 1H NMR d (CDCl3) 5.44 (m, 1H, H-7),
5.37 (d, 1H, JNH,5 = 10.2 Hz, NH), 5.26 (ddd, 1H, J3eq,4 = 4.8 Hz,
J3ax,4 = J4,5 = 10.7 Hz, H-4), 5.11 (ddd, 1H, J7,8 = 2.7 Hz, H-8), 4.81
(dd, 1H, J8,9a = 2.1 Hz, H-9a), 4.33 (dd, 1H, J6,7 = 2.2 Hz, H-6), 4.17
(dd, 1H, J9a,9b = 12.3 Hz, J8,9b = 8.0 Hz, H-9b), 4.08 (ddd, 1H,
J4,5 = J5,6 = 10.2 Hz, H-5), 3.80 (s, 3H, COOCH3), 2.52 (m, 3H, SCH2,
H-3eq), 2.13, 2.07, 2.03, 2.02, and 1.88 (each s, 15H, NHAc, 4
OAc), 1.51 (m, 2H, SCH2CH2), 1.25 (m, 18H, 9CH2), 0.88 (t, 3H,
CH2CH3). Anal. Calcd for C32H53N1O12S: C, 56.87; H, 7.90; N, 2.07.
Found: C, 56.68; H, 7.97; N, 2.00.
4.1.4. Fan(0)3-Lac (25)
To a solution of dendrimer 22 (32.8 mg, 0.0134 mmol) in MeOH
(2 mL) was added NaOMe, and the mixture was stirred overnight at
room temperature. IR-120B (H+) resin was added to the mixture to
remove Na+, and the suspension was filtered, and the filtrate was
concentrated. 0.1 M aq NaOH (3 mL) was added to the residue
and the solution was stirred overnight at room temperature.
IR-120B (H+) resin was added to the solution, and the mixture
was filtered and concentrated. Gel filtration of the residue using
Sephadex G-25 with 5% aq AcOH gave 25 quantitatively.
4.1.7. 2-(Trimethylsilyl)ethyl [methyl (5-acetamido-4,7,8,9-
½
a 1D9
ꢁ
ꢀ4.98 (c 0.16, H2O); IR (KBr) 3400 (
m
O–H), 2922 (
m
C–H), 1066
tetra-O-acetyl-3,5-dideoxy-
anosyl)onate]-(2?3)-O-(2,6-di-O-benzyl-b-
(1?4)-2,3,6-tri-O-benzyl-b- -glucopyranoside (31)
D
-glycero-
a
-D
-galacto-2-nonulopyr-
C–O); 1H NMR d (D2O) 7.30 (m, 5H, SiPh), 4.39 (d, 3H, J1 = 7.0 Hz,
D-galactopyranosyl)-
00
(m
;2
H-10), 4.33 (d, 3H, J1,2 = 7.0 Hz, H-1), 3.85–3.47 (m), 3.27 (br, 3H, H-
2), 2.39 (br, 12H, –CH2SCH2–), 1.46 (br), 0.80 (br, 6H, CH2Si); 13C
NMR d (D2O, 49.00 (MeOD)) 136.70, 134.17, 129.45, 128.17,
103.16, 102.54, 78.65, 75.48, 74.83, 74.71, 72.97, 72.77, 71.11,
70.37, 68.69, 61.17, 60.47, 35.72, 31.89, 29.56, 29.10, 25.17,
24.14, 11.81. FAB-MS Calcd for C66H116O33S3Si [M+H]+: 1561.6.
Found: m/z 1561.9. Anal. Calcd for C66H116O33S3Si1ꢂ4H2O: C,
48.51; H, 7.65; N, 0.00. Found: C, 48.52; H, 7.34; N, 0.00.
D
To a solution of lauryl thio glycoside 28 (4.20 g, 8.1 mmol) and
lactosyl diol 30 (0.432 mL, 4.27 mmol) in acetonitrile (82 mL) was
added 3 Å MS powder (8.2 g) and the mixture was stirred at room
temperature for a few hours. After the suspension had been cooled
to ꢀ35 °C, N-iodosuccinimide (4.00 g, 17.8 mmol) and trifluoro-
methanesulfonic acid (159 lL, 1.8 mmol) were added and the stir-
ring was continued at ꢀ35 °C for 4 h. The suspension was filtered
through a pad of Celite, and the filtrate was diluted with CHCl3
and washed successively with cold satd aq NaHCO3, 1 M aq sodium
thiosulfate and brine, dried over anhyd MgSO4, filtered, and evap-
orated in vacuo. Chromatographic purification of the residue by sil-
ica gel with 3:2 (v/v) toluene–EtOAc as the eluent gave known 31
(3.34 g, 54.6%). Analytical data for this compound agree with those
in previous report.20
4.1.5. Dumbbell(1)6-Lac (26)
An acetate 24 (159 mg, 0.0326 mmol) was deprotected in a
manner similar to that described for 25 to give 26 quantitatively.
½
a 2D0
ꢁ
ꢀ4.44 (c 0.76, H2O); IR (KBr) 3400 (
mO–H), 2918 (
m
C–H), 1068
C–O); 1H NMR d (D2O) 4.39 (near t, 12H, J1,2 = J0 = 9.6 Hz, H-1, H-
0
(m
,2
1
10), 3.84–3.46 (m), 3.26 (br, 6H, H-2), 2.49 (br, 24H, –CH2SCH2–),
1.49 (br), 0.63 (br), ꢀ0.12 (br, 6H, SiMe2); 13C NMR d (D2O)
103.00, 102.40, 78.51, 75.32, 74.69, 74.54, 72.81, 72.61, 70.94,
70.19, 68.54, 61.01, 60.32, 35.78, 31.84, 29.47, 28.98, 25.07,
24.25, 20.60, 20.24, 18.58, 17.50, 11.89, ꢀ2.40. MALDI-TOF-MS
Calcd for C128H240O66S6Si3 [M+Na]+: 3134.30. Found: m/z
3138.09. Anal. Calcd for C128H240O66S6Si3ꢂ6H2O: C, 47.74; H, 7.89;
N, 0.00. Found: C, 47.78; H, 7.77; N, 0.00.
4.1.8. [Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
D
-glycero-
6-tri-O-acetyl-b-
-glucopyranosyl trichloroacetoimidate (32)
a
-
D
-galacto-2-nonulopyranosyl)onate]-(2?3)-O-(2,4,
D-galactopyranosyl)-(1?4)-2,3,6-tri-O-acetyl-a-
D
Trichloroacetoimidate 32 was efficiently synthesized from 31
by the method previously reported by Tietze et al.20
4.1.6. Methyl (lauryl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
4.1.9. Pentenyl [Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-
dideoxy-2-thio-
D
-glycero-
D
-galacto-2-nonulopyranosid)onate
3,5-dideoxy-
(2?3)-O-(2,4,6-tri-O-acetyl-b-
tri-O-acetyl-b- -glucopyranoside (33)
To a solution of an imidate 32 (1.03 g, 0.853 mmol) and 4-pen-
ten-1-ol (0.432 mL, 4.27 mmol) in dichloromethane (24.0 mL) was
added 4 Å MS powder (2.0 g), and the mixture was stirred at room
temperature for a few minutes. After the mixture had been cooled
D
-glycero-
a
-
D
-galacto-2-nonulopyranosyl)onate]-
(2826 and 29)
D
-galactopyranosyl)-(1?4)-2,3,6-
To a solution of known acetate 27 (5.6 g, 10.5 mmol) in dichloro-
methane (60 mL) was added 1-dodecanethiol (10 mL, 42.0 mmol),
and the solution was cooled to 0 °C. Then, to the solution was added
D
BF3–OEt2 (215 lL, 1.71 mmol), and the stirring was continued at
0 °C for 30 min and at room temperature for 3 h. The solution was
diluted with CHCl3 and washed successively with cold water, cold
satd aq NaHCO3 and brine, dried over anhyd MgSO4, filtered, and
evaporated in vacuo. Complete separation of an anomeric mixture
of the residue was performed by silica gel column chromatography
to ꢀ25 °C, BF3–OEt2 (215
lL, 1.71 mmol) was added to the mixture
with stirring, and the stirring was continued at ꢀ25 °C for 20 min
and at ꢀ5 °C for 4 h. The mixture was filtered through a pad of Cel-
ite, and the filtrate was diluted with CHCl3 and washed succes-
sively with satd aq NaHCO3 and brine, dried over anhyd MgSO4,
filtered, and evaporated in vacuo. The residual syrup was applied
to a column of silica gel with 20:1 (v/v) CHCl3–MeOH as the eluent
to yield 33 (816 mg, 84.3%) as a foam.
with 1:2 (v/v) toluene–EtOAc to afford pure
26.2%) and b-laurylate 29 (4.3 g, 59.2%) as foam, respectively.
a
-laurylate 2826 (1.9 g,
4.1.6.1. Data for
+26.0 (c 1.09, CHCl3) [lit.26
1751 ( C@O), 1658 ( C@O), 1550 (
a
-laurylate 28. Rf 0.58 [5:1 (v/v) CHCl3–MeOH];
+21.4 (c 0.69, CHCl3)]; IR (KBr)
N–H), 1222 ( C–O–C), 1037 (mC–O–
½
a 2D8
ꢁ
½
a 2D5
m
ꢁ
Rf 0.50 [10:1 (v/v) CHCl3–MeOH]; ½a D18
ꢁ
ꢀ6.87 (c 2.02, CHCl3); IR
m
m
m
(KBr) 2949 (
m
C–H), 1745 (
m
C@O), 1674 (
m
C@O, SAc, NAc), 1554 ( N–H),
m

H. Oka et al. / Bioorg. Med. Chem. 17 (2009) 5465–5475
5473
1230 (mC–O–C), 1045 (m ;
C–O–C) cmꢀ1 1H NMR d (CDCl3) 5.78 (m,
NMR d (CDCl3) 5.49 (m, 1H, H-800), 5.39 (dd, 1H, J6 ,7 = 2.8 Hz,
00 00
00 00
1H, –CH@CH2), 5.54 (m, 1H, H-800), 5.39 (dd, 1H, J6 ,7 = 2.7 Hz,
J7 ,8 = 9.4 Hz, H-700), 5.18 (t, 1H, J2,3 = J3,4 = 9.4 Hz, H-3), 5.12 (d,
00 00
J7 ,8 = 9.3 Hz, H-700), 5.18 (t, 1H, J2,3 = J3,4 = 9.3 Hz, H-3), 5.08 (d, 1H,
1H, JNH,5 = 9.9 Hz, NH), 4.93 (dd, 1H, J1 ,2 = 8.1 Hz, J2 ,3 = 10.3 Hz,
00 00
00
0
0
0
0
JNH,5 = 10.2 Hz, NH), 5.02–4.85 (m, H-2, H-20, H-40, –CH@CH2), 4.67
H-20), 4.88 (m, H-2, H-400, –CH), 4.67 (d, 1H, H-10), 4.52 (dd, 1H,
00
(d, 1H, J1 ,2 = 10.0 Hz, H-10), 4.52 (dd, 1H, J2 ,3 = 10.2 Hz, J3 ,4 = 3.3 Hz,
H-30), 4.45 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.43 (m, 2H, H-6b, H-900), 4.18
(dd, 1H, J5,6a = 5.4 Hz, J6a,6b = 11.9 Hz, H-6a), 4.02 (m), 3.87 (m, 3H,
H-4, one of OCH2–, –CH), 3.84 (s, 3H, COOCH3), 3.63 (dd, 1H,
J3 ,4 = 3.3 Hz, H-30), 4.45 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.43 (m, H-6b,
H-900, –CH), 4.18 (dd, 1H, J6a,6b = 11.9 Hz, J5,6a = 5.4 Hz, H-6a),
0
0
0
0
0
0
0
0
00 00
4.09–3.82 (2 m), 3.84 (s, 3H, COOCH3), 3.63 (dd, 1H, J5 ,6 = 10.7 Hz,
J6 ,7 = 2.6 Hz, H-600), 3.59 (m, 1H, H-5), 3.46 (dt, 1H, Jgem = 9.7 Hz,
00 00
J5 ,6 = 10.8 Hz, J6 ,7 = 2.7 Hz, H-600), 3.59 (m, 1H, H-5), 3.49 (dt, 1H,
Jgem = 9.6 Hz, Jvic = 6.7 Hz, one of OCH2–), 2.58 (dd, 1H,
Jvic = 6.6 Hz, one of OCH2–), 2.85 (t, 2 H, J = 7.2 Hz, SCH2–), 2.58
00 00
00 00
(dd, 1 H, J3 a,3 e = 12.7 Hz, J3 e,4 = 4.6 Hz, H-300eq), 2.32 (s, 3H,
SAc), 2.24 (s, 3H, NHAc), 2.16, 2.094, 2.086, 2.086, 2.077, 2.061,
2.040, 2.040, 2.008, and 1.855 (each s, 30H, 10 OAc), 1.61 (m, 5
H, H-300ax, OCH2CH2CH2CH2CH2S), 1.39 (m, 2H, –CH2CH2CH2SAc).
FAB-MS Calcd for C51H73N1O30S1 [M+H]+: 1212, [M+Na]+: 1234.
Found: m/z: 1212, m/z: 1234. Anal. Calcd for C51H73N1O30S1: C,
50.53; H, 6.07; N, 1.16. Found: C, 50.03; H, 6.03; N, 1.14.
00
00
00
00
J3 a,3 e = 12.6 Hz, J3 e,4 = 4.5 Hz, H-300eq), 2.25, 2.16, 2.09, 2.08, 2.06,
2.04, 2.03, 2.01, and 1.86 (each s, 33H, NHAc, OAc), 2.08 (m,
OCH2CH2CH2CH@CH2), 1.66 (m, OCH2CH2–, H-300ax); 13C NMR d
(CDCl3) 170.66, 170.51, 170.48, 170.45, 170.35, 170.25, 170.17,
170.05, 169.65, 169.54, 169.48, 169.44, 167.81, 137.68, 114.91,
100.82, 100.40, 96.63, 76.19, 73.27, 72.45, 71.86, 71.70, 71.20,
70.30, 69.77, 69.20, 69.13, 67.70, 67.14, 66.78, 62.20, 62.12, 61.37,
52.98, 48.88, 37.25, 29.67, 28.44, 22.99, 21.36, 20.78, 20.67, 20.62,
20.56, 20.52, 20.45. Anal. Calcd for C49H69N1O29: C, 51.80; H, 6.12;
N, 1.23. Found: C, 51.50; H, 6.14; N, 1.19.
00
00
00
00
4.1.12. Fan(0)3-SLac-Ac (35)
To a solution of 16 (6.48 mg, 0.014 mmol) in DMF was added
thioacetate 34 (100 mg, 0.082 mmol), and the mixture was stirred
until it became clear. MeOH (0.2 mL) was added to the solution,
and the solution was stirred at room temperature for 1 h. To the
solution was added NaOMe (4.90 mg), and the mixture was stirred
overnight. After addition of AcOH (0.1 mL) to the mixture, the mix-
ture was concentrated. The residue was treated with Ac2O (2 mL)
and pyridine (2 mL) and then concentrated. The residue was di-
luted with CHCl3, and the organic solution was washed succes-
sively with 1 M aq HCl and brine, dried over anhyd MgSO4,
filtered, and evaporated in vacuo. The residual syrup was dissolved
in MeOH–Et2O, and diazomethane in Et2O was added dropwise to
the solution at room temperature. After addition of AcOH to the
solution, the solution was concentrated. A combination of chro-
matographic purification of the residue by silica gel with 30:1 (v/
v) CHCl3–MeOH as the eluent and by gel permeation chromatogra-
phy using Sephadex LH-20 with MeOH as the eluent gave 35
(41.2 mg, 80.2%) including 100,20-lactone form.
4.1.10. Pentenyl (5-acetamido-3,5-dideoxy-
2-nonulopyranosylonic acid)-(2?3)-O-b- -galactopyranosyl-(1?
4)-O-b- -glucopyranoside (1)
An acetate 33 (50.0 mg, 0.044 mmol) was dissolved in MeOH
(1.0 mL), and 1 M NaOMe in MeOH (44 L) was added dropwise
D-glycero-a-D-galacto-
D
D
l
to the solution. The reaction mixture was stirred overnight at room
temperature. IR-120B (H+) resin was added to the solution, and the
mixture was filtered and evaporated. 0.05 M aq NaOH (2 mL) was
added to the residue, and the solution was stirred at room temper-
ature for 2 h. IR-120B (H+) resin was added to the solution, and the
mixture was filtered and concentrated to afford 1 (26.0 mg, 84.4%).
½
a 1D8
ꢁ
ꢀ3.45 (c 0.96, H2O); IR (KBr) 3394 (
m
O–H), 2937 (
mC–H), 1732
(m
C@O), 1639 ( C@O), 1560 ( N–H), 1036 ( C–O–C), 619 (m
m
m
m
N–H) cmꢀ1; 1H
NMR d (D2O) 5.78 (m, 1H, –CH@CH2), 4.96 (dd, 1H, Jgem = 1.5 Hz,
Jtrans = 15.4 Hz, one of –CH@CH2), 4.96 (d, 1H, Jcis = 10.3 Hz, one of
Rf 0.3 [10:1 (v/v) CHCl3–MeOH]; IR (KBr) 2941 (
C@O), 1687 ( C@O), 1543 ( N–H), 1435 ( C–N), 1230 ( C–O–C), 1039
C–O–C) cmꢀ1 1H NMR d (CDCl3) 7.40 (m, 5H, Ph), 5.53 (m, 3H,
mC–H), 1749
–CH@CH2), 4.40 (d, 1H, J1 ,2 = 7.7 Hz, H-10), 4.35 (d, 1H, J1,2
=
0
0
(
(
m
m
m
;
m
m
m
8.1 Hz, H-1), 4.03 (dd, 1H, J2 ,3 = 9.7 Hz, J3 ,4 = 2.8 Hz, H-30), 3.88–
0
0
0
0
00
00
3.42 (m), 3.18 (t, 1H, J2,3 = 7.9 Hz, H-2), 2.63 (dd, 1H, J3 a,3 e = 12.5 Hz,
H-800), 5.39 (dd, 3H, J6 ,7 = 2.7 Hz, J7 ,8 = 9.1 Hz, H-700), 4.52 (dd,
00 00
00 00
J3 e,4 = 4.0 Hz, H-300eq), 2.02 (q, 2H, J = 7.1 Hz, –CH2CH@CH2), 1.91
00
00
1H, J2 ,3 = 10.2 Hz, J3 ,4 = 3.2 Hz, H-30), 3.84 (s, COOCH3), 2.58 (dd,
0
0
0
0
(s, 3H, NDAc), 1.77 (t, 1H, J3 a,4 = 12.1 Hz, H-300ax), 1.60 (m, 2H,
OCH2CH2CH2). Anal. Calcd for C28H47N1O19ꢂ1.5H2O: C, 46.15; H,
6.92; N, 1.92. Found: C, 46.29; H, 6.77; N, 1.82.
00
00
J3 a,3 e = 12.6 Hz, J3 e,4 = 4.6 Hz, H-300eq), 2.49 (t, Jvic = 7.0 Hz,
SCH2–), 2.43 (t, Jvic = 7.0 Hz, SCH2–), 2.25–1.85 (each s, NHAc,
OAc), 1.55 (br, OCH2CH2CH2CH2CH2S), 1.39 (br, 6H, –CH2CH2CH2S),
0.92 (br, 6H, SiCH2). FAB-MS Calcd for C162H233N3O87S3Si [M+Na]+:
3761.28. Found: m/z 3760.84.
00
00
00
00
4.1.11.
O-acetyl-3,5-dideoxy-
onate]-(2?3)-O-(2,4,6-tri-O-acetyl-b-
2,3,6-tri-O-acetyl-b- -glucopyranoside (34)
To a solution of 33 (100 mg, 0.088 mmol) in 1,4-dioxane
(209 L) was added thioacetic acid (209 L, 3.0 mmol), and the
x
-Acetylthio-pentyl [methyl (5-acetamido-4,7,8,9-tetra-
-glycero- -galacto-2-nonulopyranosyl)-
-galactopyranosyl)-(1?4)-
D
a-D
D
4.1.13. Ball(0)4-SLac-Ac (36)
D
Coupling reaction between ball-type dendrimer 17 (47 mg,
0.06 mmol) and thioacetate 34 (430 mg, 0.35 mmol) was carried
out by the method described for 35 to give Ball(0)4-Slac-Ac 36
(50 mg, 33.1%).
l
l
solution was heated to 50 °C. After addition of AIBN (24.6 mg,
0.15 mmol) to the solution, the temperature of the solution was
raised to 80 °C, and the heated solution was stirred for 3 h at the
same temperature. After cooling the solution to room temperature,
1H NMR d (CDCl3) 4.68 (d, 4 H, J1 ,2 = 8.0 Hz, H-10), 4.49 (d, 4H,
J1,2 = 8.0 Hz, H-1), 3.84 (s, COOCH3), 2.59–2.44 (m, 20 H, H-300eq,
–CH2SCH2–), 0.56 (br, 8H, SiCH2–).
0
0
cyclohexene (608 lL) was added to the solution, and the reaction
mixture was further stirred for a few minutes. The solution was
evaporated and co-evaporated with toluene. The residue was di-
luted with CHCl3, and the CHCl3 layer was washed successively
with satd aq NaHCO3 and brine, dried over anhyd MgSO4, filtered,
and evaporated in vacuo. Chromatographic purification of the res-
idue by silica gel with 1:0–30:1 (v/v) CHCl3–MeOH as the eluent
afforded 34 (105 mg, 99.1%).
4.1.14. Dumbbell(1)6-SLac-Ac (37)
Coupling reaction between dumbbell-type dendrimer 18
(47 mg, 0.06 mmol) and thioacetate 34 (430 mg, 0.35 mmol) was
carried out by the same method as that described for 35 to give
Dumbbell(1)6-Slac-Ac 37 (79 mg, 76.7%) including 100,20-lactone
form. In addition, dimer 38 (54 mg) and starting material 34
(38.4 mg) were also obtained.
Rf 0.51 [10:1 (v/v) CHCl3–MeOH]; ½a D29
ꢁ
ꢀ6.28 (c 1.29, CHCl3); IR
C@O, Sac, NAc), 1544 ( N–H),
C–O–C), 631–603 (
N–H) cmꢀ1; 1H
Rf 0.3 [10:1 (v/v) CHCl3–MeOH]; IR (KBr) 2941 (mC–H), 1749 (mC@O),
(KBr) 2959 (
m
C–H), 1756 (
m
C@O), 1689 (
m
m
****
1670 (
m
C@O), 1541 (
m
N–H), 1437 (
m
C–N), 1234 (
m
C–C), 1041 (mC–O–C
)
1
cmꢀ1; H NMR d (CDCl3) 5.50 (m, H-800), 5.36 (dd, 3H, J6 ,7 = 2.7 Hz,
00 00
1436 (
m
C–N), 1250 (
m
C–O–C), 1040 (
m
m

5474
H. Oka et al. / Bioorg. Med. Chem. 17 (2009) 5465–5475
J7 ,8 = 9.1 Hz, H-700), 5.19–4.59 (m), 4.49 (dd, 1H, J2 ,3 = 10.4 Hz,
4.1.20. Dumbbell(1)6-SLac (4)
00 00
0
0
J3 ,4 = 3.0 Hz, H-30), 4.43–3.70 (m), 3.81 (s, COOCH3), 3.62 (m, 12H,
H-5, H-600), 3.43 (m, 6H, one of OCH2–), 2.55 (dd, H-300eq), 2.46 (t,
Jvic = 7.0 Hz, SCH2–), 2.45 (t, Jvic = 7.0 Hz, SCH2–), 2.43–1.83 (m, NHAc,
OAc, –CH), 1.53 (br), 1.38 (br), 1.23 (br, SiCH2CH2CH2Si), 0.56 (br,
20H, SiCH2–), ꢀ0.09 (s, 6H, SiMe2). FAB-MS Calcd for
C320H474N6O174S6Si3 [M+Na]+: 7488.61. Found: m/z 7488.30.
Removal of all protection of Dumbbell-type dendrimer 37
(45.4 mg, 0.0365 mmol) was carried out by the same method as
that described for 2 to give water-soluble 4 in quantitative yield.
0
0
IR (KBr) 3406 (
m
O–H), 2931 (
mC–H), 1730 (mC@O), 1643 (mC@O), 1566
(m
N–H), 1032 ( C–°C), 624 (m
m
N–H), cmꢀ1; 1H NMR d (D2O) 4.46 (near d,
6H, H-10), 4.36 (near d, 6H, H-1), 4.08 (near d, 6H, H-30), 3.99–3.46
(m), 3.26 (br, 6H, H-2), 2.67 (near d, 3H, H-300eq), 2.50 (br, 24H,
–CH2SCH2–), 1.96 (s, 18H, NDAc), 1.85, 1.57, and 1.41 (3 br), 0.63
(br, 20H, Si CH2), ꢀ0.06 (br, 6H, SiMe2).
4.1.15. Fan(0)3-amide-SLac-OAc (39)
Coupling reaction between Fan(0)3-amide-Br dendrimer 19
(47 mg, 0.06 mmol) and thioacetate 34 (430 mg, 0.35 mmol) was
carried out by the same method as that described for 35 to give
39 (155 mg, 65.7%).
d 2.23 (near dd, J3 a,3 e = 12.9 Hz, J3 e,4 = 4.8 Hz, H-300eq of lac-
00
00
00
00
*
tone) included about 3%.
FAB-MS Calcd for C194H342N6O114S6Si3 [M+H]ꢀ: 4857.9. Found:
1H NMR d (CDCl3) 7.39 (m, 5H, Ph), 4.67 (d, 3H, J1 ,2 = 8.0 Hz, H-
m/z 4859.2.
0
0
10), 4.52 (dd, 3H, J2 ,3 = 10.2 Hz, J3 ,4 = 3.2 Hz, H-30), 3.84 (s,
COOCH3), 3.59 (m, 3H, one of OCH2–), 3.21 (m, 6H, NCH2–), 2.58
0
0
0
0
4.1.21. Fan(0)3-amide-SLac (5)
(dd, 3H, J3 a,3 e = 12.3 Hz, J3 e,4 = 4.3 Hz, H-300eq), 2.47 (m, 12H,
SCH2ꢀ), 0.79 (m, 6H, SiCH2–).
00
00
00
00
Removal of all protection of Fan-amide-type dendrimer 39
(106 mg, 0.026 mmol) was carried out by the same method as that
described for 2 to give water-soluble 5 in quantitative yield.
1H NMR d (D2O) 7.26 (m, 5H, Ph), 4.40 (d, 3H, J1 ,2 = 7.5 Hz, H-10),
4.29 (d, 3H, J1,2 = 7.5 Hz, H-1), 2.61 (br, 3H, H-300eq), 2.34 (m, 12H,
–CH2SCH2–), 0.65 (br, 6H, CH2Si). MALDI-TOF-MS Calcd for
C117H200N6O60S3SiNa [M+Na]+: 2798.1. Found: m/z 2799.3.
4.1.16. Ball(0)4-amide-SLac-OAc (40)
0
0
Coupling reaction between Ball(0)4-amide-Br dendrimer 20
(33 mg, 0.06 mmol) and thioacetate 34 (350 mg, 0.29 mmol) was
carried out by the same method as that described for 35 to give
40 (28 mg, 15.5%).
1H NMR d (CDCl3) 4.67 (d, 4 H, J1 ,2 = 8.0 Hz, H-10), 4.44 (d, 4H,
J1,2 = 8.0 Hz, H-1), 3.84 (s, COOCH3), 3.18 (m, 8H, NCH2–), 2.48 (q,
16H, J = 6.8 Hz, SCH2–), 0.51 (m, 8H, SiCH2–).
0
0
4.1.22. Ball(0)4-amide-SLac (6)
Removal of all protection of Ball-amide-type dendrimer 40
(22 mg, 0.004 mmol) was carried out by the same method as that
described for 2 to give water-soluble 6 (9.0 mg, 64%).
1H NMR d (D2O) 4.50 (d, 4H, J1 ,2 = 7.9 Hz, H-10), 4.43 (d, 4H,
4.1.17. Dumbbell(1)6-amide-SLac-OAc (41)
Coupling reaction between Dumbbell(1)6-amide-Br dendrimer
21 (48 mg, 0.03 mmol) and thioacetate 34 (400 mg, 0.33 mmol)
was carried out by the same method as that described for 35 to
give 41 (85 mg, 34.8%).
0
0
00
00
J1,2 = 7.9 Hz, H-1), 3.27 (br, 8H, NCH2), 2.72 (dd, 4H, J3 a,3 e = 12.5 Hz,
J3 e,4 = 4.2 Hz, H-300eq), 2.55 (m, 16H, SCH2), 2.22 (m, 8H, CH2), 2.00
00
00
(s, 12H, NDAc), 1.83 (t, 4H, J3 a,3 e = J3 a,4 = 12.0 Hz, H-300ax), 0.53
(br, 8H, CH2Si). MALDI-TOF-MS Calcd for C148H260N8O80S4SiNa
[M+Na]+: 3611.04. Found: m/z 3610.44.
00
00
00
00
1H NMR data could not be assigned due to intramolecular
lactonization.
4.1.23. Dumbbell(1)6-amide-SLac (7)
4.1.18. Fan(0)3-SLac (2)
Removal of all protection of Dumbbell-amide-type dendrimer
41 (85 mg, 0.010 mmol) was carried out by the same method as
that described for 2 to give water-soluble 7 (44 mg, 77%).
1H NMR d (D2O) 4.51 (br, 6H, H-10), 4.41 (br, 6H, H-1), 4.12 (br,
6H, H-30), 2.53 (br, 24H, –CH2SCH2–), 0.57 (m, 20H, –CH2Si), 0.03
(br, 6H, SiCH3). MALDI-TOF-MS Calcd for C230H408N12O12S6Si3Na
[M+Na]+: 5561.4. Found: m/z 5562.4.
To
a solution of fully protected dendrimer 35 (26 mg,
0.0070 mmol) in MeOH (2 mL) was added NaOMe, and the mixture
was stirred overnight at room temperature. IR-120B (H+) resin was
added to the mixture, and the mixture was filtered and concen-
trated. 0.025 M aq NaOH (2 mL) was added to the residue, and
the solution was stirred overnight at room temperature. IR-120B
(H+) resin was added to the mixture, and the suspension was fil-
tered and concentrated. The residual syrup was purified by gel per-
meation chromatography using Sephadex G-25 with 5% aq AcOH to
afford 2 in quantitative yield.
4.2. Viruses
Influenza viruses A/PR/8/34 (H1N1), A/Aichi/2/68 (H3N2), and
A/Memphis/1/71 (H3N2) were cultured in 11-day-old embryonic
chicken eggs, purified, and concentrated as described previously.27
Viral hemagglutinin (HA) units were determined in microtiter
plates using 0.5% guinea pig erythrocytes.
IR (KBr) 3396 (
N–H), 1072 ( C–O–C), 1034 (
m
O–H), 2927 (
m
C–H), 1730 (
m
C@O), 1633 (
mC@O), 1566
(m
m
mC–O–C), 706 (
m
N–H), 619 (m
N–H) cmꢀ1; 1H
NMR d (D2O) 7.30 (m, 5H, SiPh), 4.45 (br, 3H, H-10), 4.32 (br, 3H, H-
1), 4.07 (near d, 3H, H-30), 3.98–3.46 (m), 3.24 (br, 3H, H-2), 2.66
(near d, 3H, H-300eq), 2.38 (br, 12H, –CH2SCH2–), 1.95 (s, 9H, NDAc),
1.84, 1.46, and 1.32 (3m), 0.81 (br, 6H, CH2Si).
*d 2.33 (near dd, H-300eq of lactone) included about 8%.
FAB-MS Calcd for C99H167N3O57S3Si [M+H]ꢀ: 2433.9. Found: m/z
2434.1.
4.3. Hemagglutination inhibition assay (HAI)
The hemagglutination inhibition (HAI) assay was carried out
using 96-well microtiter plates as described previously.28 Phos-
phate buffer saline (pH 6.5) containing 0.01% gelatin was used as
a dilution buffer. Human erythrocytes were used as indicator cells.
4.1.19. Ball(0)4-SLac (3)
Removal of all protection of Ball-type dendrimer 36 (50 mg,
0.010 mmol) was carried out by the same method as that described
for 2 to give water-soluble 3 (30 mg, 93.8%).
Virus suspension (22 HA units in 25
lL of PBS) was added to each
well containing an inhibitor in twofold serial dilutions with the
dilution buffer. The plates were incubated for 1 h at 4 °C. After
0.05 ml of 0.5% (v/v) chicken erythrocytes in PBS had been added
to the plates, the plates were kept at 4 °C for 1 h. The maximum
dilution of the samples showing complete inhibition of the hemag-
glutination was defined as the hemagglutination inhibition titer.
1H NMR d (D2O) 4.46 (d, 4H, J1 ,2 = 7.0 Hz, H-10), 4.38 (d, 4H,
0
0
00
00
00
00
J1,2 = 7.5 Hz, H-1), 2.68 (dd, 4H, J3 a,3 e = 12.9 Hz, J3 e,4 = 4.3 Hz, H-
300eq), 1.82 (m, 4H, H-300ax), 0.66 (br, 6H, CH2Si). MALDI-TOF-MS
Calcd for C124H216N4O76S4SiNa [M+Na]+: 3158.4. Found: m/z
3159.4.

H. Oka et al. / Bioorg. Med. Chem. 17 (2009) 5465–5475
5475
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We are grateful to Snow Brand Milk Products Co., Ltd for provid-
ing 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
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