Chemoenzymatic synthesis of artificial glycopolypeptides containing multivalent sialyloligosaccharides with a γ-polyglutamic acid backbone and their effect on inhibition of infection by influenza viruses

Article abstract of DOI:10.1016/j.bmc.2006.11.006

Highly water-soluble, artificial glycopolypeptides with a γ-polyglutamic acid (γ-PGA) backbone derived from Bacillus subtilis sp. and multivalent sialyloligosaccharide units have been chemoenzymatically synthesized as potential polymeric inhibitors of infection by bird and human influenza viruses. 5-Trifluoroacetamidopentyl β-N-acetyllactosaminide and 5-trifluoroacetamidopentyl β-lactoside were enzymatically synthesized from LacNAc and lactose, respectively, by cellulase-mediated condensation with 5-trifluoroacetamido-1-pentanol. After deacetylation, the resulting 5-aminopentyl β-LacNAc and β-lactoside glycosides were coupled to the α-carboxyl groups of the γ-PGA side chains. The artificial glycopolypeptides carrying LacNAc and lactose were further converted to Neu5Acα2-(3/6)Galβ1-4Glcβ and Neu5Acα2-(3/6)Galβ1-4GlcNAcβ sialyloligosaccharide units by α2,3- and α2,6-sialyltransferase, respectively. The interaction of these glycopolypeptides with various influenza virus strains has been investigated by three different methods. Glycopolypeptides carrying Neu5Acα2,6LacNAc inhibited hemagglutination mediated by influenza A and B viruses, and their relative binding affinities for hemagglutinin were 10²- to 10⁴-fold higher than that of the naturally occurring fetuin control. A glycopolypeptide carrying Neu5Acα2,6LacNAc inhibited infection by A/Memphis/1/71 (H3N2) 93 times more strongly than fetuin, as assessed by cytopathic effects on virus-infected MDCK cells. The avian virus [A/duck/Hong kong/4/78 (H5N3)] bound strongly to Neu5Acα2,3LacNAc/Lac-carrying glycopolypeptides, whereas the human virus [A/Memphis/1/71 (H3N2)] bound to Neu5Acα2,6LacNAc in preference to Neu5Acα2,6Lac. Taken together, these results indicate that the binding of viruses to terminal sialic acids is markedly affected by the structure of the asialo portion, in this case either LacNAc or lactose, in the sugar chain of glycopolypeptides.

Full text of DOI:10.1016/j.bmc.2006.11.006

Bioorganic & Medicinal Chemistry 15 (2007) 1383–1393
Chemoenzymatic synthesis of artificial glycopolypeptides containing
multivalent sialyloligosaccharides with a c-polyglutamic acid
backbone and their effect on inhibition of infection
by influenza viruses
Makoto Ogata,a Takeomi Murata,b Kouki Murakami,c Takashi Suzuki,c
Kazuya I. P. J. Hidari,c Yasuo Suzukid and Taichi Usuia,b,
*
aScience of Biological Resource, The United Graduate School of Agricultural Science, Gifu University,
Yanagido 1-1, Gifu 501-1193, Japan
bDepartment of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Ohya 836,
Suruga ward, Shizuoka 422-8529, Japan
cDepartment of Biochemistry, University of Shizuoka, School of Pharmaceutical Sciences and COE Program in the 21st century,
Shizuoka, Japan
dCollege of Life and Science, Chubu University, Japan
Received 6 September 2006; revised 3 November 2006; accepted 6 November 2006
Available online 9 November 2006
Abstract—Highly water-soluble, artificial glycopolypeptides with a c-polyglutamic acid (c-PGA) backbone derived from Bacillus
subtilis sp. and multivalent sialyloligosaccharide units have been chemoenzymatically synthesized as potential polymeric inhibitors
of infection by bird and human influenza viruses. 5-Trifluoroacetamidopentyl b-N-acetyllactosaminide and 5-trifluoroacetamid-
opentyl b-lactoside were enzymatically synthesized from LacNAc and lactose, respectively, by cellulase-mediated condensation with
5-trifluoroacetamido-1-pentanol. After deacetylation, the resulting 5-aminopentyl b-LacNAc and b-lactoside glycosides were cou-
pled to the a-carboxyl groups of the c-PGA side chains. The artificial glycopolypeptides carrying LacNAc and lactose were further
converted to Neu5Aca2-(3/6)Galb1–4Glcb and Neu5Aca2-(3/6)Galb1–4GlcNAcb sialyloligosaccharide units by a2,3- and a2,6-si-
alyltransferase, respectively. The interaction of these glycopolypeptides with various influenza virus strains has been investigated by
three different methods. Glycopolypeptides carrying Neu5Aca2,6LacNAc inhibited hemagglutination mediated by influenza A and
B viruses, and their relative binding affinities for hemagglutinin were 102- to 104-fold higher than that of the naturally occurring
fetuin control. A glycopolypeptide carrying Neu5Aca2,6LacNAc inhibited infection by A/Memphis/1/71 (H3N2) 93 times more
strongly than fetuin, as assessed by cytopathic effects on virus-infected MDCK cells. The avian virus [A/duck/Hong kong/4/78
(H5N3)] bound strongly to Neu5Aca2,3LacNAc/Lac-carrying glycopolypeptides, whereas the human virus [A/Memphis/1/71
(H3N2)] bound to Neu5Aca2,6LacNAc in preference to Neu5Aca2,6Lac. Taken together, these results indicate that the binding
of viruses to terminal sialic acids is markedly affected by the structure of the asialo portion, in this case either LacNAc or lactose,
in the sugar chain of glycopolypeptides.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
face. The viruses recognize not only sialic acids (Sia) on
the receptors but also particular sugar chain structures,
such as sialyllacto-series type I (Siaa2-(3/6)Galb1–3Glc-
NAcb1) and type II (Siaa2-(3/6)Galb1–4GlcNAcb1)
structures.1 Influenza viruses vary in their recognition
of different types (Neu5Ac or Neu5Gc) and linkages
(a2–3 or a2–6) of Sia residues.2 Human influenza A
viruses, which have been isolated from humans over
the past 30 years, preferentially recognize a2,6-linked
Neu5Ac residues.1 The host cell specificity of viruses
is dependent on the linkage between Sia and the
Influenza A and B viruses infect host cells through the
binding of viral hemagglutinins (HAs) to sialoglycopro-
teins or sialoglycolipids on receptors on the host cell sur-
Keywords: Glycopolypeptides; Influenza virus; Inhibition; c-Polyglu-
tamic acid; Sialyloligosaccharides.
*
Corresponding author. Tel./fax: +81 54 238 4873; e-mail:
actusui@agr.shizuoka.ac.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.006

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M. Ogata et al. / Bioorg. Med. Chem. 15 (2007) 1383–1393
penultimate galactose, as well as on the number of Sia
residues and the core structure. Multivalent interactions
must occur between viral HAs and cell-surface Sia resi-
dues, which must then be amplified by so-called ‘glyco-
side cluster effects.’3
The structures of the aminoalkyl spacer-linked disaccha-
1
ride glycosides 1 and 2 were confirmed by H- and 13C
NMR analyses. The O-linked b-anomeric bond between
the glycosyl and aglycon moieties was confirmed by the
1
following H- and 13C NMR assignments. The protons
of the O-linked b-anomeric bond between the glycosyl
and aglycon moieties showed lower chemical shifts with
Various synthetic glycopolymers carrying multivalent
Sia residues that target viral HAs have been prepared
as influenza virus inhibitors by using polyacrylamide
(PA),3–9 poly(acrylacid) (PAA),10,11 polystyrene,12
chitosan,13 and cyclic peptide14 as the polymer back-
bone. However, synthetic glycopolymers potentially
pose several problems for in vivo use, such as low
solubility, significant cytotoxicity,15 and immunogenici-
ty.16 From this viewpoint, we have previously reported
that artificial glycopolymers containing multivalent
sialooligosaccharides with a poly(a-L-glutamic acid)s
(a-PGA) backbone are useful for inhibiting infection
by influenza viruses, as assessed by measuring cyto-
pathic inhibitory effects in virus-infected cells.17 Our
strategy of molecular design is to construct an amphi-
philic structure, taking into account immunogenicity,
by arranging hydrophobic main chains and hydrophilic
pendant oligosaccharides via convenient synthetic
routes.
larger coupling constants (compound 1, d 4.53 ppm (J1,2
=
7.0 Hz); compound 2, d 4.48 ppm (J1,2 = 7.9 Hz)). In the
13C NMR spectra, the O-b-linked C-1b signal was char-
acterized by higher chemical shifts (compound 1, d
103.8 ppm; compound 2, d 104.8 ppm). The resulting
disaccharide glycosides were easily converted to 5-amin-
opentyl b-N-acetyllactosaminide (3) and 5-aminopentyl
b-lactoside (4) by hydrolysis in an alkaline solution with
yields in the range of 96–99%.
2.1.2. Synthesis of spacer-linked asialoglycopolypeptides
carrying LacNAc or lactose. The amino function of the
resulting disaccharide glycosides reacted with the car-
boxyl group of c-PGA in the presence of the condensa-
tion reagents BOP and HOBt, as previously described.19
In the synthesis of glycopolypeptides carrying LacNAc,
two c-PGAs of different molecular weight (77,000 and
990,000) were used. The reaction solution was applied
to a column of Sephadex G-25M PD-10 to separate
the glycosylated c-PGA from the low-molecular-weight
reactants. The degree of substitution (DS) in the mole
fraction of the substituted residues in the asialoglyco-
polypeptides 5a and 5b was 61% and 58%, respectively,
In this study, we propose a simple synthesis of a new
type of asialo-type polymer starting from N-acetyllac-
tosamine or lactose. Scheme 1 represents the pathway
from N-acetyllactosamine or lactose via the following
three steps: (1) enzymatic synthesis of a spacer-linked
disaccharide glycoside; (2) coupling of the resulting
glycoside to c-polyglutamic acid (c-PGA); and (3) sialy-
lation of the resulting polymer to produce highly water-
soluble glycopolymers carrying clustered identical
sialyldisaccharide segments. The inhibitory effects of
these glycopolypeptides on infection by bird and human
influenza viruses have been subsequently investigated
by measuring the cytopathic outcome in virus-infected
Madine–Darby canine kidney (MDCK) cells.
1
as calculated from the relative intensities of H NMR
signals due to peptide methylene protons and those
due to spacer-linked methylene protons in aglycon.
The structures of synthesized glycopolymers were con-
firmed by 1H- and 13C NMR analyses. In the 1H
NMR spectrum of 5a, characteristic signals at d
3.19 ppm were assigned to aglycon e-methylene protons
and those at d 2.41 ppm were assigned to peptide c-
methylene protons. The analogue asialoglycopolypep-
tide 6 carrying a lactose residue was synthesized in a
similar way, as summarized in Table 1.
2. Results
2.1.3. Synthesis of sialoglycopolypeptides carrying
a-Neu5Ac-(2,3/6)-b-LacNAc or a-Neu5Ac-(2,3/6)-b-lac-
toside. Asialoglycopolypeptides 5a,5b, and 6 were sialy-
lated, respectively, to 7a, 7b, and 9, and 8a, 8b, and 10
by a2,3- and a2,6-sialyltransferase (Scheme 1). After
separation through a column of Sephadex G-25M PD-
10, the target glycopolypeptides were obtained. The
structures of the synthesized sialoglycopolypeptides
2.1. Convenient synthetic route to a new type of artificial
glycoconjugate polymer
2.1.1. Synthesis of aminoalkyl disaccharide glycoside. A
condensation reaction between LacNAc and 5-trifluoro-
acetamido-1-pentanol was performed using a partially
purified enzyme preparation from Trichoderma reesei.18
The molar ratio of LacNAc to 5-trifluoroacetamido-1-
pentanol was 1:1.5, and the total substrate concentra-
tion in the reaction mixture was 70% (w/v). The reaction
mixture was easily fractionated with a charcoal–Celite
column. The target 5-trifluoroacetamidopentyl b-N-
acetyllactosaminide (1) was obtained in a low yield of
1.1% based on the initial amount of LacNAc. In a sim-
ilar way, 5-trifluoroacetamidopentyl b-lactoside (2) was
synthesized by a condensation reaction between lactose
and 5-trifluoroacetamido-1-pentanol. The target prod-
uct was obtained by a Silica gel column in 1.0% yield
based on the initial amount of lactose.
1
were confirmed by H- and 13C NMR analyses.
In the 1H NMR spectrum of 7a, characteristic signals at
d 2.76 (dd, 1H, J3ax,3eq 11.9 J3eq,4 3.0 Hz, H-300eq) and d
1.81 (t, 1H, J3ax,3eq 11.9 J3ax,4 11.9 Hz, H-300ax) were as-
signed to the H-300 proton. In 8a, d 2.67 (dd, 1H, J3ax,3eq
12.2 J3eq,4 4.2 Hz, H-300eq) and d 1.74 (t, 1H, J3ax,3eq 12.2
J3ax,4 12.2 Hz, H-300ax) were assigned to the H-300 pro-
ton. The 1H NMR spectrum also showed that the degree
of sialylation was 96–100% from the integration data of
the proton signals (Table 2). The degree of substitution
of neutral sugar derivatives (NS) and sialyl sugar deriv-
atives (Sia) based on DP of c-PGA as 100% (Table 2).

M. Ogata et al. / Bioorg. Med. Chem. 15 (2007) 1383–1393
1385
Scheme 1. (1) Enzymatic synthesis of spacer-linked disaccharide glycoside, (2) coupling of the resulting glycoside with c-polyglutamic acid (c-PGA),
and (3) sialylation to highly water-soluble glycopolymers carrying clustered identical sialyldisaccharide segments.
Table 1. Synthesis of asialoglycopolypeptides with different degrees of polymerization of glutamic acid residues
Products
Compound
c-PGA
DPa mg
510 15.1 119
9,90,000 6557 15.0 118
77,000 510 16.5 130
BOPb HOBtc Amino-sugard Yield DSe
(mg)
(mg)
(mg)
(mg) (%)
MW
77,000
Poly (5-aminopentyl b-N-acetyllactosaminide/c-PGA) 5a
5b
15
15
140
139
17.0
24.0
61
58
Poly (5-aminopentyl b-lactoside/c-PGA)
6
16
140
29.6
69
a Degree of polymerization of glutamic acid residues.
b Benzotriazol-1-yloxvtris-(dimethylamino)phosphonium hexafluorophosphate.
c l-Hydroxybenzotriazole hydrate.
d 5-Aminopentyl b-N-acetyllactosaminide for 5a–5b and 5-aminopentyl b-lactoside 6.
e Degree of substitution of sugar derivatives based on DP of c-PGA as 100%. Calculated from 1H NMR data at 25 ꢁC.
In the 13C NMR spectra of 7a and 8a, the respective C-
30 and C-60 signals were distinguished by a downfield
position with chemical shifts at d 77.9 and d 66.1. These
data indicate that the galactosyl residues of the sugar
chains are regiospecifically sialylated in the a2–3 and
a2–6 linkages.
lation from the integration data of the resulting proton
signals (Table 2). The properties of the glycopolypep-
tides synthesized are summarized in Table 2.
2.2. Interaction of synthetic glycopolypeptides with influ-
enza viruses
The structures of the sialoglycopolypeptides7b, 8b, 9
2.2.1. Hemagglutination inhibition (HI) assay of the
glycopolypeptides using influenza viruses. Various
substances carrying sialyloligosaccharides inhibit
1
and 10 were similarly confirmed by H and 13C NMR
analyses. It was possible to evaluate the extent of sialy-

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M. Ogata et al. / Bioorg. Med. Chem. 15 (2007) 1383–1393
Table 2. Glycopolypeptides for inhibiting influenza virus infection
Back bone
Sugar moiety
Compound
DPa
DSe (%)
kDad
NSb
Siac
c-PGA
Galb1–4GlcNAcb–
5a
5b
510
6557
61
58
—
—
210
2600
Galb1–4Glcb–
Neu5Aca2–3Galb1–4GlcNAcb–
6
7a
510
510
69
3
—
65
210
330
Neu5Aca2–6Galbl–4GlcNAcb–
7b
8a
8b
6557
510
0
0
0
58
68
58
3800
330
6557
3800
Neu5Aca2–3Galb1–4Glcb–
Neu5Aca2–6Galbl–4Glcb–
9
510
510
21
30
48
39
290
280
10
a Degree of polymerization of glutamic acid residues.
b Neutral sugar derivatives substituted.
c Sialyl sugar derivatives substituted (=Sia contents).
d Calculated kDa.
e Degree of substitution of sugar derivatives based on DP of c-PGA as 100%. Calculated from 1H NMR data at 25 ꢁC.
hemagglutination mediated by viral hemagglutinin. We
therefore tested the inhibitory effect of the glycopolypep-
tides on hemagglutination by influenza viruses [A/Mem-
phis/1/71 (H3N2), A/PR/8/34 (H1N1), and B/Lee/40], as
summarized in Table 3. The Neu5Aca2,6LacNAc-carry-
ing glycopolymers 8a and 8b predominantly inhibited
the hemagglutinin of influenza viruses A/Memphis/1/71
(H3N2) and B/Lee/40. These glycopolypeptides showed
102- to 104-fold higher affinities for the viral hemagglu-
tinins relative to the control fetuin. The Neu5Aca2,6-
Lac-carrying glycopolymer 10 was observed to bind A/
Memphis/1/71 (H3N2) and B/Lee/40 more weakly than
compound 8a.
itory effects, even at concentrations as high as 1000 lg/
ml.
2.2.3. Binding specificity of influenza viruses to glycopep-
tides. The binding specificity of avian [A/duck/Hong
kong/4/78 (H5N3)] and human [A/Memphis/1/71
(H3N2)] influenza viruses to glycopolypeptides was
determined by a solid-phase binding assay.20,21 Avian
influenza viruses are known to preferentially bind to oli-
gosaccharides that terminate in an a2,3-linked Neu5Ac,
whereas human viruses preferentially bind to those that
end in an a2,6-linked Neu5Ac. The viruses demonstrat-
ed the anticipated binding specificity to the synthesized
glycopeptides (Fig. 1). The avian virus predominantly
bound to 7a, 7b, and 9 carrying Neu5Aca2,3LacNAc/
Lac (Fig. 1a), whereas the human virus preferentially
bound to 8a and 8b carrying Neu5Aca2,6LacNAc and
bound to10 carrying Neu5Aca2,6Lac to a lesser extent
(Fig. 1b). The effect of the molecular weight of the arti-
ficial glycopolypeptides on the binding activity was also
assessed using 7a and 7b, and 8a and 8b, which had dif-
ferent molecular weights (7a and 8a, 330 kDa; 7b and
8b, 3800 kDa) but roughly equivalent Sia contents
(around 60%). Binding of 8a and 8b by A/Memphis/1/
71 (H3N2) increased in a molecular-weight-dependent
manner, but that of 7a and 7b by A/duck/Hong kong/
4/78 (H5N3) did not. This observation indicates that
binding by the human virus may be influenced not only
by the carbohydrate structure of the asialo moiety in the
sugar unit, but also by molecular weight. Neither virus
strain showed binding to asialoglycopolypeptides.
By contrast, the Neu5Aca2,3LacNAc and Neu5Aca2,3-
Lac-carrying glycopolymers 7a and 9 inhibited hemag-
glutination
by
A/PR/8/34
(H1N1).
These
glycopolypeptides showed 30- to 34-fold higher affinities
relative to fetuin. Compounds 8a and 10 were observed
to bind A/PR/8/34 (H1N1) very weakly. The asialogly-
copolypeptides 5a, 5b, and 6 did not affect influenza
virus-mediated hemagglutination.
2.2.2. Inhibition of influenza virus infection by glycopoly-
peptides. Next, we tested whether our series of synthetic
glycopolypeptides inhibited infection by the influenza
virus A/Memphis/1/71 (H3N2) in focus-forming assays.
The virus was incubated with the glycopolypeptides at
the indicated concentrations, and inoculated onto
MDCK cells. The results are summarized in Table 4.
Infection of A/Memphis/1/71 (H3N2) was inhibited by
8a carrying Neu5Aca2,6LacNAc (IC50 69 lg/ml), but
not 7a carrying Neu5Aca2,3LacNAc. Similar results
were also observed for 10 carrying Neu5Aca2,6Lac
(IC50 317 lg/ml), but its activity was weaker than 8a.
The inhibition activity of 8a in comparison with 10
indicates that an internal core carbohydrate residue,
such as LacNAc, in the sugar chain is important for
A/Memphis/1/71 (H3N2) to bind to terminal Neu5Ac
residues. The inhibitory activity of glycopolypeptide 8a
was 93 times higher than that of fetuin. The desialylated
glycopolymers (5a and 6) and c-PGAs showed no inhib-
3. Discussion
Artificial glycopolymers generated to possess a single
type of Neu5Ac linkage (either a2,3 or a2,6) could be
an ideal tool to study the molecular interactions between
receptors and viruses. We therefore designed a series of
glycopolypeptides consisting of three parts: glycan,
spacer, and polypeptide backbone. The N-acetyllactos-
amine and lactose residues were at first enzymatically

M. Ogata et al. / Bioorg. Med. Chem. 15 (2007) 1383–1393
Table 3. Inhibition of influenza virus hemagglutination by sialoglycopolypeptides
1387
Glycopolypeptides
Compound
HI activity (nM)
A/Memphis/1/71 (H3N2) A/PR/8/34 (H1N1)
B/Lee/40
c-PGAa
NDb
ND
ND
Poly (5-aminopentyl b-LacNAc/c-PGA)
5a
5b
ND
ND
ND
e
—
ND
—
Poly (5-aminopentyl b-Lac/c-PGA)
6
ND
ND
ND
Poly (Neu5Aca2–3LacNAc b-5-aminopentyl/c-PGA)
7a
7b
ND
16c(102)d
95 (34)
—
189(17)
—
Poly (Neu5Aca2–6LacNAc b-5-aminopentyl/c-PGA)
8a
8b
12(136)
0.13 (12,500)
1515(2.1)
—
24(136)
—
Poly (Neu5Aca2–3Lac b-5-aminopentyl/c-PGA)
Poly (Neu5Aca2–6Lac b-5-aminopentyl/c-PGA)
Fetuin
9
862(1.9)
112(15)
1628(1)
108(30)
1785(1.8)
3255(1)
431 (7.6)
54 (60)
10
3255(1)
a c-PGA: MW = 77,000.
b ND: not detected (no activity).
c Minimum concentrations required for complete inhibition of hemagglutination.
d Relative inhibitory potency. (All data normalized to those of fetuin. Higher values indicate greater inhibitory potency).
e ‘—’ not determined.
Table 4. IC50 of glycopolypeptides in focus-forming assays of influenza A virus [A/Memphis/1/71(H3N2)]
Glycopolypeptides
Compound
A/Memphis/1/71 (H3N2)
b
IC50 (lg/ml)
IC50 (Sia lM)
c-PGAa
>1000
>1000
>1000
>1000
69
>1000
>1000
>1000
>1000
72
Poly (5-aminopentylb-LacNAc/c-PGA)
Poly (5-aminopentylb-Lac/c-PGA)
Poly (Neu5Aca2–3LacNAcb-5-aminopentyl/c-PGA)
Poly (Neu5Aca2–6LacNAcb-5-aminopentyl/c-PGA)
Poly (Neu5Aca2–3Lacb-5-aminopentyl/c-PGA)
Poly (Neu5Aca2–6Lacb-5-aminopentyl/c-PGA)
Fetuin
5a
6
7a
8a
9
>1000
317
6448
>1000
225
—
10
a c-PGA: MW = 77,000.
b Concentration of Sia units.
connected to 5-trifluoroacetamido-1-pentanol. Recently,
we found that endo-b-glycosidase, a kind of cellulase
from T. reesei, catalyzes two types of reaction, transgly-
cosylation and condensation.18 Thus, this enzymatic
catalysis was harnessed for the present synthesis of spac-
er-linked trifluoroacetamidoalkyl N-acetyllactosaminyl
and lactosyl glycosides. The efficiency of the reaction
is not always high, but this method has two advantages:
first, the excess of unreacted LacNAc substrate, which is
a valuable substrate, is recovered by straightforward
column chromatography and can be reutilized for syn-
thesis; and second, the O-glycosidation process stereo-
specifically gives only the b-glycoside without the need
for any protection or deprotection steps.
a-Neu5Ac-(2,3/6)-b-Lac by a2,3- and a2,6-sialyltrans-
ferase. We have previously reported that artificial glyco-
polypeptides with a poly(a-L-glutamic acid) backbone
can be used as acceptors of a CMP-Neu5Ac donor by
utilizing a2,3- or a2,6-sialyltransferase.17 This method
was applicable to the elongation of sugar chains in the
present glycopolypeptides with a c-PGA backbone.
Sialylation of the LacNAc unit was almost quantitative,
but sialylation of the lactose unit occurred at low effi-
ciency, around 60–70% (Table 2). This indicates that
the sialylation in such glycopolymers occurs preferen-
tially on a LacNAc structure than on a lactose structure
in the asialo-portion in the sugar chain.
Fetuin, a glycoprotein that contains three Neu5Aca2,6
and Neu5Aca2,3 residues, is used as a control against
which to measure the interaction between influenza
viruses and host cell-surface receptors. This molecule
binds both human and animal types of influenza virus,
because it contains two different Neu5Ac linkages,
Neu5Aca2,6 and Neu5Aca2,3.22,23 Among the glyco-
polypeptides tested in the hemagglutination inhibition
assay, 8b carrying multivalent Neu5Aca2,6LacNAc
units inhibited the hemagglutination of A/Memphis/1/
After deacylation of the resulting disaccharide glyco-
sides, the amino function was easily coupled to the
carboxyl group of c-PGA as the backbone of each gly-
copolymer. The DS (%) of 5-aminopentyl disaccharide
glycosides in the resulting asialoglycopolypeptides
reached a high proportion of 58–69% by controlling
the coupling reaction (Table 1). The asialoglycopolypep-
tides were finally sialylated to obtain sialoglyco-
polypeptides carrying a-Neu5Ac-(2,3/6)-b-LacNAc or

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M. Ogata et al. / Bioorg. Med. Chem. 15 (2007) 1383–1393
by Sia-conjugated PA,3–9,24 PAA,10,11 dendrimers,15
A/DK/HK/4/78 (H5N3)
a
sialylphosphatidylethanolamine derivatives,25 sialyllac-
tose-conjugated polystyrene,12 lyso-GM3-conjugated
PGA,26 GM3 lactose27 and sialylLacNAc-conjugated
PA.8 The IC50 of glycopolymer 8a used in the present
study is roughly equal to that of polymers synthesized
by Tuchida et al.12
0.4
γ-PGA
0.35
0.3
0.25
0.2
5a
6
7a
7b
8a
9
0.15
0.1
The relationship between viral binding activity and the
core determinant of the sugar chain in glycopolymers
is not clear. In our study, the contribution of asialo-por-
tions in sugar chains was examined in a solid-phase
binding assay. The avian influenza viruses bound almost
equally to both 7a and 7b carrying Neu5Aca2,3Lac-
NAc, and to 9 carrying Neu5Aca2,3Lac, suggesting that
the asialo-portion of the disaccharide unit does not con-
tribute to the binding activity of the glycopolymers
(Fig. 1a). In contrast to this result, the fact that human
virus binds much more strongly to 8a and 8b than to
10 indicates that the asialo-portion (LacNAc) in the
sugar chains has an important role in binding of the
virus to the terminal Neu5Ac residues (Fig. 1b). The
effect of molecular weight (length) of the backbone
was also shown to be critical in our glycopolymers
because binding by the human influenza virus was
enhanced in a molecular-weight-dependent manner,
whereas that of the avian influenza virus was not. Paul-
son and colleagues have also demonstrated that binding
of influenza virus to Sia residues is influenced by the
asialo-portion of the carbohydrate structure in experi-
ments based on inhibiting the adsorption of A/Mem-
phis/102/72(H) to erythrocytes using natural and
synthetic monovalent sialosides.28–30
0.05
0
10
0
500
1000
1500
2000
2500
Glycopolypeptdes conc. (ng/ml)
b
A/Memphis/1/71 (H3N2)
0.4
γ-PGA
0.35
0.3
0.25
0.2
5a
6
7a
8a
8b
9
0.15
0.1
0.05
0
10
0
500
1000
1500
2000
2500
Gycopolypeptides conc. (ng/ml).
Figure 1. The direct binding activity of avian and human influenza
viruses to glycopolypeptides was determined by a previously published
method.20 (a) The avian [A/DK/HK/4/78 (H5N3)] virus predominantly
bound to 7a, 7b and 9 carrying a2,3-sialoglycopolypeptides. c-PGA,
asialoglycopolypeptides (5a and 6), and a2,6-sialoglycopolypeptides
(8a and 10) were not bound to A/DK/HK/4/78 (H5N3) virus. (b) The
human [A/Memphis/1/71 (H3N2)] virus preferentially bound to 8a and
8b carrying Neu5Aca2,6LacNAc and bound to10 carrying Neu5A-
ca2,6Lac to a lesser extent. c-PGA, asialoglycopolypeptides (5a and 6)
and a2,3-sialoglycopolypeptides (7a and 9) were not bound to A/
Memphis/1/71 (H3N2) virus.
We have previously reported that glycopolymers with a
poly(a-L-glutamic acid) backbone carrying multivalent
sialyl oligosaccharide units have relatively low immuno-
genicity.17 However, the supply of poly(a-L-glutamic
acid) as a backbone, which is chemically synthesized,
is limited. The objective of our work is to develop a con-
venient chemo-enzymatic procedure suitable for large-
scale preparation of glycopolymers. Sufficient amounts
of the backbone material are needed for practical syn-
thesis. PGA, a natural substance produced from B. sub-
tilis sp., is commercially available in large amounts. The
physiological merits of PGA as the backbone of glyco-
polymers in terms of cytotoxicity and immunogenicity
are expected. Generally, conventional glycopolymers
have a problem of toxicity caused by a backbone such
as PA.15,31 In our previous study, acrylamide monomer,
which is known to be a potent neutrotoxin,32 and PAA-
Na were found to be cytotoxic to MDCK cells under the
conditions used for the tests.17 It has also been shown
that sugar-bovine serum albumin (BSA) and PA conju-
gates act as antigens or immunogens.
71 (H3N2) most potently at a concentration of 0.13 nM
(or 5 · 10ꢀ4 mg/ml), and its relative binding affinity was
12,500-fold higher than that of fetuin itself (Table 3).
Compound 8b had a 92-fold higher affinity relative to
8a. This indicates that a a2,6-sialoglycopolymer with a
high-molecular-weight PGA backbone shows enhanced
binding to hemagglutinin proteins. In contrast to this re-
sult, glycopolypeptides 7a and 9 with multivalent
Neu5Aca2,3LacNAc units bound to A/PR/8/34
(H1N1) in preference to A/Memphis/1/71 (H3N2) or
B/Lee/40. From these results, it can be seen that the
present a2,6- and a2,3-sialylglycopolymers are useful
for analyzing the preferred receptor species for three
kinds of viruses based on the relative affinity of viral
HA. In the focus-forming assay, 8a and 10 inhibited
infection by A/Memphis/1/71 (H3N2) at an IC50 of 69
and 317 lg/ml, respectively (Table 4). A clear difference
due to the structure of the inner disaccharide unit
(LacNAc or lactose) was observed. To date, infections
by influenza viruses have been successfully inhibited
4. Conclusion
We developed a simple synthesis of artificial glycopoly-
peptides suitable for large-scale preparation. It should
be emphasized that our PGA-based glycopolymers have
an extremely high solubility in water (>10% w/v), as

M. Ogata et al. / Bioorg. Med. Chem. 15 (2007) 1383–1393
1389
compared with PA-based glycopolymers (<1%), and
that they are remarkably heat-stable with no aggrega-
tion, even in boiling water. Therefore, PGA-based gly-
copolymers have ideal characteristics as potential
in vivo polymeric inhibitors.
using a JEOL JMS DX-303HF mass spectrometer cou-
pled to a JEOL DA-800 mass data system. An accelerat-
ing voltage of 10 kV and a mass resolution of 1000 were
employed. A sample in distilled water (1 ll) was loaded
onto a probe tip and mixed with glycerol (1 ll) as a ma-
trix. 1H and 13C NMR spectra were recorded on a JEOL
JNM-LA 500 spectrometer at 25 ꢁC. Chemical shifts are
expressed in d relative to sodium 3-(trimethylsilyl) propi-
onate as an external standard. The amount of protein
was determined using a Bio-Rad protein assay kit.
5. Experimental
5.1. General methods
5.1.1. Materials. Cellulase (cellulose XL-522, crude en-
zyme) from Trichoderma reesei was purchased from
Nagase ChemteX Co. p-Nitrophenyl b-lactoside (Lac
b-pNP) and N-acetyllactosamine (LacNAc) were pre-
pared by our previously described methods.33 c-PGA-
Na (MW 77,000, 9,90,000) from B. subtilis sp. was a kind
gift from Meiji Food Materia Co. CMP-b-D-N-acetyl-
neuraminic acid disodium salt (CMP-b-Neu5Ac, 2Na)
was a kind gift from Yamasa Corporation (Chiba, Ja-
pan). a2,3-(N)-sialyltransferase (rat recombinant, Spo-
doptera frugiperda) and a2,6-(N)-sialyltransferase (rat
recombinant, S. frugiperda) were purchased from Cal-
biochem–Novabiochem (San Diego, CA). Rabbit anti-
influenza A virus antiserum (anti-P-50) was raised
against an influenza A virus carrying the HA gene from
strains A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2).34
Human influenza A and B virus strains, A/PR/8/34
(H1N1), A/Memphis/1/71 (H3N2), B/Lee/40, and A/
DK/HK/4/78 (H5N3), were grown in the allantoic sacs
of 10-day-old embryonated eggs and purified by sucrose
density gradient centrifugation. Viral hemagglutination
units (HAUs) of purified viruses were determined as de-
scribed previously.35 MDCK cells were propagated in
EMEM supplemented with 10% FBS. All other reagents
were of the highest quality commercially available and
were used without further purification.
5.4. Synthesis of 5-trifluoroacetamido-1-pentanol
5-Amino-1-pentanol (10 g, 97 mmol) was dissolved in
dry pyridine (20 ml) at 0 ꢁC. Trifluoroacetic anhydride
(25 ml, 180 mmol) was added and the resulting mixture
was stirred magnetically for 1 h at room temperature.
The reaction was terminated by adding crushed ice, neu-
tralized with saturated NaHCO3aq (200 ml), and then
concentrated to a syrup. It was dissolved in 20 ml of
CHCl3/CH3COCH3 = 8/2 and then loaded onto a Silica
Gel 60 N column (B 4.5 · 35 cm). The column was
developed with the same solvent at a flow rate of
10 ml/min and a fraction size of 25 ml/tube. Fractions
33–120 were pooled and concentrated. 5-Trifluoroace-
tamido-1-pentanol was obtained in a total yield of
94% (18 g).
30
D
½aꢁ +2.7ꢁ (c 0.1, water); FAB-mass: m/z 200 [M+H]+
1
(matrix: glycerol); H NMR (D2O, 500 MHz): d 3.60
(t, 2H, H-a), 3.33 (t, 2H, H-e), 1.61 (m, 2H, H-x),
1.57 (m, 2H, H-b), 1.37 (m, 2H, H-c); 13C NMR
(D2O, 500 MHz):d 161.8 (CF3CONH–), 119.9
(CF3CONH–), 64.3 (C-a), 42.5 (C-e), 33.6 (C-b), 30.2
(C-x), 25.1 (C-c).
5.5. Synthesis of trifluoroacetamidopentyl disaccharide
glycosides
5.2. Enzyme assay
5.5.1. 5-Trifluoroacetamidopentyl b-N-acetyllactosami-
nide (1). The crude cellulase from T. reesei was partially
purified by removing unwanted b-D-galactosidase by
our previously described method.18 A mixture contain-
ing 52.2 mmol of LacNAc, 78.4 mmol of 5-trifluoroace-
tamido-1-pentanol, and partially purified enzyme (6200
U of Lac b-pNP hydrolytic activity) in 52.2 ml of
50 mM sodium acetate buffer (pH 5.0) was incubated
for 144 h at 40 ꢁC. The reaction was terminated by heat-
ing at 100 ꢁC for 10 min, and the supernatant obtained
from centrifugation (17,000 rpm, 10 min) was loaded
onto a charcoal–Celite column (B 4.5 · 100 cm) equili-
brated with distilled water. Subsequently, the absorbed
portion was eluted with a linear gradient of 0–25% eth-
anol in a total volume of 10 L, followed by 80% ethanol,
at a flow rate of 5.0 ml/min, and a fraction size of 60 ml/
tube. The neutral sugar content of the eluted fractions
was measured at 485 nm by the phenol–sulfuric acid
method. An aliquot from fractions 53–101 was concen-
trated and lyophilized: LacNAc was recovered in a yield
of 86% (17.2 g). An aliquot from fractions 133–181 was
then concentrated and dissolved in 5 ml of CHCl3/
CH3OH/H2O = 7/3/0.5 and then loaded onto a Silica
Gel 60 N column (B 4.5 · 50 cm). The column was
To measure Gal b-pNP and Lac b-pNP hydrolytic activ-
ities, a mixture containing 25 ll of a 10 mM substrate
solution (Gal b-pNP and Lac b-pNP) in 50 mM sodium
acetate buffer (pH 5.0, 70 ll) and an appropriate
amount of enzyme (5 ll) was incubated for 20 min at
40 ꢁC. One-tenth of the reaction mixture was removed
at 5-min intervals throughout the time course of the
experiment and the reaction was stopped by adding it
to 1.0 M Na2CO3 (190 ll) in a microplate. The amount
of p-nitrophenol liberated was determined by measuring
the absorbance at 405 nm using a microplate reader
(Biolumin 960, Amersham Pharmacia, Sweden). One
unit of enzyme was defined as the amount releasing
1 lmol of p-nitrophenol per minute.
5.3. Analytical methods
HPLC analysis was carried out using a Mightysil Si60
column (4.6 · 250 mm, KANTO CHEMICAL Co.)
with a JASCO Intelligent system liquid chromatograph
and detection at 210 nm. The column was eluted with
90% CH3CN at a flow rate of 1.0 ml/min at 40 ꢁC.
FAB-mass analysis was carried out in positive ion mode

1390
M. Ogata et al. / Bioorg. Med. Chem. 15 (2007) 1383–1393
developed with the same solvent at a flow rate of 10 ml/
min and a fraction size of 25 ml/tube. An aliquot from
fractions 70–100 was then concentrated and lyophilized:
compound 1 was obtained in a total yield of 1.1%
(322 mg) based on the initial amount of LacNAc.
room temperature, it was loaded onto a Sephadex G-
25 column (B 2.5 · 55 cm) equilibrated with H2O at a
flow rate of 1.0 ml/min and fraction size of 2.0 ml/tube.
An aliquot from fractions 60–71 was concentrated and
lyophilized. 5-Aminopentyl b-N-acetyllactosaminide 3
was obtained in a total yield of 99% (82 mg).
30
½aꢁ ꢀ14.4ꢁ (c 0.1, water); FAB-mass: m/z 565 [M+H]+
D
30
½aꢁ ꢀ15.5ꢁ (c 0.1, water); FAB-mass: m/z 469 [M+H]+
1
(matrix: glycerol); H NMR (D2O, 500 MHz): d 4.53
D
1
0
0
(d, 1H, J1,2 7.0 Hz, H-1), 4.47 (d, 1H, J1 2 7.7 Hz, H-
10), 3.99 (dd, 1H, J5,6b 1.5, J6a,6b 12.5 Hz, H-6b), 3.93
(1H, H-40), 3.90 (m, 1H, H-ab), 3.83 (dd, 1H, J5,6a 5.0,
J6a,6b 12.5 Hz, H-6a), 3.78–3.75 (2H, H-60a, H-60b),
3.73–3.70 (4H, H-50, H-4, H-3, H-2), 3.67 (dd, 1H,
(matrix: glycerol); H NMR (D2O, 500 MHz): d 4.52
0
0
(d, 1H, J1,2 8.0 Hz, H-1), 4.47 (d, 1H, J1 2 7.7 Hz, H-
10), 3.98 (dd, 1H, J5,6b 2.2, J6a,6b 12.2 Hz, H-6b), 3.92
(1H, H-40), 3.90 (m, 1H, H-ab), 3.82 (dd, 1H, J5,6a 5.2,
J6a,6b 12.2 Hz, H-6a), 3.78–3.74 (2H, H-60a, H-60b),
3.73–3.69 (4H, H-50, H-4, H-3, H-2), 3.66 (dd, 1H,
J2 3 10, J3 4 3.4 Hz, H-30), 3.59 (m, 1H, H-aa), 3.58
0
0
0 0
(1H, H-5), 3.54 (dd, 1H, J1 2 7.7, J2 3 10 Hz, H-20),
3.33 (t, 2H, H-e), 2.03 (s, 3H, CH3CONH–), 1.61 (m,
2H, H-x), 1.58 (m, 2H, H-b), 1.36 (m, 2H, H-c); 13C
NMR (D2O, 500 MHz):d 177.2 (CH3CONH–), 161.8
(CF3CONH–), 119.9 (CF3CONH–), 105.7 (C-10), 103.8
(C-1), 81.3 (C-4), 78.2 (C-50), 77.6 (C-5), 75.3 (C-3),
75.3 (C-30), 73.8 (C-20), 73.0 (C-a), 71.4 (C-40), 63.8
(C-60), 62.9 (C-6), 57.9 (C-2), 42.5 (C-e), 31.0 (C-b),
30.2 (C-x), 25.2 (C-c), 24.9 (CH3CONH–).
J2 3 10, J3 4 3.4 Hz, H-30), 3.60 (m, 1H, H-aa), 3.59
0
0
0
0
0
0
0 0
(1H, H-5), 3.53 (dd, 1H, J1 2 7.7, J2 3 10 Hz, H-20),
2.88 (t, 2H, H-e), 2.03 (s, 3H, CH3CONH–), 1.60 (m,
2H, H-x), 1.59 (m, 2H, H-b), 1.37 (m, 2H, H-c); 13C
NMR (D2O, 500 MHz): d 177.2 (CH3CONH–), 105.7
(C-10), 103.8 (C-1), 81.3 (C-4), 78.2 (C-50), 77.6 (C-5),
75.3 (C-3), 75.2 (C-30), 73.8 (C-20), 73.0 (C-a), 71.3 (C-
40), 63.8 (C-60), 62.9 (C-6), 57.9 (C-2), 42.4 (C-e), 31.0
(C-b), 30.5 (C-x), 25.0 (C-c), 25.0 (CH3CONH–).
0
0
0 0
5.5.2. 5-Trifluoroacetamidopentyl b-lactoside 2. A mix-
ture containing 151 mmol of lactose, 151 mmol of 5-tri-
fluoroacetamido-1-pentanol, and partially purified
enzyme (4500 U of Lac b-pNP hydrolytic activity) in
151 ml of 50 mM sodium acetate buffer (pH 5.0) was
incubated for 144 h at 40 ꢁC. The reaction was terminat-
ed by heating at 100 ꢁC for 10 min, and the supernatant
obtained from centrifugation (17,000 rpm, 10 min) was
concentrated to a syrup. It was dissolved in 25 ml of
CHCl3/CH3OH/H2O = 7:3:0.5 and then loaded onto a
Silica Gel 60 N column (B 4.5 · 50 cm). The column
was developed with the same solvent at a flow rate of
10 ml/min and fraction size of 25 ml/tube. An aliquot
from fractions 65–103 was concentrated and lyophilized.
Compound 2 was obtained in a total yield of 1.0%
(849 mg) based on the initial amount of lactose.
5-Aminopentyl b-lactoside4 was obtained by alkaline
hydrolysis in a manner similar to the preparation of 5-
Aminopentyl b-N-acetyllactosaminide described above.
30
½aꢁ ꢀ7.0ꢁ (c 0.1, water); FAB-mass: m/z 428 [M+H]+
D
1
(matrix: glycerol); H NMR (D2O, 500 MHz): d 4.49
0
0
(d, 1H, J1,2 8.0 Hz, H-1), 4.45 (d, 1H, J1 2 7.6 Hz, H-
10), 3.98 (dd, 1H, J5,6b 1.3, J6a,6b 12.2 Hz, H-6b), 3.93
(1H, H-40), 3.92 (m, 1H, H-ab), 3.80 (dd, 1H, J5,6a 4.0,
J6a,6b 12.2 Hz, H-6a), 3.77–3.76 (2H, H-60a, H-60b),
3.73 (1H, H-50), 3.72–3.62 (4H, H-aa, H-30, H-4, H-3),
3.60 (1H, H-5), 3.54 (dd, 1H, J1 2 7.6, J2 3 9.5 Hz, H-
20), 3.30 (t, 1H, J1,2 8.0, J2,3 8.0 Hz, H-2), 2.97 (t, 2H,
H-e), 1.69–1.64 (4H, H-b, H-x), 1.46 (m, 2H, H-c);
13C NMR (D2O, 500 MHz):d 105.7 (C-10), 104.8 (C-1),
81.2 (C-4), 78.2 (C-50), 77.6 (C-5), 77.3 (C-3), 75.7 (C-
2), 75.3 (C-30), 73.8 (C-20), 72.9 (C-a), 71.4 (C-40), 63.8
(C-60), 62.9 (C-6), 42.3 (C-e), 31.0 (C-b), 29.7 (C-x),
24.9 (C-c).
0
0
0 0
30
½aꢁ ꢀ2.5 ꢁ (c 0.1, water); FAB-mass: m/z 524 [M+H]+
D
1
(matrix: glycerol); H NMR (D2O, 500 MHz): d 4.48
0
0
(d, 1H, J1,2 7.9 Hz, H-1), 4.45 (d, 1H, J1 2 7.7 Hz, H-
10), 3.98 (dd, 1H, J5,6b 1.8, J6a,6b 12.5 Hz, H-6b), 3.93
(1H, H-40), 3.92 (m, 1H, H-ab), 3.80 (dd, 1H, J5,6a 5.2,
J6a,6b 12.5 Hz, H-6a), 3.81–3.76 (2H, H-60a, H-60b),
3.73 (1H, H-50), 3.72 (m, 1H, H-aa), 3.69–3.62 (3H,
5.7. Synthesis of asialoglycopolypeptides with c-PGA
backbones
Poly(5-aminopentyl b-N-acetyllactosaminide/c-PGA) 5a
was synthesized as follows. The compositions of amin-
opentyl disaccharides, c-PGAs, and reagents used for
the coupling reaction are summarized in Table 1. The
DS in the mole fraction of substituted residues in the
glycopolypeptides was calculated as a percentage from
H-30, H-4, H-3), 3.57 (1H, H-5), 3.54 (dd, 1H, J1 2 7.7,
0
0
J2 3 9.8 Hz, H-20), 3.34 (t, 2H, H-e), 3.30 (t, 1H, J1,2
7.9, J2,3 7.9 Hz, H-2), 1.66 (m, 2H, H-b), 1.61 (m, 2H,
H-x), 1.40 (m, 2H, H-c); 13C NMR (D2O, 500 MHz):d
161.8 (CF3CONH–), 119.9 (CF3CONH–), 105.7 (C-10),
104.8 (C-1), 81.2 (C-4), 78.2 (C-50), 77.6 (C-5), 77.3
(C-3), 75.7 (C-2), 75.3 (C-30), 73.8 (C-20), 73.1 (C-a),
71.4 (C-40), 63.8 (C-60), 62.9 (C-6), 42.5 (C-e), 31.1 (C-
b), 30.2 (C-x), 25.2 (C-c).
0
0
1
the relative intensities of the H NMR signal areas of
peptide c-methylene protons and aglycon e-methylene
protons. c-PGA (MW 77,000, 15.1 mg) was dissolved
in 1.3 ml of 100 mM Na2CO3/Na2HCO3 buffer (pH
10.0). BOP (119 mg) and HOBt (15 mg) in Me2SO
(3.5 ml) were then added and the resulting mixture was
stirred magnetically for 15 min at room temperature.
5-Aminopentyl b-N-acetyllactosaminide (140 mg) in
100 mM Na2CO3/Na2HCO3 buffer (pH 10.0, 0.9 ml)
was added with continuous stirring for 24 h under the
5.6. Synthesis of aminopentyl disaccharide glycosides
5-Trifluoroacetamidopentyl b-N-acetyllactosaminide
(100 mg, 0.18 mmol) was dissolved in 1.0 M NaOH
(1.2 ml). After the mixture was incubated for 60 min at

M. Ogata et al. / Bioorg. Med. Chem. 15 (2007) 1383–1393
1391
same conditions. The reaction mixture was loaded onto
a Sephadex G-25M PD-10 column (Amersham Biosci-
ences Corp., NJ, USA) equilibrated with 100 mM PBS
(pH 7.4). The high-molecular-weight fraction collected
was dialyzed against distilled water for 3 days and
lyophilized to afford compound 5a (17.0 mg).
reaction mixture was loaded onto a Sephadex G-25 M
PD-10 column equilibrated with 100 mM PBS (pH
7.4). The high-molecular-weight fraction collected was
dialyzed against distilled water for 3 days and lyophi-
lized to afford compound 7a (6.4 mg).
1H NMR (D2O, 500 MHz): d 4.55 (d, 1H, J1 2 7.7 Hz,
H-10), 4.51 (d, 1H, J1,2 6.5 Hz, H-1), 4.32–4.21 (1H, a-
methine, c-PGA), 4.13–3.57 (21H, from sugar, H-a),
0
0
1H NMR (D2O, 500 MHz): d 4.52 (d, 1H, J1,2 6.8 Hz,
H-1), 4.47 (d, 1H, J1 2 8.0 Hz, H-10), 4.31–4.21 (1H, a-
methine, c-PGA), 3.98 (dd, 1H, J5,6b 1.5, J6a,6b
12.2 Hz, H-6b), 3.93 (1H, H-40), 3.87 (m, 1H, H-ab),
3.83 (dd, 1H, J5,6a 4.6, J6a,6b 12.2 Hz, H-6a), 3.80–3.71
(7H, H-60a, H-60b, H-50, H-aa, H-4, H-3, H-2), 3.67
0
0
00
00
00
00
3.18 (2H, H-e), 2.76 (dd, 1H, J3 ax;3 eq 11.9, J3 eq;4
3.0 Hz, H-300eq), 2.41 (2H, c-methylene, c-PGA), 2.18–
1.85 (2H, b-methylene, c-PGA), 2.03 (s, 6H,
CH3CONH00–, CH3CONH–), 1.81 (t, 1H, J3 ax;3 eq
00
00
(dd, 1H, J2 3 10.0, J3 4 3.1 Hz, H-30), 3.58 (1H, H-5),
11.9, J3 ax;4 11.9 Hz, H-300ax), 1.55 (2H, H-x), 1.50
(2H, H-b), 1.30 (2H, H-c); 13C NMR (D2O, 500 MHz):d
177.8 (CH3CONH–), 177.6 (CH3CONH00–), 177.6
(HOOC-, c-PGA), 175.9 (HOOC-00), 105.4 (C-10),
103.8 (C-1), 102.6 (C-200), 81.1 (C-4), 78.3 (C-50), 77.9
(C-30), 77.6 (C-5), 75.7 (C-600), 75.2 (C-3), 74.5 (C-800),
73.0 (C-a), 72.2 (C-20), 71.1 (C-400), 70.9 (C-700), 70.3
(C-40), 65.4 (C-900), 63.8 (C-60), 62.9 (C-6), 57.9 (C-2),
56.4 (a-methine, c-PGA), 54.5 (C-500), 42.4 (C-300), 42.1
(C-e), 34.3 (c-methylene, c-PGA), 31.0 (C-b), 30.8 (C-
x), 29.8 (b-methylene, c-PGA), 25.3 (C-c), 25.1
(CH3CONH–), 24.9 (CH3CONH00–).
0
0
0
0
00
00
3.55 (dd, 1H, J1 2 8.0, J2 3 10.0 Hz, H-20), 3.19 (t, 2H,
H-e), 2.41 (2H, c-methylene, c-PGA), 2.18–1.96 (2H,
b-methylene, c-PGA), 2.03 (s, 3H, CH3CONH–), 1.55
(2H, H-x), 1.51 (2H, H-b), 1.31 (m, 2H, H-c); 13C
NMR (D2O, 500 MHz):d 177.1 (HOOC-, c-PGA),
177.1 (CH3CONH–), 105.7 (C-10), 103.8 (C-1), 81.3
(C-4), 78.2 (C-50), 77.6 (C-5), 75.3 (C-3), 57.9 (C-2),
75.2 (C-30), 73.8 (C-20), 73.1 (C-a), 71.4 (C-40), 63.8
(C-60), 62.9 (C-6), 55.8 (a-methine, c-PGA), 42.1 (C-e),
34.4 (c-methylene, c-PGA), 31.1 (C-b), 30.7 (C-x),
29.8 (b-methylene, c-PGA), 25.2 (C-c), 25.1
(CH3CONH–). Compound 5b gave similar NMR data.
0
0
0 0
Poly(Neu5Aca2–3LacNAc b-5-aminopentyl/c-PGA) 7b
was enzymatically synthesized from compound 5b in a
manner similar to that used to prepare compound 7a.
Compound 7b gave similar NMR data.
Poly(5-aminopentyl b-lactoside/c-PGA) 6 was obtained
in a manner similar to that used to prepare compound
5a (see Table 1).
1H NMR data of compound 6 (D2O, 500 MHz): d 4.47
Poly(Neu5Aca2–6LacNAc b-5-aminopentyl/c-PGA) 8a
was similarly synthesized from compound 5a using rat
recombinant a2,6-(N)-sialyltransferase (40 mU/ml) to
afford compound 8a (6.1 mg).
0
0
(d, 1H, J1,2 8.0 Hz, H-1), 4.45 (d, 1H, J1 2 8.3 Hz, H-
10), 4.31–4.20 (1H, a-methine, c-PGA), 3.97 (dd, 1H,
J5,6b 1.9, J6a,6b 11.6 Hz, H-6b), 3.93 (1H, H-40), 3.90
(m, 1H, H-ab), 3.80 (dd, 1H, J5,6a 5.8, J6a,6b 11.6 Hz,
H-6a), 3.78–3.76 (2H, H-60a, H-60b), 3.74 (1H, H-50),
3.72 (m, 1H, H-aa), 3.68–3.62 (3H, H-30, H-4, H-3),
1H NMR (D2O, 500 MHz): d 4.55 (d, 1H, J1,2 6.7 Hz,
H-1), 4.45 (d, 1H, J1 2 7.6 Hz, H-10), 4.33–4.21 (1H, a-
methine, c-PGA), 4.01–3.52 (21H, from sugar, H-a),
0
0
3.59 (1H, H-5), 3.55 (t, 1H, J1 2 8.3, J2 3 8.3 Hz, H-20),
3.31 (t, 1H, J1,2 8.0, J2,3 8.0 Hz, H-2), 3.20 (t, 2H, H-e),
2.41 (2H, c-methylene, c-PGA), 2.19–1.85 (2H, b-meth-
ylene, c-PGA), 1.63 (2H, H-x), 1.53 (2H, H-b), 1.36
(m, 2H, H-c); 13C NMR data of compound 6 (D2O,
500 MHz):d 177.8 (HOOC-, c-PGA), 105.7 (C-10),
104.9 (C-1), 81.2 (C-4), 78.2 (C-50), 77.6 (C-5), 77.3 (C-
3), 75.7 (C-2), 75.3 (C-30), 73.8 (C-20), 73.2 (C-a), 71.4
(C-40), 63.8 (C-60), 62.9 (C-6), 55.4 (a-methine,
c-PGA), 42.1 (C-e), 34.5 (c-methylene, c-PGA), 31.2
(C-b), 30.9 (C-x), 29.5 (b-methylene, c-PGA), 25.2 (C-c).
0
0
0 0
00
00
00
00
3.19 (2H, H-e), 2.67 (dd, 1H, J3 ax;3 eq 12.2, J3 eq;4
4.2 Hz, H-300eq), 2.40 (2H, c-methylene, c-PGA), 2.18–
1.85 (2H, b-methylene, c-PGA), 2.06 (s, 3H,
CH3CONH00–), 2.03 (s, 3H, CH3CONH–), 1.74 (t, 1H,
J3 ax;3 eq 12.2, J3 ax;4 12.2 Hz, H-300ax), 1.56 (2H, H-x),
1.51 (2H, H-b), 1.31 (2H, H-c); 13C NMR (D2O,
500 MHz):d 177.7 (CH3CONH–), 177.6 (CH3CONH00–
), 177.2 (HOOC-, c-PGA), 175.9 (HOOC-00), 106.3 (C-
10), 103.7 (C-1), 102.8 (C-200), 83.5 (C-4), 77.3 (C-50),
76.4 (C-5), 75.4 (C-600), 75.3 (C-3), 75.2 (C-30), 74.4 (C-
800), 73.6 (C-20), 73.0 (C-a), 71.2 (C-40), 71.2 (C-400),
70.9 (C-700), 66.1 (C-60), 65.5 (C-900), 63.2 (C-6), 57.7
(C-2), 56.4 (a-methine, c-PGA), 54.7 (C-500), 42.7 (C-
300), 42.1 (C-e), 34.3 (c-methylene, c-PGA), 31.0 (C-b),
30.8 (C-x), 29.8 (b-methylene, c-PGA), 25.2 (C-c),
25.2 (CH3CONH–), 24.9 (CH3CONH00–).
00
00
00
00
5.8. Synthesis of sialoglycopolypeptides with c-PGA
backbones
Poly(Neu5Aca2–3LacNAc b-5-aminopentyl/c-PGA) 7a
was enzymatically synthesized from compound 5a as
follows. A mixture containing 5.0 mg of compound 5a,
16.0 mM CMP-b-Neu5Ac, 40 mU/ml of rat recombi-
nant a2,3-(N)-sialyltransferase, 2.5 mM MnCl2, 0.1%
BSA, and 10 U/ml of calf intestine alkaline phosphatase
(Boehringer-Mannheim, Mannheim, Germany) in
50 mM MOPS buffer (pH 7.4) was incubated at 37 ꢁC
for 48 h in a total volume of 1.0 ml. After heating at
100 ꢁC and centrifugation, the supernatant from the
Poly(Neu5Aca2–6LacNAc b-5-aminopentyl/c-PGA) 8b
was enzymatically synthesized from compound 5b in a
manner similar to that used to prepare compound 8a.
Compound 8b gave similar NMR data.
Poly(Neu5Aca2–3Lac b-5-aminopentyl/c-PGA) 9 was
enzymatically synthesized from compound 6 as follows.

1392
M. Ogata et al. / Bioorg. Med. Chem. 15 (2007) 1383–1393
A mixture containing 5.5 mg of compound 6, 16.0 mM
CMP-b-Neu5Ac, 40 mU/ml of rat recombinant a2,3-
(N)-sialyltransferase, 2.5 mM MnCl2, 0.1% BSA, and
10 U/ml calf intestine alkaline phosphatase (Boehrin-
ger-Mannheim, Mannheim, Germany) in 50 mM MOPS
buffer (pH 7.4) was incubated at 37 ꢁC for 48 h in a total
volume of 1.1 ml. After heating at 100 ꢁC and centrifu-
gation, the supernatant from the reaction mixture was
loaded onto a Sephadex G-25M PD-10 column equili-
brated with 100 mM PBS (pH 7.4). The high-molecu-
lar-weight fraction collected was dialyzed against
distilled water for 3 days and lyophilized to afford com-
pound 9 (6.7 mg).
ly.8 Phosphate-buffered saline (PBS, pH 6.5) was used as
a dilution buffer. Human erythrocytes were used as indi-
cator cells. Virus suspension (4 HA units in 0.025 ml of
PBS) was added to each well containing the artificial gly-
copolypeptides (1000 to 0.244 lg/ml) in a twofold serial
dilution in dilution buffer. After incubation for 1 h at
4 ꢁC, 0.05 ml of 0.5% (v/v) guinea-pig suspension eryth-
rocytes was added to the plates, and allowed to settle for
2 h at 4 ꢁC. The maximum dilution of the samples show-
ing complete inhibition of hemagglutination was defined
as the HI titer. As a control, the HA inhibitory activity
of fetuin (10 mg/ml) was assayed at the same time.
5.10. Solid-phase binding assay
1H NMR (D2O, 500 MHz): d 4.53 (d, 1H, J1 2 7.6 Hz,
H-10), 4.47 (d, 1H, J1,2 7.3 Hz, H-1), 4.34–4.19 (1H, a-
methine, c-PGA), 4.13–3.58 (20H, from sugar, H-a),
3.30 (t, 1H, J1,2 7.3 Hz, J2,3 7.3 Hz, H-2), 3.20 (2H, H-
0
0
Direct binding of the viruses and the glycopeptides was
determined as described previously.20 In brief, each gly-
copeptide (2 lg/ml) in PBS was serially diluted twofold
with PBS. Fifty microliters of each glycopeptide dilution
was added to the wells of microtiter plates (Corning
Polystyrene Universal-BIND Microplate, USA) and
incubated at 4 ꢁC for 2 h. The plates were irradiated un-
der UV light at 254 nm for 10 min. The glycopolymer
solution was then removed and the plates were blocked
with 0.3 ml of PBS containing 0.25% lipid-free BSA at
room temperature for 12 h, as described previously.21
After being washed with PBS five times, the plates were
incubated with a solution containing influenza virus
(128 HAU in PBS) on ice for 5 h. The plates were
washed with PBS containing 0.01% Tween 20 (T-PBS)
three times, and antibodies to the virus were added.
After incubation for 2 h on ice, the plates were washed
three times with ice-cold T-PBS and then incubated with
horseradish peroxidase (HRP)-conjugated protein A
(Organon Teknika N.V. Cappel Products, Turnhout,
Belgium, diluted 2000-fold in PBS containing 0.25% lip-
id-free BSA) on ice for 2 h. The plates were washed with
ice-cold T-PBS four times and then incubated with OPD
(o-phenylenediamine, Sigma, USA) in PBS containing
0.01% H2O2 for 10 min at room temperature. The reac-
tion was stopped with 0.05 ml of 1 N HCl and the absor-
bance was determined at 492 nm.
e), 2.76 (dd, 1H, J3 ax;3 eq 11.3, J3 eq;4 3.0 Hz, H-300eq),
2.42 (2H, c-methylene, c-PGA), 2.18–1.90 (2H, b-meth-
ylene, c-PGA), 2.03 (s, 3H, CH3CONH00–), 1.82 (t, 1H,
00
00
00
00
J3 ax;3 eq 11.3, J3 ax;4 11.3 Hz, H-300ax), 1.63 (2H, H-x),
1.53 (2H, H-b), 1.36 (2H, H-c); 13C NMR (D2O,
500 MHz): d 177.7 (CH3CONH00–), 177.7 (HOOC–, c-
PGA), 175.9 (HOOC00–), 105.5 (C-10), 104.9 (C-1),
102.5 (C-200), 81.1 (C-4), 78.3 (C-50), 77.9 (C-30), 77.6
(C-5), 77.3 (C-3), 75.7 (C-2), 75.7 (C-600), 74.5 (C-800),
73.2 (C-a), 72.2 (C-20), 71.1 (C-400), 70.9 (C-700), 70.3
(C-40), 65.4 (C-900), 63.8 (C-60), 62.9 (C-6), 56.4 (a-
methine, c-PGA), 54.5 (C-500), 42.3 (C-300), 42.1 (C-e),
34.3 (c-methylene, c-PGA), 31.2 (C-b), 30.9 (C-x),
29.8 (b-methylene, c-PGA), 25.3 (C-c), 24.9
(CH3CONH00–).
00
00
00
00
Poly(Neu5Aca2–6Lac b-5-aminopentyl/c-PGA) 10 was
also synthesized from compound 6 using rat recombi-
nant a2,6-(N)-sialyltransferase (40 mU/ml) in a similar
manner to afford compound 10 (6.8 mg).
1H NMR (D2O, 500 MHz): d 4.47 (d, 1H, J1,2 8.6 Hz,
H-1), 4.43 (d, 1H, J1 2 7.6 Hz, H-10), 4.32–4.20 (1H, a-
methine, c-PGA), 3.99–3.52 (20H, from sugar, H-a),
3.33 (t, 1H, J1,2 8.6 Hz, J2,3 8.6 Hz, H-2), 3.21 (2H, H-
0
0
e), 2.72 (dd, 1H, J3 ax;3 eq 11.9, J3 eq;4 4.0 Hz, H-300eq),
2.42 (2H, c-methylene, c-PGA), 2.23–1.90 (2H, b-meth-
ylene, c-PGA), 2.03 (s, 3H, CH3CONH00–), 1.75 (t, 1H,
5.11. Inhibition of influenza virus infection by
glycopeptides
00
00
00
00
J3 ax;3 eq 11.9, J3 ax;4 11.9 Hz, H-300ax), 1.63 (2H, H-x),
1.52 (2H, H-b), 1.36 (2H, H-c); 13C NMR (D2O,
500 MHz): d 177.7 (CH3CONH00–), 177.7 (HOOC–, c-
PGA), 175.9 (HOOC00–), 106.1 (C-10), 105.8 (C-1),
104.9 (C-200), 82.5 (C-4), 77.4 (C-50), 77.3 (C-3), 76.4
(C-5), 75.7 (C-2), 75.6 (C-600), 75.3 (C-30), 74.5 (C-800),
73.8 (C-20), 73.2 (C-a), 71.3 (C-40), 71.2 (C-400), 71.1
(C-700), 66.3 (C-60), 65.5 (C-900), 63.0 (C-6), 56.4 (a-
methine, c-PGA), 54.6 (C-500), 42.8 (C-300), 42.1 (C-e),
34.4 (c-methylene, c-PGA), 31.2 (C-b), 30.9 (C-x),
29.8 (b-methylene, c-PGA), 25.2 (C-c), 24.9
(CH3CONH00–).
Each glycopeptide (1 mg/ml) in serum-free medium
(SFM) (hybridoma-SFM complete DPM, Invitrogen
Corp. NY, USA) was serially diluted twofold with
SFM. Seventy-five microliters of each glycopeptide dilu-
tion was mixed with 75 ll of influenza virus suspension
(100 FFU) in SFM and then incubated at 4 ꢁC for 1 h.
Confluent monolayers of Madine–Darby canine kidney
(MDCK) cells in 96-well microplates (Corning Costar
Corporation, Cambridge, MA) were inoculated with
100 ll of the mixture at room temperature. After 1 h
at 34 ꢁC, the inoculum was removed from each plate,
and the monolayers were washed three times with PBS
and incubated for 16 h at 34 ꢁC in 100 ll of SFM. The
monolayers in each well were washed three times with
PBS, fixed with 50 ll of methanol at room temperature
for 5 min, and washed three more times with PBS. Infec-
tion foci of cells were detected by a focus-forming assay,
00
00
00
00
5.9. Hemagglutination inhibition assay
The hemagglutination inhibition (HI) assay was carried
out using 96-well microtiter plates as described previous-

M. Ogata et al. / Bioorg. Med. Chem. 15 (2007) 1383–1393
1393
as described previously,36,37 using Anti-NP monoclonal
antibody and horseradish peroxidase-conjugated goat
anti-mouse immunoglobulin G plus M (IgG+M) anti-
body. Infected cells were defined as the mean of three
counts of blue-strained cells within one well. Virus infec-
tion was determined as focus-forming units (FFU). The
concentration causing 50% inhibition of FFU was deter-
mined by plotting the percentage inhibition against the
concentration of each glycopeptide.
15. Reuter, J. D.; Myc, A.; Hayes, M. M.; Gan, Z.; Roy, R.;
Qin, D.; Yin, R.; Piehler, L. T.; Esfand, R.; Tomalia, D.
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R. G.; Khorlin, A. Bioorg. Khim. 1986, 12, 100.
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I.-P. J. K.; Suzuki, T.; Suzuki, Y.; Kobayashi, K.; Ashida,
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Acknowledgments
We are grateful to the Yamasa Corporation for the gen-
erous gift of the CMP-Neu5Ac. This work was support-
ed by a Grant-in-Aid for scientific research (Nos.
16380077 and 16580072) from the Ministry of Educa-
tion, Science, Sports, and Culture of Japan, and a re-
search grant from Cooperation of Innovative
Technology and Advanced Research in Evolutional
Area (CITY AREA) –in the Central Shizuoka Area-,
Ministry of Education, Culture, Sports, Science and
Technology (MEXT) of Japan.
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