Synthesis of an artificial glycoconjugate polymer carrying P(k)-antigenic trisaccharide and its potent neutralization activity against Shiga-like toxin

Article abstract of DOI:10.1016/S0968-0896(99)00129-7

Fluorescence-labeled glycoconjugate polymers carrying carbohydrate segments of a globotriaosyl ceramide (Gb₃) were synthesized and subjected to biological assays using Escherichia coli O-157 strains and Shiga-like toxins (Stx-I and Stx-II). For

Full text of DOI:10.1016/S0968-0896(99)00129-7

Bioorganic & Medicinal Chemistry 7 (1999) 2053±2062
Synthesis of an Arti®cial Glycoconjugate Polymer Carrying
Pk-Antigenic Trisaccharide and its Potent Neutralization Activity
against Shiga-like Toxin
Hirofumi Dohi, a Yoshihiro Nishida, a Mitsuharu Mizuno, a Masashige Shinkai, b
Takeshi Kobayashi, b Tae Takeda, c Hirotaka Uzawa d and Kazukiyo Kobayashi a,*
aDepartment of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa-ku,
Nagoya 464-8603, Japan
bDepartment of Biotechnology, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan
cDepartment of Infectious Disease Research, National Children's Medical Research Center, Taishido, Setagaya, Tokyo 154, Japan
dNational Institute of Materials and Chemical Research, 1-1 Higashi, Tsukuba, 305-8565, Japan
Received 16 November 1998; accepted 6 May 1999
AbstractÐFluorescence-labeled glycoconjugate polymers carrying carbohydrate segments of a globotriaosyl ceramide (Gb3) were
synthesized and subjected to biological assays using Escherichia coli O-157 strains and Shiga-like toxins (Stx-I and Stx-II). For the
¯uorescence labeling, a new polymerizable ¯uorescent monomer with a TBMB carbonyl chromophore (Ex. 325 nm, Em. 410 nm)
was designed. A glycosyl monomer of the trisaccharide segment of Gb3 was prepared from p-nitrophenyl b-lactoside and copoly-
merized with acrylamide and the ¯uorescent monomer to prepare a ¯uorescence-labeled glycoconjugate copolymer carrying [a-d-
galactopyranosyl-(1!4)-b-d-galactopyranosyl]-(1!4)-b-d-glucopyranoside. The polymer showed potent neutralization activity
against Stx-I and also binding activity onto E. coli O-157 strains. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
In the present study, our interest was focused on a Pk-
antigenic trisaccharide, [a-d-galactopyranosyl-(1!4)-
b-d-galactopyranosyl]-(1!4)-b-d-glucopyranoside. This
trisaccharide constitutes the carbohydrate chain of Gb3
ceramide which is known to be a ligand of Shiga-like
toxins (Stx-I and Stx-II, or verotoxins).11±14 We describe
in this paper the syntheses of a series of ¯uorescence-
labeled glycoconjugate polymers carrying the tri-
saccharide and its components (a-d-galactopyranoside,
and b-lactoside). A new polymerizable ¯uorescence
compound was also designed and incorporated into
each glycoconjugate polymer. Their biological activities
were assayed using Shiga-like toxins (Stx-I and Stx-II)
and Escherichia coli 0-157 strains.
Arti®cial glycoconjugate polymers carrying biologically
active carbohydrates as pendant groups constitute a
new class of biomimetic and biomedical materials.1
They have provided access to many new methodologies
in cell cultivation,2 tumor diagnosis,3 and detection and
trapping of viruses and toxins.4,5 Their wide range of
utility can be ascribed primarily to the widely occurring
carbohydrate-binding proteins on the surfaces of cells,
bacteria, and viruses. Moreover, multivalency or cluster
e€ects of the carbohydrates integrate the binding
anity of glycoconjugate polymers to carbohydrate-
binding proteins and contribute much to extend their
potential utility.6±8 In our continuous e€orts to synthe-
size and apply arti®cial glycoconjugate polymers, we
reported several convenient ways to incorporate
biologically active oligosaccharides into polystyrenes,
polyacrylamides, and polyglutamates.9,10
Results and Discussion
Synthesis of a polymerizable glycosyl monomer of the
Gb3 trisaccharide segment
Syntheses of a Gb3 ceramide and its analogues have
already been accomplished by several groups.15 The
* Corresponding author.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00129-7

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H. Dohi et al. / Bioorg. Med. Chem. 7 (1999) 2053±2062
reported synthetic methods, designed for the syntheses
of glycosyl ceramides, could not be applied conveniently
for the present purpose to prepare glycoconjugate poly-
mers, in which introduction of a polymerizable group at
the anomeric center becomes a key step. In the present
study we adopted a synthetic strategy starting from
pNP-b-lactoside 1 (Scheme 1) towards p-nitrophenyl
(pNP) glycoside of the Gb3 trisaccharide, since the nitro
group is convertible into an amino group leading to the
desired glycoconjugate polymer.16
out using SnCl2 and AgClO4 as activators19 and 6-O-
acetylated galactose derivative 5 as the donor. After
being puri®ed on silica gel column chromatography, 40-
O-a-d-galactosyl lactoside 6 was obtained in 62% yield
(a:b=>95:5, no b-glycosidic linkage could be detected in
the 1H NMR analysis). In the above glycosylation, 6-O-
acetylated donor 5 was used to aim at the a-selective
glycosylation owing to the 6-O-acyl participation.20,21
The donor was conveniently synthesized from methyl
2,3,4,6-tetra-O-benzyl-d-galactopyranoside via acid-
catalyzed regioselective acetolysis at C-1 and C-6
(Scheme 2). De-protection of 6 using sodium methoxide
in methanol (room temperature, 2 h) followed by
hydrogenolysis with palladium hydroxide in methanol
gave a trisaccharide 7 with a terminal amino group,
which was then converted into the desired monomer 8
carrying the Gb3 trisaccharide segment.
Chemical manipulations on 1 were, however, found to
have to be approached cautiously since the pNP group
was labile under both basic and acidic conditions
employed for the usual benzylation (benzylbromide,
NaH/DMF), isopropylidenation (re¯uxing in dimethoxy
propane±acetone, p-toluenesulfonic acid), and deiso-
propylidenation (heating in 70% acetic acid) processes.
Isopropylidenation of 1 was thus carried out using tri-
methylsilyl chloride (TMSCl) and acetone.17 The reac-
tion proceeded regioselectively to a€ord 2 in a nearly
quantitative yield. After per-O-acetylation, de-O-iso-
propylidenation was performed using TMSCl and ethyl-
ene glycol18 in MeOH (room temperature) to a€ord a
key compound 3 as a crystalline solid (74% yield,
mp=148ꢀC). The OH-30 group of 3 was protected by a
pivaloyl group, and the galactosylation for 4 was carried
Design and synthesis of a new polymerizable ¯uorescent
monomer
Fluorescence-labeled glycoconjugate polymers are
expected to provide useful tools to investigate carbohy-
drate±protein interactions.22 A convenient method to
incorporate a ¯uorescence chromophore into glyco-
conjugate polymers has already been reported by Nish-
imura et al., who applied an N-allyl dansyl derivative as
Scheme 1. Synthesis of a polymerizable glycosyl monomer carrying Gb3 trisaccharide segment.

H. Dohi et al. / Bioorg. Med. Chem. 7 (1999) 2053±2062
2055
Scheme 2. Synthesis of a glycosyl donor 5.
a polymerizable ¯uorescence monomer.23 More recently,
Kanie and Wong et al. applied BODIPY chromophore
pyridine. The selective 6-O-acylation should be ascribed
to the bulky substituent (tert-Bu) of TBMB carboxylic
acid. The ¯uorescence monomer 12, obtained as a
colorless powder, was found to be soluble in water,
chloroform, methanol, dimethyl sulfoxide, and di-
methylformamide, which are popularly used for poly-
merization reactions. The solubility in both water
and methanol is particularly useful in separating the
unreacted monomer from the polymer during the
reprecipitation in methanol and dialysis in water.
The aqueous solution of the monomer shows ¯uores-
cence maximum at Ex. 325 nm and Em. 410 nm, the
intensity of which is comparable to that of quinine sul-
fate (Ex. 350 nm, Em. 450 nm). The ¯uorescence was
found to be stable in the pH range between 4.5 and 12.0
(the intensity change within 5%).
in the preparation of a ¯uorescence-labeled lyso GM3
/
poly-l-glutamic acid conjugate.24 Dansyl and BODIPY
¯uorescence is known to be sensitive to micro environ-
mental changes including solvents and pH. The
changeable ¯uorescence character is advantageous to
monitor the binding events between carbohydrates and
proteins in a simple manner. The unstable ¯uorescence
intensity, however, often incurs problems in a quantita-
tive assay such as that presented in this paper. Here, we
utilize a new ¯uorescence chromophore of TBMB car-
boxylic acid (2-tert-butyl-2-methyl-1,3-benzodioxole-4-
carboxylate) that shows strong and stable ¯uorescence
intensity under various conditions.25 The stable ¯uores-
cence is considered to re¯ect the characteristic structure
of TBMB ¯uorophore. It has neither an amino nor a
polyacene group, which is common for other ¯uores-
cence agents, but has bulky alkyl substituents (tert-Bu
and Me) in the vicinity which may have a role in pro-
tecting the chromophore from an intermolecular inter-
action with solvents and other chromophores to cause
micro environmental changes.
Fluorescence-labeled glycoconjugate polymers carrying
Gb3 carbohydrate segments
The ¯uorescence monomer 12 (less than 0.1 mol equiv
to glycosyl monomers) was used as a `seasoning' in
the radical copolymerization between 8 (1 mol equiv)
and acrylamide (2.9 mol equiv). After being pre-
cipitated in methanol, dialyzed (Mw 3500 cut-o€) in
water for 3 days and lyophilized, a ¯uorescence-labeled
glycoconjugate polymer carrying the Gb3 segment (gly-
copolymer III in Figure 1) was obtained as a colorless
powder. Copolymerization with other glycosyl monomers
gave the corresponding ¯uorescence glycopolymers I
In order to prepare a TBMB carboxylate monomer
which is copolymerizable with the glycosyl monomer 8
in aqueous solution, TBMB carboxylic acid was coupled
with allyl a-d-galactopyranoside (Scheme 3). The chemi-
cal conversion was performed using racemic TBMB car-
bonyl ¯uoride26 and allyl a-d-galactopyranoside in
Scheme 3. Preparation of a new polymerizable ¯uorescence monomer 12 bearing TBMB carboxylate chromophore.

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H. Dohi et al. / Bioorg. Med. Chem. 7 (1999) 2053±2062
Figure 1. Chemical structures of ¯uorescence-labeled glycoconjugate polymer I±III. The molar ratio of each polymer component is cited in Table 1.
and II, respectively. The molar ratio of each component
in these polymers was determined as summarized in
Table 1 based on the 1H NMR spectra (see Experi-
mental). The polymers show respective ¯uorescence
maximum at Ex. 325 nm and Em. 409 nm similar to that
of the ¯uorescence monomer. The ¯uorescence intensity
was found to show excellent linearity against the con-
centration in the range at least between 0.001 and 0.10
mg/mL for each ¯uorescence-labeled polymer.
labeled RCA-lectin allowed us to determine the anity
1
constants of I and II to be 1.6Â104 and 2.1Â105 M
and those of the corresponding non-¯uorescent poly-
mers as 1.7Â104 and 1.6Â105 M 1, respectively (Table
2). Though some increase in the Ka value was observed
for ¯uorescence polymer II compared with the non-
¯uorescence II, the ¯uorescence chromophore was
found not to impair the binding with this lectin. The
change in TBMB carboxylate ¯uorescence upon the
lectin binding was too small in the present case to
determine the Ka values (Fig. 2).
Biological activities of glycoconjugate polymers I±III
In¯uence of ¯uorescence chromophore on lectin binding.
In order to evaluate the possible in¯uence of a TBMB
The data in Table 2 also indicate that all glycoconjugate
polymers I±III have higher binding anity with RCA120
than monomeric allyl a-d-galactopyranoside. This can
be rationalized by the carbohydrate cluster e€ect as
described before. Polymer II has apparently higher
binding anity than polymer I, re¯ecting the higher
binding anity of this lectin to b-d-galactosides. The
carbonyl chromophore on
a carbohydrate±protein
interaction, association constants (Ka) of I and II with
RCA120 lectin (b-d-galactose binding lectin) were com-
pared with those of the corresponding non-labeled
polymers (Table 2). The binding assays using FITC-
Table 1. Radical copolymerization of ¯ourescence monomer 12 with glycoside monomer and acrylamidea
Polymer no. Glycoside monomer Monomer 12 Acrylamide APSb
TMEDAc
Yield
mg (%) of glycosyl unit
Mol fractiond
Relative ¯uorescence
intensity (50 mg/mL)
Ex. 325 nm, Em. 410 nm
mg (mmol)
mg (mmol)
mg (mmol)
mg
mL
I
II
III
40.2 (0.18)
100 (0.22)
50.0 (0.08)
8.0 (0.018)
9.6 (0.022)
3.4 (0.008)
64.7 (0.91)
78.0 (1.10)
16.5 (0.23)
12.7
15.1
3.5
55.3
59.8
14.0
77 (68)
161 (86)
35 (50)
0.10
0.14
0.20
29
25
13
All reactions were carried out in degassed water at 30ꢀC for 1.5±2.0 h.
APS=ammonium peroxodisulfate, 5 mol%.
TMEDA=N,N,N0,N0-tetramethylethylenediamine, 30 mol%.
Determined by 1H NMR spectra.
a
b
c
d

H. Dohi et al. / Bioorg. Med. Chem. 7 (1999) 2053±2062
2057
Table 2. Association constants of ¯uorescence-labeled and non-
labeled glycoconjugate polymers with RCA120 lectin
in kidney tissues.27 In the present section, the biological
activity of polymers I±III was assayed using human
kidney cells (ACHN cells) against Stx-I and Stx-II.
ACHN cells cannot survive in the presence of Shiga-
like toxins. The number of cells alive in the presence of
each toxin and a glycoconjugate polymer was estimated
(see Experimental for details) as a measure of neu-
tralization activity of the polymer against Shiga-like
toxins. Since these polymers have no ceramide group,
we expected that the biological assay would a€ord clear
insight into the carbohydrate±toxin interaction. The
assay has revealed that the polymer III has powerful
neutralization activity (CD50=0.001±0.005 mg/mL, in
1 a
Polymers
Ka [M ]
Allyl a-d-galactopyranoside
Non FL-polymer I
FL-polymer I
5.7Â103
1.7Â104
1.6Â104
Non FL-lac polymer II
FL-lac polymer II
1.6Â105
2.1Â105
FL-polymer III
1.5Â105
a
Values are expressed as per molar sugar unit. Derived from Stern±
Volmer plots for ®ve diluted solutions of polymers against FITC-
labeled lectin solution (0.01 mg/mL phosphate bu€er) (see Experi-
mental for details).
9
the order of 1Â10 M per trisaccharide unit) against
Stx-I (Fig. 3). Polymers I and II showed no activity at
all against Stx-I nor against Stx-II. Moreover, the poly-
mer III was found to have no activity against Stx-II,
though Gb3 ceramide is thought to be a weak ligand
also of Stx-II. This result means that the trisaccharide
segment of Gb3 is the real ligand of Stx-I but not of
Stx-II.
¯uorescence polymer III, however, shows a similar
binding anity to that of polymer II, though it has a
non-reducing terminal a-d-galactopyranoside linkage.
This suggests that the lectin binding can occur also at
the internal lactose residue or may be simply due to the
di€erence in the aglycon structure between II and III.
Toone et al. reported that the binding constant of
monomeric Gb3 triaoside with Stx-I was in the order of
Ka=1Â103 M 1 using 1-O-methyl globotriaoside.27 The
low anity constant, however, could not explain the
species-selective adhesion of this toxin onto kidney cells
(estimated to be in the order of Ka=1Â109 M 1). The
Neutralization activity against Shiga-like toxins for
human ACHN cells. A globotriaosyl ceramide Gb3 is
known to be a ligand of Shiga-like toxins (Stx-I and
Stx-II).10±14 The selective renal toxicity of these toxins is
assumed to re¯ect the high abundance of Gb3 ceramide
Figure 2. Fluorescence change of FITC (a) and TBMB carboxylate (b) chromophores upon the polymer II-RCA120 lectin binding. In (a), an aliquot
of a ¯uorescence polymer-II solution was added to the bu€er solution of FITC-labeled lectin, while in (b), an aliquot of non-¯uorescent lectin
solution was added to the solution of TBMB-labeled polymer-II.

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H. Dohi et al. / Bioorg. Med. Chem. 7 (1999) 2053±2062
Figure 3. Neutralization activity of polymer-III against Shiga-like toxin-I (a) and Shiga-like toxin-II (b). Absorbance at 540 nm represents the
number of ACHN cells alive in the presence of Stx-I or Stx-II and polymer-III. Blank indicates the condition free from both toxin and polymer, and
Stx-I only indicates the absence of the polymer-III.
strong neutralization activity of polymer III suggests
that the polymer III inhibits the toxin±cell adhesion
e€ectively by clustering trisaccharides around the poly-
mer. Stx-I is known to construct a subunit structure
composed of an A-subunit centered in ®ve copies of a B-
subunit. Provided that each of the ®ve B-subunits has
the same binding ability with the Gb3 trisaccharide, it
may be concluded that the binding of Stx-I is totally
enhanced by carbohydrate clusters on cell surfaces and
e€ectively inhibited by this polymer.
(OD660=1.5). In this assay, non-toxic E. coli O-157
strains having no plasmids of Shiga-like toxin produced
were used to rule out possible polymer binding medi-
ated by the toxins. A non-glycosylated polyacrylamide
was also subjected to the same assay to estimate an
expected non-speci®c binding ability of the copolymer-
ized acrylamide moiety due to multivalency hydrogen
bonding expressed in the polymer.
The assay data are summarized in Figure 4 which shows
the polymer amounts (mg/mL of E. coli suspension,
OD660=1.5) adsorbed onto each E. coli strain. Under
the examined conditions, non-glycosylated poly-
acrylarnide was also adsorbed onto every strain, and
Adhesion activity onto E. coli O-157 strains. Recogni-
tion of cell membrane glycolipids or glycoproteins is an
essential event also for microorganisms to initiate colo-
nization and infections onto host organs.28 Several types
of carbohydrate receptors (adhesins) have already been
identi®ed for E. coli strains, which include a mannose-
type adhesin and a P-type adhesin recognizing Gb3 and
Gb4, globosyl ceramides.14 To our best knowledge,
however, the type of adhesin has not yet been identi®ed
for E. coli O-157 strains. Fluorescence-labeled glyco-
conjugate polymers are expected to provide useful tools
for determining the species-speci®c interactions of car-
bohydrates with certain cells, bacteria, and viruses. In
this section, ¯uorescence polymers I, II and III are sub-
jected to a binding assay using E. coli O-157 strains as a
preliminary test to speculate on the existence of adhe-
sins on these bacterial cells.
The assay was carried out using simple and facile pro-
tocols as follows. Each of three E. coli O-157 strains
(NM, H-45, H-7) was incubated in a medium containing
a ¯uorescent glycoconjugate polymer for 90 min, col-
lected by centrifugation, washed and solubilized. The
incubation period was set at 90 min in order to investi-
gate the polymer binding at the initial stage, when the
adhesion may not have reached saturation. The ¯uores-
cence intensity was measured and applied to linear plots
(0.001±1.0 mg/mL) standardized for the polymer con-
centration to calculate the amounts of ¯uorescence
polymer (mg/mL) adsorbed onto the E. coli strain
Figure 4. Relative adhesion activity of each polymer onto E. coli
strains determined from the ¯uorescence intensity of strains.

H. Dohi et al. / Bioorg. Med. Chem. 7 (1999) 2053±2062
2059
this may be due to the non-speci®c binding onto ubi-
quitous surface lipoproteins or lipopolysaccharides of
E. coli via multivalence hydrogen bonding as expected
above. Polymer I carrying a-d-galactopyranoside was
found to show similar, or even less binding than poly-
acrylamide. This indicates that the clustering mono-
saccharides in I have no binding anity to any strains
or even interfere with the non-speci®c binding of the
polyacrylamide moiety. On the other hand, b-lactosides
in polymer II and Gb3 trisaccharide in polymer III
showed apparent positive e€ects on the binding to every
strain, particularly the latter to B;H7 and O-157;NM
strains. These results imply a receptor-mediated binding
mechanism arising from the existence or inducement of
adhesion proteins of the P-type. The weak but sub-
stantial binding property of b-lactosides in polymer II
may be explained in terms of the `isoreceptor ligand'28
for P-type adhesion which recognizes weakly not only
the Gb3 trisaccharide and Gb4 tetrasaccharide but also
the internal lactose moiety.
FITC-RCA120 lectin from Ricinus communis and its
non-labeled form were purchased from Sigma Chemical
Company. All other chemicals for the syntheses of
¯uorescence glycoconjugate polymers were purchased
from Wako Pure Chemical Industries Co., Ltd., and
Tokyo Kasei Kogyo in Tokyo. All E. coli O-157
strains studied here are non-pathogenic ones producing
no Shiga-like toxins. The cultures of each E. coli O-157
strain (H7, H45, and NM) were kindly o€ered by
Professor M. Ohta of Medical School of Nagoya Uni-
versity. Syntheses of ¯uorescence monomer 12 and
¯uorescent glycoconjugate polymers I±III were per-
formed as follows.
p-Nitrophenyl 30,40-O-isopropylidene-ꢀ-lactoside 2. Tri-
methylchlorosilane (16 mL) was added to a suspension
of p-nitrophenyl b-lactoside (4 g, 8.6 mmol) in acetone
(50 mL). The mixture was stirred for 4 h under N2
stream. The mixture was azeotroped repeatedly with
toluene:EtOH (20:1, mL) to a€ord 2 (4.2 g, 97%) as a
white crystal; [a]d 48.1ꢀ (c 1.0; MeOH); mp>290ꢀC
(decomposed); IR (cm 1): 1516 (NO2), 1086, 1065
1
(ketal). H NMR (d ppm, 500 MHz, CDCl3): 8.15 and
Conclusion
7.13 (dÂ2, 4H of pNP group), 5.18 (d, 1H, J1,2=7.8 Hz,
GlcH-1), 4.40 (d, 1H, J1,2=8.3 Hz, GalH-1), 4.24
(broad-s, 1H, GalH-4), 4.10 (broad-dd, 1H, GalH-3),
1.42 and 1.26 (sÂ2, 6H CH3 of isopropylidene).
FABMS: 656 [M+m-nitrobenzylalcohol+1]+.
We have shown a general synthetic approach to a ¯uor-
escence-labeled glycoconjugate polymer carrying the
Gb3 trisaccharide segment. The ¯uorescence monomer
can be used as a `seasoning' in the radical copolymer-
ization to prepare ¯uorescence-labeled glycoconjugate
polymers in aqueous solution. The strong and stable
¯uorescence intensity allowed us to investigate the
adhesion activity of these polymers onto E. coli O-157
strains as a preliminary test to speculate on the existence
of adhesins. The test has revealed that the polymer
carrying Pk-antigenic trisaccharides has high binding
anity with every E. coli O-157 strain studied here.
Moreover, the same polymer showed potent neutraliza-
tion activity against Shiga-like toxin-I for human
ACHN cells but no activity against Shiga-like toxin-II.
This means that the Gb3 trisaccharide is the real carbo-
hydrate ligand of Stx-I. For Stx-II, the lipophilic
ceramide structure of Gb3 may play a major role in the
toxin±Gb3 binding, or there may exist a certain oligo-
saccharide structure in globo- or ganglio-series as the
real carbohydrate ligand of Stx-II.
p-Nitrophenyl 2,3,6,20,60-penta-O-acetyl-ꢀ-lactoside 3.
Triethylamine (5.0 mL) and 4-N,N-dimethylaminopyr-
idine (10 mg) were added to a solution of 2 in Ac2O:
pyridine (25:50, mL) and stirred for 24 h. The mixture
was azeotroped with toluene:EtOH (10:1) and diluted
with CHCl3. The organic layer was washed with 1 N
aq HCl, satd aq NaHCO3, and water, dried over
MgSO4, and concentrated. After silica gel column pur-
i®cation, excess ethylene glycol (1 mL) was added to a
solution of penta-O-acetylated product (200 mg, 0.31
mmol) in CH2Cl2:MeOH (5:5, mL) followed by addi-
tion of trimethylchlorosilane (50 mL) and the solution
was stirred for 5 h. The mixture was concentrated and
diluted with CHCl3. The organic layer was washed with
water, dried over MgSO4, and concentrated. Puri®ca-
tion of the residue by silica gel column chromatography
(toluene:EtOAc=3:1) a€orded 3 (141 mg, 74%) as a
colorless crystal; [a]d 19.8ꢀ (c 1.0; CHCl3); mp 147±
150ꢀC; IR (cm 1): 3481 (OH), 1747, 1234 (Ac), 1522
Experimental
1
(NO2). H NMR (d ppm, 500 MHz, CDCl3): 8.20 and
1
Materials and Methods. H NMR (200 and 500 MHz)
7.06 (dÂ2, 4H, H of pNP group), 5.20 (d, 1H, J1,2=7.4
Hz, GlcH-1), 4.37 (d, 1H, J1,2=7.5 Hz, GalH-1), 3.87
(broad-d, 1H, GalH-4), 3.62 (broad-d, 1H, GaH-3),
2.15, 2.12, 2.09Â2, 2.06 (sÂ5, 15H, CH3 of acetyl group)
FABMS: 674 [M+1]+.
spectra were recorded at 40ꢀC in CDC13 using an
internal tetramethylsilane (TMS) standard unless other-
wise stated. Fluorescence spectra were measured with a
JASCO FP-550A spectro¯uorometer at room tempera-
ture. IR was measured in the form of KBr disc for solid
samples or ®lm on KBr for liquid samples. TLC was
performed on silica gel 60-F254 (Merck) detectable
under UV lamp or 5% H2SO4 in EtOH. Silica gel col-
umn chromatography was performed on silica gel 60
(Merck 0.063±0.200 mm) using toluene±ethyl acetate as
eluting solvents. N-p-Vinylbenzoyl b-lactosyl amine,2
racemic TBMB carboxylic acid26 and allyl a-d-galacto-
pyranoside29 were prepared by reported methods.
p-Nitrophenyl 2,3,6,20,60-penta-O-acetyl-30-O-pivaloyl-ꢀ-
lactoside 4. Pivaloyl chloride (0.43 mL, 3.5 mmol) was
added to a solution of compound 3 (1.00 g, 1.66 mmol),
triethylamine (0.68 mL, 2.49 mmol), and 4-N,N-di-
methylaminopyridine (10 mg) in CH2Cl2 (100 mL) at
0ꢀC and stirred for 1.5 h. The mixture was washed with
water, dried over MgSO4, and concentrated. Silica gel
column chromatography (toluene:EtOAc=5:1) a€orded

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H. Dohi et al. / Bioorg. Med. Chem. 7 (1999) 2053±2062
4 (1.00 g, 82%) as a colorless crystal; [a]d 19.8ꢀ (c 1.0;
6 h. The mixture was neutralized with Amberlyst 15E-ion
exchange resin, ®ltered, and concentrated. After silica
gel column puri®cation, the de-acylated product was
dissolved in 10 N HCl (10 mL, 100 mmol) and MeOH (5
mL) containing 10% Pd(OH)2 on carbon and hydro-
genated at room temperature for 6 h. The ®ltrate
through a pad of Celite was evaporated to a€ord 7 (29
mg, 92%) as a syrup; IR (cm 1) 3419 (OH), 1228, 1076
CH2Cl2); mp 175±179ꢀC; IR (cm 1): 3515 (OH), 1745,
1
1254 (Ac), 1707, 1232 (Piv), 1520 (NO2). H NMR (d
ppm, 500 MHz, CDCl3): 8.20 and 7.07 (dÂ2, 4H, H of
pNP group), 5.19 (d, 1H, J1,2=7.8 Hz, GlcH-1), 4.48 (d,
1H, J1,2=8.3 Hz, GalH-1), 4.84 (dd, 1H, J2,3=10.0 Hz,
J3,4=3.0 Hz, GalH-3), 4.02 (broad-d, 1H, GalH-4), 2.12,
2.09Â2, 2.06, 2.04 (sÂ5, 15H, CH3, of acetyl group), 1.19
(s, 9H, CH3 of pivaroyl group). FABMS: 757 [M]+.
1
(NH2). H NMR (d ppm, 500 MHz, D2O): 6.83 and
6.64 (dÂ2, 4H, H of pAP group), 4.82 (d, 1H, J1,2=8.1
0
0
0
6-O-Acetyl-2,3,4-tri-O-benzyl-D-galactopyranosyl ¯uoride
5. 6-O-Acetyl-2,3,4-tri-O-benzyl-d-galactopyranose 11
was prepared starting from methyl d-galactopyranoside
according to conventional methods for benzylation
(benzylbromide, NaH in DMF), acid-catalyzed acetolysis
(0.1% H2SO4, in acetic anhydride for 2 h), and deacetyl-
ation using 2-aminoethanol in DMSO±ethyl acetate mix-
ture(roomtemperature,48h).Thecrudesyrupycompound
11 was used for the next 1-¯uorination as follows.
Hz, GlcH-1), 4.77 (d, J1 ,2 =3.2 Hz, GalH-1 ), 4.35 (d,
1H, J1,2=7.7 Hz, GalH-1). FABMS: 749 [M+m-nitro-
benzylalcohol+1]+.
p-N-Acryloylamidophenyl O-[ꢁ-D-galactopyranosyl-(1!4)-
ꢀ-D-galactopyranosyl]-(1!4)-ꢀ-D-glucopyranoside 8. A
solution of 7 (25 mg, 40 mmol) and triethylamine (22 mL,
120 mmol) in MeOH (3 mL) was cooled at 0ꢀC. After 10
min, acryloyl chloride (4 mL, 50 mmol) in CH2Cl2 (0.5
mL) was added, and the mixture was stirred at room
temperature for 1.5 h. The mixture was concentrated
and puri®ed by TSK gel HW-40S column chroma-
tography (water) to a€ord 8 (21 mg, 83%) as a white
amorphous powder; IR (cm 1) 3298 (OH), 1660
(amide), 1033, 827 (ole®n). 1H NMR (d ppm, 500 MHz,
D2O): 7.54 and 7.23 (dÂ2, 4R aromatic H), 6.48 (dd,
1H, H-ole®n), 6.40 (dd, 1H, H-trans), 5.95 (dd, 1H,
Hexa¯uoropropene±diethylamine complex (Tokyo Kasei,
0.29 mL, 3.5 mmol) was added to a solution of 6-O-
acetyl-2,3,4-tri-O-benzyl-d-galactopyranose (300 mg,
0.53 mmol) in toluene (15 mL) at 0ꢀC and stirred for
2 h. The mixture was washed with satd aq NaHCO3
and water, dried over MgSO4, and concentrated. Silica gel
column chromatography (toluene:EtOAc=20:1) a€or-
1
ded 5 (289 mg, 95%; a/b=70/30 determined H NMR)
H-cis), 5.19 (d, 1H, J1,2=7.8 Hz, GlcH-1), 5.15 (d,
0
1
0
0
as a syrup; H NMR (d ppm, 500 MHz, CDCl3): 7.25±
J1 ,2 =3.6 Hz, GalH-1 ), 4.62 (d, 1H, GalH-1). FABMS:
7.44 (m, 15H, aromatic H), 5.73 and 5.47 (dÂ2, 1H,
J1,2=3.3 Hz, J1,F=53 Hz, H-1a), 5.32 and 5.06 (dÂ2,
1H, J1,2=6.7 Hz, J1,F=53 Hz, H-1b).
803 [M+m-nitrobenzylalcohol+1]+.
Allyl 6-O-TBMB carbonyl-ꢁ-D-galactopyranoside 12.
Racemic TBMB carboxylic acid (96.9 mg, 0.41 mmol)
was dissolved in CH2Cl2 (3 mL) and treated with hexa-
¯uoropropene±diethylamine complex (100.6 mg, 0.45
mmol) at room temperature for 30 min. After being
evaporated, the residue was puri®ed on silica gel column
(toluene:ethyl acetate=1:1) to a€ord syrupy TBMB
carbonyl ¯uoride16 (96.5 mg, 99%). To the mixture of
allyl a-d-galactopyranoside (100 mg, 0.45 mmol) and
4-N,N-dimethylaminopyridine (5 mg) in dry pyridine
(10 mL) was added TBMB carbonyl ¯uoride (96.5 mg,
0.41 mmol) in pyridine (1 mL). The mixture was kept at
room temperature for 6 h and concentrated in vacuo.
The residue was chromatographed on silica gel (toluene:
ethyl acetate=3:1 to 1:10). The fractions containing 12
were collected and concentrated. The residue was dis-
solved in water (15 mL) and then lyophilized to a€ord a
white glassy powder (62.4 mg, 45%); IR (cm 1) 3365
(OH), 1618, 1351, 1330 (PhC(O)OR), 1081, 1058 (ketal).
1H NMR (d ppm, 200 MHz, CDCl3): 7.34 (broad, 1H
J=8.8 Hz, Ph-H), 6.80±6.88 (m, 2H, Ph-H), 5.76±6.00
(m, 1H, C±CHˆC), 5.08±5.35 (m, 2H, C CˆCH2), 5.00
(d, 1H, J1,2=3.5 Hz, GalH-1), 4.4±4.7 (m, 2H, GalH-
6R,S), 1.62 (s, 3H, CH3 of ketal), 1.08 (s, 9H, tert-Bu of
ketal). FABMS: 439 [M+1]+. UV (H2O, 0.1 mM):
p-Nitrophenyl O-[6-O-acetyl-2,3,4-tri-O-benzyl-ꢁ-D-galacto-
pyranosyl-(1!4)-2,6-di-O-acetyl-3-O-pivaloyl-ꢀ-D-gal-
actopyranosyl)]-(1!4)-2,3,6-tri-O-acetyl-ꢀ-D-glucopyran-
oside 6. Flame-dried 4 A molecular sieves were added to
a stirred mixture of silver perchlorate (402 mg, 0.65 mmol)
and SnCl2 (368 mg, 0.65 mmol) in dry CH2Cl2 (10 mL).
The mixture was cooled to 0ꢀC and a solution of 4 (113
mg, 0.16 mmol) in dry CH2Cl2 (2.5 mL) was added.
After 10 min, a solution of 5 (278 mg, 0.49 mmol) in dry
CH2Cl2 (2.5 mL) was added, and the mixture was
stirred for 10 h. The mixture was ®ltered through a pad
of Celite. The ®ltrate was washed with satd aq NaHCO3
and water, dried over MgSO4, and concentrated. Silica
gel column chromatography (toluene:EtOAc=5:1)
a€orded 6 (124 mg, 62%) as a syrup; IR (cm 1): 1747,
1
1228 (Ac and Piv), 1519 (NO2). H NMR (d ppm, 500
MHz, CDCl3): 8.20 and 7.03 (dÂ2, 4H H of pNP),
7.15±7.44 (m, 15H, aromatic H of benzyl group), 4.60±
4.74 (dÂ6, 6H, CH2 of benzyl group), 5.16 (d, 1H,
0
0
J1,2=7.3 Hz, GlcH-1), 4.70 (d, 1H, J1 2 =1.6 Hz, GalH-
10), 4.47 (d, 1H, J1,2=8.3 Hz, GalH-1), 4.82 (dd, 1H,
J2,3=10.1 Hz, J3,4= 3.0 Hz, GalH-3), 3.84 (broad-s, 1H,
GalH-4), 2.06Â2, 2.03, 2.00Â2, 1.97 (sÂ6, 18H, CH3 of
acetyl group), 1.14 (s, 9H, CH3 of pivaloyl group).
FABMS: 1148 [M+1]+.
1
325 nm (e=5000, TBMB carboxylate Lb transition),
1
242 nm (shoulder), 222 nm (e=17000, La transition).
Fluorescence (H2O, 0.001 mM): Ex. (max) 325 nm, Em.
(max) 410 nm.
p-Aminophenyl O-[ꢁ-D-galactopyranosyl-(1!4)-ꢀ-D-gal-
actopyranosyl]-(1!4)-ꢀ-D-glucopyranoside 7. To a stirred
solution of 6 (60 mg, 50 mmol) in MeOH (3 mL) was
added NaOMe (10 mg), and the mixture was stirred for
Fluorescence-labeled glycoconjugate polymers I±III. A
solution of allyl a-d-galactopyranoside (40.2 mg, 0.18

H. Dohi et al. / Bioorg. Med. Chem. 7 (1999) 2053±2062
2061
mmol), acrylamide (64.7 mg, 0.91 mmol) and ¯uor-
escent monomer 12 (8 mg, 0.018 mmol) in water (2 mL)
was degassed under reduced pressure. To the mixture
was added N,N,N0,N0-tetramethylethylenediamine (55.3
mg) in one portion and then ammonium peroxo-
disulfate (12.7 mg). The mixture was again degassed,
and saturated with nitrogen gas, and the polymerization
tube was sealed under reduced pressure, and incubated
at 30ꢀC for 3 h. The solution was poured into methanol
(20 mL) in a centrifugation tube, and the mixture was
centrifuged (5ꢀC, 5000 rpm). The precipitate dissolved
in water was again poured into methanol. The pre-
cipitate was dissolved in water (20 mL), dialyzed for 2
days in water (Mw 3500 cut o€), and lyophilized to
a€ord a white powder of polymer I (77 mg, 68%); IR
(cm 1) 3392 (OH), 2933, 1668 (amide), 1617, 1350, 1327
and change in the ¯uorescence intensity at the con-
centration, respectively. The ¯uorescence measurement
was carried out for FITC-RCA lectin solution in 10
2
mM phosphate bu€er (1.04Â10 mg/mL, pH 7.2) at
room temperature (air-conditioned at 20ꢀC). After 0.3
mL aliquot of each polymer solution (10 mg/mL) was
added to the lectin solution, the mixture was stabilized
for 10 min and then the ¯uorescence spectra were mea-
sured. Attempts to determine the Ka based on the
¯uorescence change of TBMB carboxylate failed due to
the weak ¯uorescence change upon the lectin addition
(Fig. 3b).
Neutralization activity of glycoconjugate polymers I±III
against Stx-I and Stx-II. Non-¯uorescence polymers I±
III and polyacrylamide were employed for the assay.
1
(PhC(O)OR), 1078, 1058 (ketal). H NMR (d ppm, 500
The mixture of each Stx-I (250 pg/mL phosphate bu€er
2
MHz, D2O, 50ꢀC): 5.0 (H-1 of sugar), 3.6±4.1 (H-2±H-6
of sugar), 3.4 (-CH2-O-), 2.3±2.4 (-CH2-CH-, main
chain), 1.7±1.9 (-CH2-CH-, main chain). UV (H2O,
solution (PBS)) and the diluted polymer solution (10
±
10 14 mg/mL) was preincubated in a well at 37ꢀC for 1 h.
To the well were added ACHN cells (65 mL of 8Â105
cells/mL in Eagle's medium containing 10% FCS), and
the mixture was incubated at 37ꢀC for 3 days. The mix-
ture was treated with 0.014% neutral red solution (100
mL) at 37ꢀC for 1 h, washed with PBS (2Â200 mL), and
then mixed with 0.5 N HCl±35% EtOH (100 mL). The
absorbance at 540 nm was measured for each polymer
at di€erent concentrations.
1
1 mg/mL): 322 nm (TBMB carboxylate, Lb), 245 nm,
1
220 nm (TBMB carboxylate, La). Fluorescence (H2O,
0.1 mg/mL): Em. (max) 325 nm, Ex. (max) 409 nm. The
molar fractions of monomer components are summar-
ized in Table 1.
In the same manner as described above, polymers II and
III were prepared from N-p-vinylbenzoyl b-lactosyl
amine and p-acryloylamidophenyl 4-O-[3-O-a-d-galacto-
pyranosyl-b-d-galactopyranosyl]-b-d-glucopyranoside,
respectively. The molar ratio of every reagent and
monomer was summarized in Table 1. Polymer II: IR
(cm 1) 3370 (OH), 2931, 1670 (amide), 1617, 1349,
Adhesion activity of I±III onto E. coli O-157. Each of
the E. coli O-157 strains (OD660=1.5) in a medium
(3 mL) containing each of the polymer solutions
(0.1 mg/mL 0.1 N phosphate bu€er pH 7.2, 3 mL) was
incubated at 37ꢀC for 90 min. The mixture was cen-
trifuged (3000 rpm, 15 min), and the precipitate was
washed twice with the same bu€er. To the residue was
added 5.5 N NaOH (100 mL) and kept for 30 min for
solubilization. The phosphate bu€er (2.4 mL) was
added, and the ¯uorescence intensity at Ex. 320 nm and
Em. 410 nm was measured. The amount of ¯uorescence
polymers adsorbed onto each E. coli strain was deter-
mined using standard plots for the ¯uorescence intensity
versus the concentration of polymers (0.00±0.1 mg/mL).
1
(PhC(O)OR), 1078, 1058 (ketal). H NMR (d ppm, 500
MHz, D2O, 50ꢀC): 7.3±7.5 (Phenyl), 5.3 (GlcH-1), 4.6
(GalH-1), 3.7±4.0 (H-2±H-6 of sugars), 2.3±2.4 (-CH2-
CH-, main chain), 1.7±1.9 (-CH2-CH-, main chain).
Fluorescence (H2O, 0.1 mg/mL): Em. (max) 325 nm,
Ex. (max) 409 nm. Polymer III: IR (cm 1) 3394 (OH),
2931, 1668 (amide), 1618, 1351, 1328 (PhC(O)OR),
1
1078, 1059 (ketal). H NMR (d ppm, 500 MHz, D2O,
50ꢀC): 7.0±7.5 (Ph-H), 5.2 (GlcH-1b), 5.05 (GalH-1a),
4.4 (GalH-1b), 3.6±4.2 (H-2±H-6 of sugars), 2.3±2.6
(-CH2-CH-, main chain), 1.7±1.9 (-CH2-CH-, main
chain). Fluorescence (H2O, 0.1 mg/mL): Em. (max) 325
nm, Ex. (max) 409 nm.
Acknowledgements
This work is supported in part by grants from the Min-
istry of Education, Science, and Culture, Japan (Priority
Areas K. K.), and also from Takeda Science Founda-
tion and Tatematsu Foundation (Y. N.). The authors
are grateful to Dr Yasuo Suzuki of Shizuoka Pre-
fectural University for helpful discussions during this
study.
Association constants (Ka) of each glycoconjugate
polymer with RCA120 lectin. Based on the ¯uorescence
changes in FITC-labeled lectin (Ex. 490 nm, Em. 518
nm), Ka value was determined for ¯uorescence and non-
¯uorescence glycoconjugate polymers according to the
reported method10±13 and expressed as the value per
molar sugar unit. The molarity of the sugar unit was
calculated for each polymer as listed in Table 1. Stern±
Volmer plot (Fo Á‰SŠ=ÁF versus [S]) was employed to
determine the maximum ¯uorescence change ÁFmax and
Ka as follows:
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