Synthesis and antibody-binding studies of a series of parasite fuco-oligosaccharides

Article abstract of DOI:10.1016/j.bmc.2005.02.009

Complex multifucosylated oligosaccharides are structural elements of glycoprotein and glycolipid subsets of larval, egg, and adult stages of Schistosoma, the parasitic worms that cause schistosomiasis, a serious disease affecting more than 200 million peo

Full text of DOI:10.1016/j.bmc.2005.02.009

Bioorganic & Medicinal Chemistry 13 (2005) 3553–3564
Synthesis and antibody-binding studies of a series
of parasite fuco-oligosaccharides
Anne-Marie M. van Roon,a,b, Begon˜a Aguilera,c, Francisco Cuenca,c
a
c
a
´
Alexandra van Remoortere, Gijsbert A. van der Marel, Andre M. Deelder,
Herman S. Overkleeftc and Cornelis H. Hokkea,*
aDepartment of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, PO Box 9600,
2300 RC Leiden, The Netherlands
bDepartment of Biophysical Structural Chemistry, Leiden Institute of Chemistry, PO Box 9502, 2300 RA Leiden, The Netherlands
cDepartment of Bio-Organic Synthesis, Leiden Institute of Chemistry, PO Box 9502, 2300 RA Leiden, The Netherlands
Received 29 October 2004; accepted 8 February 2005
Available online 12 March 2005
Abstract—Complex multifucosylated oligosaccharides are structural elements of glycoprotein and glycolipid subsets of larval, egg,
and adult stages of Schistosoma, the parasitic worms that cause schistosomiasis, a serious disease affecting more than 200 million
people in the tropics. The fucosylated structures are thought to play an important role in the immunology of schistosomiasis.
Defined schistosomal oligosaccharides that enable immunological studies are difficult to obtain from natural sources. Therefore,
we have chemically synthesized spacer-linked GlcNAc, Fuca1-3GlcNAc, Fuca1-2Fuca1-3GlcNAc, and Fuca1-2Fuca1-2Fuca1-
3GlcNAc. This series of linear oligosaccharides was used to screen a library of anti-schistosome monoclonal antibodies by surface
plasmon resonance spectroscopy. Interestingly, the reactive antibodies could be grouped according to their specificity for the differ-
ent oligosaccharides tested, showing that these oligosaccharides form different immunological entities based on the number and link-
age of the fucose residues. Subsequently, the thus defined monoclonal antibodies were used to visualize the expression of the
corresponding oligosaccharide epitopes by adult Schistosoma mansoni worms.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
have provided evidence that carbohydrate structures
also play a role in the pathogenesis of the disease, for in-
stance in granuloma formation2–4 and in the character-
istic Th2 polarization of the cellular immune response
(for a review see Thomas and Harn).5 Obviously, it is
desirable to determine exactly which carbohydrate struc-
tures are involved in each of these processes. This may
lead to an improvement of current immunodiagnostic
methods and possibly to the development of carbohy-
drate based therapeutics.
Schistosomes are parasitic helminths that cause a
chronic, debilitating and some times fatal disease called
schistosomiasis, of which at least 200 million people in
the tropics suffer. Schistosomes have a complex life cycle
involving a fresh water snail intermediate host and a ver-
tebrate definitive host. Although the human definitive
host develops a strong immune response after infection,
this usually does not lead to elimination of the parasite
or sterile immunity. It is generally accepted that in a
schistosomal infection the major humoral immune re-
sponse is targeted against carbohydrates (for a review
see Hokke and Deelder).1 Furthermore, several studies
Numerous analytical studies, often based on sensitive
mass spectrometric approaches, have revealed that
schistosome glycosylation is extremely heterogeneous
and complex6 (and references cited therein). Several rela-
tively simple, well studied tri- or tetrasaccharide ele-
ments expressed on schistosome glycoconjugates are
the trisaccharide Lewis X (Lex, Galb1-4[Fuca1-3]Glc-
NAc),7 and the fucosylated LacdiNAc (GalNAcb1-
4GlcNAc, LDN) derivatives such as GalNAcb1-
4[Fuca1-3]GlcNAc (LDNF) and GalNAcb1-4[Fuca1-
Keywords: Schistosomiasis; Oligosaccharide synthesis; Monoclonal
antibodies; Immunolocalization.
*
Corresponding author. Tel.: +31 71 526 5065; fax: +31 71 526
6907; e-mail: c.h.hokke@lumc.nl
Both authors contributed equally.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.009

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A.-M. M. van Roon et al. / Bioorg. Med. Chem. 13 (2005) 3553–3564
2Fuca1-3]GlcNAc (LDNDF),8–10 of which the latter
structure contains a characteristic Fuca1-2Fuc se-
quence. Using neo-glycoconjugates obtained by en-
zyme-assisted synthesis, it has been established that in
schistosome-infected individuals or animals, an intense
antibody response is elicited against LDNDF.11,12 These
neo-glycoconjugates were also used to show that
LDNDF is a relatively strong inducer of IL-10 produc-
tion in peripheral blood mononuclear cells.13
Table 1. Structure and names of synthetic carbohydrates conjugated
to BSA via a spacer
Name
Abbreviation Symbol
Structures used in this screening
GlcNAc
Gn
2
α1-3
α1-3
Fuca1-3GlcNAc
FGn
FFGn
10
11
Fuca1-2Fuca1-3GlcNAc
α1-2
α1-3
Multifucosylated structures containing the unique
Fuca1-2Fuca1-2Fuca1-3GlcNAc FFFGn
15
α1-2
α1-2
β1-4
Fuca1-2Fuc linkage, such as
Fuca1-2Fuca1-3-
GalNAcb1-4[ Fuca1-2Fuca1-2Fuca1-3]GlcNAc, were
found on O-glycans from the glycocalyx of schistosomal
cercariae.14 The major egg glycolipids of Schistosoma
(S.) mansoni contain stretches of multifucosylated struc-
tures on a repeating b1-4 linked GlcNAc backbone.15
Using defined monoclonal antibodies (Mabs) it has been
determined by immunofluorescence and blotting tech-
niques that, as part of the LDNDF antigen, also in adult
worms the Fuca1-2Fuc element occurs.16,10
β1-4
Galb1-4[Fuca1-3]GlcNAcb1-
3Galb1-4Glc
LNFPIII
LDNDF
β1-3
α1-3
Other structures discussed
β1-4
α1-3
GalNAcb1-4[Fuca1-2Fuca1-
3]GlcNAc
α1-2
β1-4
α1-3
β1-4
Fuca1-3GalNAcb1-4GlcNAc
FLDN
It is extremely difficult to obtain pure compounds from
complex, heterogeneous glycoconjugate mixtures of lim-
ited natural sources like schistosome larvae, adult
worms, or eggs. Therefore, it is of importance to be able
to obtain potentially immunogenic carbohydrates via
chemical synthesis.17,18 Well-defined synthetic carbohy-
drate structures allow direct, detailed studies of their
immunogenicity and are useful as probes in the charac-
terization of antibodies. Previously, we have obtained
Fuca1-3GalNAcb1-4[Fuca1-
3]GlcNAc
FLDNF
α1-3
α1-3
j GlcNAc, n Fuc, h GalNAc, d Gal, s Glc.
provided with an amino spacer to allow (after deprotec-
tion) conjugation to a carrier protein. The donor of
choice was ethyl 2-O-tert-butyldimethylsilyl-3,4-O-iso-
propylidene-1-thio-b-D-fucopyranoside. The TBDMS
group can be selectively removed in the presence of
the other protecting groups, after coupling to the
acceptor.
numerous
schistosome-related
anti-carbohydrate
Mabs.19–21 These Mabs are valuable tools in the immu-
nodiagnosis of schistosomiasis.22,20 In addition, anti-
carbohydrate Mabs can be used to visualize the
expression of their corresponding antigens throughout
the different life stages of the parasite.10,16,23–25 Using
a stepwise synthetic scheme, we have now chemically
synthesized a series of linearly fucosylated spacer-linked
oligosaccharides containing 0, 1, or 2 of the specific a1-
2-linked Fuc residues: GlcNAc(Gn 2), Fuca1-3GlcNAc
(FGn, 10), Fuca1-2Fuca1-3GlcNAc (FFGn, 11), and
Fuca1-2Fuca1-2Fuca1-3GlcNAc (FFFGn, 15) (Table
1). These oligosaccharides were used to screen a large li-
brary of anti-schistosomal Mabs by surface plasmon
resonance (SPR) spectroscopy. Subsequently, the thus
characterized Mabs served as probes to detect their cor-
responding antigens in adult S. mansoni worms by fluo-
rescence microscopy.
The synthesis of 2 and acceptor 3 was accomplished as
follows (Scheme 1). 2-N-acetyl-(3,4,6-tri-O-acetyl-1,2-
dideoxy-a-D-glucopyrano)-[2,1-d]-2-oxazoline26
was
treated with 6-N-benzyloxycarbonyl-hexanol in the pres-
ence of TMSOTf to afford compound 1 (79%). Subse-
quent de-O-acetylation, followed by the removal of the
6-N-benzyloxycarbonyl protecting group by hydrogeno-
lysis, afforded deprotected spacer-linked 2 (Gn) in 91%
yield. Alternatively, the introduction of a 4,6-O-benzyl-
idene group gave glucosamine acceptor 3 in 92% yield.
Fucosyl donor 4 was obtained from ethyl 3,4-O-isopro-
pylidene-1-thio-b-D-fucopyranoside by silylation with
TBDMSCl and imidazole (89%). Condensation of
acceptor 3 with donor 4 was performed under the Oga-
wa conditions, which have been reported to be suitable
for the selective formation of a-fucosidic linkages.27
Thus, reactions of compound 3 with 4 promoted by cop-
per(II) bromide and tetrabutylammonium bromide gave
disaccharide 5 in 85%. Removal of the TBDMS protect-
ing group with TBAF in THF afforded disaccharide
acceptor 6 which was condensed with donor 4 under
the same conditions as described above to give trisac-
charide 7 (52%, a/b 12:1). In this case, the reaction
was not completely stereoselective and the a/b mixture
could be separated by silica gel chromatography. In
analogy, trisaccharide 7 was partially deprotected to
2. Results
2.1. Synthesis of the fucosylated carbohydrate structures
The protected oligosaccharides were assembled using a
stepwise strategy. Starting from an appropriately pro-
tected glucosamine acceptor, the target di-, tri-, and tet-
rasaccharide can be obtained by repeated elongation
with a suitable fucose donor carrying a temporary pro-
tective group at 2-OH. The selected glucosamine accep-
tor, 6-[N-(benzyloxycarbonylamino)hexyl]-2-acetamido-
4,6-O-benzylidene-2-deoxy-b-D-glucopyranoside
was

A.-M. M. van Roon et al. / Bioorg. Med. Chem. 13 (2005) 3553–3564
3555
moted by tetrabutylammonium bromide gave disaccha-
ride donor 18 in 68% yield. Donor 18 and disaccharide
acceptor 6 were condensed in the presence of copper(II)
bromide and tetrabutylammonium bromide to give tet-
rasaccharide 13 in 52% as a a/b mixture (2.3:1), which
could be separated by column chromatography. The
overall yield of the preparation of tetrasaccharide 13
using the linear strategy was 7.2% (starting from disac-
charide 6), while the convergent strategy afforded tetra-
saccharide 13 in 24.5% overall yield.
OH
O
OR1
O
HO
HO
a
O(CH2)6NH2
R2O
R3O
O(CH2)6NHZ
NHAc
2
NHAc
SEt
O
1: R1, R2, R3 = Ac
3: R1, R2 = CHPh, R3 = H
OTBDMS
b
O
O
4
c
Ph
O
O
O
OH
O
O
O(CH2)6NHZ
NHAc
2.2. Preparation of neoglycoconjugates and coupling to a
Biacore sensor chip
HO
O
O
O(CH2)6NH2
OR
e
NHAc
O
O
O
OH
10
OH
HO
Glycosides 2, 10, 11, and 15 were coupled to bovine ser-
um albumin (BSA) to obtain neoglycoproteins, which
could then easily be immobilized onto a SPR chip
(CM5 sensor chip, Biacore). To this end, glycosides 2,
10, 11, and 15 were transformed into their correspond-
ing squaric acid amide esters by reaction with diethyl
squarate in MeOH/phosphate (1:1), pH 7. The subse-
quent coupling to BSA proceeded in 0.1 M NaHCO3,
pH 9. Twenty molar equivalents of glycoside were added
to BSA and the reaction was allowed to proceed for
5 days. The average coupling degree of the sugars to
BSA was determined with MALDI-TOF MS (Table 2).
5: R = TBDMS
6: R = H
d
4, c
O
O
Ph
OH
O
O
O
O(CH2)6NHZ
HO
O
O(CH2)6NH2
NHAc
NHAc
O
O
e
O
O
O
O
OH
HO
O
OR
O
OH
O
O
OH
HO
11
The neoglycoconjugates were immobilized on a Biacore
CM5 sensor chip via the standard peptide chemistry
method (Biacore user manual) to a level of approxi-
mately 10,000 response units (RU). BSA was conjugated
on one flow channel of each of the sensor chips to serve
as a control for non-specific binding and to correct for
the background signal.
7: R = TBDMS
d
8: R = H
O
4, c
OTBDMS
9: R=
O
O
Scheme 1. Reagents and conditions: a. (1) NaOMe, MeOH; (2) H2,
Pd/C, EtOH, HCl 1 M, rt 91 %; b. (1) NaOMe, MeOH, rt; (2)
(MeO)2CHPh, p-TsOH, CH3CN, rt, 92%. c. CuBr2, Bu4NBr, DMF–
CH2Cl2 (1:1), 5, 85%; 7, 52% (a/b 12:1); 9, 17%. d. TBAF, THF, rt, 6,
88%; 8, 94%. e. (1) HOAc 60%, 50 ꢁC; (2) H2, Pd/C, EtOH, HCl 1 M,
10, 68%; 11, 45%.
2.3. Binding of Mabs to BSA neoglycoconjugates by
surface plasmon resonance
A panel of 831 Mabs, all previously derived from fu-
sions with spleen cells of mice infected with schisto-
somes,21 was screened for interaction with the
neoglycoconjugates on the SPR sensor chip. The Mabs,
which interacted with one or more of the neoglycopro-
teins are listed in Table 3. The concentration of each
of these Mabs was determined by ELISA and responses
were re-examined at a standardized concentration of
5 lg mLꢀ1 to be able to compare the relative binding
capacities. The positive Mabs were grouped according
to their interaction with one or more neoglycoconju-
gates (Table 3). Mabs from group I specifically inter-
acted with Gn, whereas Mabs from group II
recognized the FGn structure. Furthermore, some Mabs
were found that interacted with both structures contain-
ing the schistosome-specific Fuca1-2Fuc linkage. Final-
ly, one Mab was found that bound only to the
tri-fucosyl structure (group IV). None of the Mabs inter-
acted with BSA (data not shown). As an additional
control, each of the Mabs of group I–IV was tested
for interaction with lacto-N-fucopentaose III (LNFPIII,
Table 1), a fuco-pentasaccharide containing the Lex tri-
saccharide at its terminus. None of the Mabs in Table 3
interacted with LNFPIII (data not shown).
give acceptor 8. Chain elongation of trisaccharide 8 with
donor 4, afforded tetrasaccharide 9 in only 17% yield.
Treatment of compounds 6 and 7 with acetic acid 60%
at 50 ꢁC and subsequent hydrogenolysis under Pd/C in
the presence of traces of acid, furnished unprotected
disaccharide 10 (FGn) and trisaccharide 11 (FFGn),
respectively. Unfortunately, tetrasaccharide 9 decom-
posed when treated with acetic acid 60% at 50 ꢁC.
Therefore, another fucosyl donor, ethyl 2,3,4-tri-O-benz-
yl-1-thio-b-D-fucopyranoside28 was coupled to trisac-
charide 8 under Ogawa conditions (route A, Scheme
2). Under these conditions, tetrasaccharide 13 could be
obtained in 16% yield. The use of other activators (for
instance, Br2, Bu4NBr, or AgOTf) did not afford better
results. Tetrasaccharide 13 could be successfully depro-
tected by acid treatment followed by hydrogenolysis to
give compound 15 (FFFGn). In order to improve the
synthesis of tetrasaccharide 13, a 2+2 block strategy
(route B, Scheme 2) was considered as alternative for
the stepwise strategy. To this end, coupling of bromide
16, prepared from thioethyl donor 12, with ethyl
3,4-O-isopropylidene-1-thio-b-D-fucopyranoside 17 pro-

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A.-M. M. van Roon et al. / Bioorg. Med. Chem. 13 (2005) 3553–3564
route B
route A
Ph
O
O
Ph
O
O
Ph
O
O
O
O
O
O
O
O
O(CH2)6NHZ
NHAc
O(CH2)6NHZ
NHAc
O(CH2)6NHZ
NHAc
O
O
a
O
O
O
O
OH
O
O
O
O
O
O
SEt
OBn
O
6
O
O
OH
e
O
O
O
OBn
O
O
8
BnO
OBn
12
OBn
SEt
BnO
O
O
13
O
O
O
OBn
b
OBn
BnO
OH
O
18
Br
HO
O
O
O(CH2)6NHR2
O
NHAc
O
OBn
d
O
O
OBn
BnO
OH
HO
16
O
OH
HO
SEt
OH
O
OR1
OR1
O
R1O
O
17
14: R1 = Bn; R2 = Z
15: R1, R2 = H
c
Scheme 2. Reagents and conditions: a. CuBr2, Bu4NBr, DMF–CH2Cl2 (1:1), 16%. b. HOAc 60%, 50 ꢁC, 57%. c. H2, Pd/C, EtOH, HCl 1 M, 70%. d.
(1) 12, Br2, CH2Cl2, 0 ꢁC; (2) Bu4NBr, DMF–CH2Cl2, rt, 68%. e. CuBr2, Bu4NBr, DMF–CH2Cl2 (1:1), 52% (a/b 2.3:1).
Table 2. Mass spectrometric data indicating the coupling degree of
glycosides to BSA
Table 3. Interaction of Mabs with neoglycoconjugates by surface
plasmon resonance
Glycoside
Mass (Da) of BSA
neoglycoconjugate
Average coupling
degree
Maba
Isotype
Gn
FGn
FFGn
FFFGn
Group I
2
70,688
72,700
73,158
74,441
11
11
10
9
114-2B5-A
114-4C1-A
292-6G2-A
294-4C4-A
314-1G5-A
ndb
IgM
nd
++
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
10
11
15
+++
+
IgM
IgM
+++
+++
Group II
128-1E7-Cc
128-3G12-A
291-3A4-Ac
IgM
IgG3
IgM
—
—
—
+
+
+
—
—
—
—
—
—
2.4. Immunofluorescence assays
Next, the identified Mabs were used to locate their cor-
responding natural antigens in the schistosome. To this
end, frozen liver sections that contain eggs and worms,
prepared from hamsters infected with S. mansoni, were
probed with Mabs of each of the four groups in an
immunofluorescence microscopy assay. Typical staining
patterns for each group are presented in Figure 1. Inter-
estingly, Mab 294-4C4-A only interacted with the tegu-
mental outer region of the adult worm, in contrast to the
other Mabs, which stained structures inside the parasite.
The staining patterns for Mabs from group II and III
are rather similar and resemble the duct-related patterns
specified in a previous study by Thors and Linder.29 It
seems that the staining of Mab 291-3A4-A is more de-
fined than that of Mab 258-3E3. Finally, staining with
Mab 114-5B1-A resulted in completely different fluores-
cence patterns of canal like structures which have been
previously described as part of the excretory system of
the adult worms.30
Group III
257-4D8
258-3E3
IgM
IgM
—
—
—
—
++
++
+
++
Group IV
114-5B1-Ad
IgG1
—
—
—
+++
a The Mabs were screened at a concentration of 5 lg/mL, + represents
100–500 RU after a 5 min dissociation period, ++ represents 500–
1000 RU and +++ represents >1000 RU.
b Isotype of the antibody has not been determined.
c Also interacts with Fuca1-3GalNAcb1-4[Fuca1-3]GlcNAc (FLDNF)
(Vermeer, personal communication).
d Also
(LDNDF).10
interacts
with
GalNAcb1-4[Fuca1-2Fuca1-3]GlcNAc
well known where and when these structures are ex-
pressed and, most importantly, which elements exactly
confer the different immunogenic properties to the
glycoconjugates.
This is especially true for particular multifucosyl-
ated oligosaccharides that contain Fuca1-2Fuc and
Fuca1-2Fuca1-2Fuc elements that are a1-3-linked to a
GlcNAc moiety, which seem to be characteristic for
schistosomes.14,15 We have synthesized a set of linear
3. Discussion
Schistosomes produce a wide variety of immunogenic
fucosylated carbohydrate structures.1 Thus far it is not

A.-M. M. van Roon et al. / Bioorg. Med. Chem. 13 (2005) 3553–3564
3557
Figure 1. Immunofluorescence staining of frozen sections of S. mansoni-infected hamster livers incubated with Mabs from the four different groups:
(a) 294-4C4-A, (b) 291-3A4-A, (c) 258-3E3, and (d) 114-5B1-A. Different structures within the worms were stained by Mabs from the different
groups. Staining patterns of Mabs from group II and III are rather similar, however not identical. It cannot be determined from these pictures which
exact cellular structures the two Mabs 291-3A4-A and 258-3E3 bind. In contrast, staining with Mab 114-5B1-A (group IV) resulted in a completely
different fluorescence pattern. Canal like structures were stained, which have previously been described as part of the excretory system of the adult
worms.30 Incubation with fresh culture medium as a negative control did not result in fluorescence of the sections (data not shown).
oligosaccharides containing this Fuca1-2Fuc element
and investigated the interaction with a large panel of
anti-schistosomal Mabs.
Fuca1-3GlcNAc. The third group of Mabs binds to oli-
gosaccharides with at least two terminal fucoses in
Fuca1-2Fuc linkage, the common element in FFGn
and FFFGn. These Mabs did not interact with the
Fuca1-3GlcNAc structure, indicating that in this struc-
tural context a single terminal fucose is not sufficient
for binding. Moreover, the fucoses should be at a termi-
nal position in a linear structure as these Mabs did not
interact with branched LDNDF either (Vermeer, per-
sonal communication). Mab 114-5B1-A from group IV
significantly interacted only with the trifucosylated
FFFGn, indicating that this Mab requires two consecu-
tive a1-2 linked fucoses.
A stepwise synthetic approach was used successfully to
synthesize the desired mono- and difucosylated com-
pounds FGn and FFGn. For the synthesis of the final
target structure FFFGn, a 2+2 block synthetic strategy
showed to be more efficient. In both strategies thioglyco-
side donors were activated with CuBr2/Bu4NBr, a
method shown to be very selective for the formation
of a-glycosidic linkages,27 although in our hands this
method was not always completely stereoselective.
Interestingly, for all of the tested structures one or more
Mabs were found (Table 3). The Mabs could be divided
into four groups. Mabs from group I selectively inter-
acted with Gn, suggesting that terminal GlcNAc is an
antigenic structure. This is somewhat surprising since
GlcNAc is ubiquitously expressed in higher as well as
lower organisms. In mammals however, GlcNAc usually
occurs as an internal monosaccharide rather than a ter-
minal residue. Mabs from group II bound to FGn (for
an overview of the discussed carbohydrate structures
see Table 1). The disaccharide Fuca1-3GlcNAc is also
part of the Lex trisaccharide antigen. However, none
of the FGn-positive Mabs bound to Lex (in the context
of LNFPIII), suggesting that the additional b1-4-linked
Gal in Lex blocks the interaction of the Mabs with
Some of the presented Mabs have been found to bind to
other carbohydrate structures as well. Mabs 128-1E7-C
and 291-3A4-A from group II cross-react with FLDNF
(Vermeer, personal communication), suggesting they
recognize a fucose a1-3 linked to HexNAc, since both
Fuca1-3GalNAc (terminal disaccharide of FLDNF)
and Fuca1-3GlcNAc (FGn) are bound. However, it
may be expected that clear quantitative differences in
the binding of these cross-reacting Mabs to their respec-
tive antigens occur. Unfortunately, we cannot obtain
quantitative SPR data with the current experimental set-
up, and therefore it is not possible to elaborate on the
affinity and kinetic characteristics of the Mabs with
respect to their different possible binding partners.
However, in a direct comparison of the binding to

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A.-M. M. van Roon et al. / Bioorg. Med. Chem. 13 (2005) 3553–3564
FLDNF- and FLDN-conjugates by SPR, 128-1E7-C
gave an at least 3-fold better response for FLDNF than
for FLDN, which indicates that F-LDN-F is the more
accurate representation of the epitope of 128-1E7-C.
This conclusion is further supported by the observation
that pre-incubation with keyhole limpet haemocyanin
(KLH), which expresses the F-LDN-F element,31 inhib-
its the binding of 128-1E7-C to schistosome glycolip-
ids.16 Notably, Mab 114-5B1-A was previously shown
to bind to LDNDF.10 In the current study, Mab 114-
5B1-A interacted with FFFGn. Initial binding of 114-
5B1-A to FFGn could also be observed, but the SPR
response returned to backgrounds levels within the 5-
min equilibration period before measurement, indicating
that 114-5B1-A has an insignificant, but detectable affin-
ity for FFGn. From the primary carbohydrate sequence
these characteristics are difficult to explain. Therefore
we have examined the spatial orientation of these carbo-
hydrate structures using a web-based tool (Sweet)32 de-
signed to construct three-dimensional models of
carbohydrates. The overlay of the models of each struc-
ture (Fig. 2) suggests clearly that the terminal Fuca1-
2Fuc moiety in the linear, curve-shaped FFFGn is ori-
ented similarly as the Fuca1-2Fuc element in the
branched LDNDF, thus explaining why Mab 114-5B1-
A can bind both FFFGn and LDNDF. In FFGn how-
ever, this terminal Fuca1-2Fuc moiety may not be well
available for binding by 114-5B1-A since the model of
FFGn overlays with only the first three reducing-end
residues of FFFGn (not shown).
antibodies could be mapped and fine-tuned. It should
be noted that neoglycoconjugate structures might only
be limited representations of the natural glycoprotein
and -lipid structures expressed by the parasites. The
Mabs described in this study are however very useful
tools to discriminate between different fucosylated
structures with small but relevant structural and
(immuno)biological differences.
The expression of the fucosylated carbohydrate struc-
tures within the adult worms was studied by immunoflu-
orescence assays, similar to previous studies on other
sets of related fucosylated and non-fucosylated glyco-
conjugates.30,23,33,16,10 The results of the current micro-
scopic study show major differences between the
staining patterns obtained with Mabs from each of the
groups, except group II and III (Fig. 1). The Mabs
may for instance be applied as immuno-affinity tools
to purify glycoproteins or glycolipids carrying prede-
fined glycosylation patterns from crude schistosome ex-
tracts. In conclusion, the presented results provide a
good starting point for further studies to assess the role
of these carbohydrate structures in the immunology of
schistosomiasis, utilizing both the synthesized structures
and the corresponding Mabs.
4. Experimental section
4.1. General
By combining the data obtained from this and previous
studies using related oligosaccharide structures, the epi-
topes of a number of anti-schistosomal monoclonal
TLC analysis was performed on Merck Alumina silica
gel F254 plates, with detection by means of UV absorp-
tion (254 nm) and/or charring with ammonium molyb-
date (25 g/L) and ceric ammonium sulfate (10 g/L) in
10% aq H2SO4 or a ninhydrine solution or 0.2% orcinol
in 20% methanolic H2SO4. Column chromatography
was performed with Fluka Silica Gel (230–400 mesh).
Unless otherwise indicated, all the reagents were pur-
chased from commercial sources and used without fur-
ther purification. EtOAc and Pet. Ether 60–80 were of
technical grade and distilled before used. CH2Cl2
was distilled over CaH2, the rest of the solvents were
of p.a. grade (Baker) and stored over molecular
1
sieves. H and 13C NMR spectra were recorded on a
Bruker AC 200 (200 and 50 MHz), Bruker AV 400
(400 and 100 MHz) or Bruker DMX 600 (600 and
150 MHz) as indicated. Optical rotations were measured
on a Propol automatic polarimeter (sodium D line
k = 589 nm). High resolution mass spectra were re-
corded on a Q-star Applied Biosystems Q-TOF instru-
ment (TOF-section). MALDI-TOF mass spectrometric
analyses were performed on a Reflex III mass spectro-
meter or an Ultraflex TOF/TOF mass spectrometer
(Bruker Daltonics).
4.2. 6-[N-(Benzyloxycarbonylamino)hexyl]-2-acetamido-
3,4,6-tri-O-acetyl-2-deoxy-b-D-glucopyranoside (1)
Figure 2. Overlay of modeled structures of FFFGn (gray) and
LDNDF (black). The Fuca1-2Fuc Moiety of LDNDF overlays almost
perfectly with the terminal Fuca1-2Fuc moiety of FFFGn, this might
be the explanation for the binding of Mab 114-5B1-A to both these
structures. The picture was created using PyMol Molecular Graphics
System (DeLano, W.L. 2002, http://www.pymol.org).
A mixture of known26 2-methyl-(3,4,6-tri-O-acetyl-1.2-
dideoxy-a-D-glucopyrano)-[2,1-d]-2-oxazoline (4.67 g,
14.2 mmol) and 6-N-(benzoyloxycarbonylamino)-1-hex-
anol (5.3 g, 21.11 mmol) was coevaporated three times

A.-M. M. van Roon et al. / Bioorg. Med. Chem. 13 (2005) 3553–3564
3559
with 1,2-dichloroethane, and then dissolved in dry
˚
CH2Cl2 (170 mL). Molecular sieves 4 A were added
were added. The reaction mixture was stirred overnight
at room temperature and then neutralized with triethyl-
amine and evaporated. The crude was purified by col-
umn chromatography (CH2Cl2–MeOH 30:1) to give 3
(0.86 g, 92%). Rf (CH2Cl2–MeOH 10:1): 0.65. [a]D
and the mixture stirred at room temperature for
15 min. Then TMSOTf (0.52 mL, 2.82 mmol) was added
and stirring was continued for 40 h. The reaction mix-
ture was then neutralized with triethylamine and washed
with aq satd NaHCO3 and water, dried (MgSO4), fil-
tered, and evaporated. The crude was purified by col-
umn chromatography (Pet. Ether–EtOAc 1:1) to give 1
(6.53 g, 79%). Rf (CH2Cl2–MeOH 10:1): 0.65. [a]D
ꢀ13.4 (c 2.5, CH2Cl2). Lit. [a]D ꢀ12.7 (c 1, CH3Cl).
1H NMR (400 MHz, CDCl3): d 7.35–7.28 (m, 5H, Ar),
6.28 (d, 1H, J = 8.7 Hz, NHAc), 5.29 (t, 1H,
J = 9.9 Hz, H-3), 5.15–5.05 (m, 4H, CH2Ph, H-4,
NHZ), 4.63 (d, J = 8.2 Hz, H-1), 4.24 (dd, 1H, J =
4.6 Hz, J = 12.3 Hz, H-6), 4.10 (br d, 1H, J = 12.1 Hz,
H-6), 3.83 (m, 2H, CH2O, H-2), 3.63 (m, 1H, H-5),
3.46 (m, 1H, CH2O), 3.18 (m, 2H, CH2NH), 2.07 (s,
3H, Ac), 2.01 (s, 6H, 2 Ac), 1.92 (s, 3H, Ac), 1.53 (m,
4H, (CH2)2), 1.32 (m, 4H, (CH2)2). 13C NMR ( CDCl3):
d 170.5, 170.2, 169.2 (CO), 156.4 (CONH), 136.4, 128.2,
127.7, 127.6 (Ar), 100.2 (C-1), 77.2, 71.3, 68.5 (C-3, C-4,
C-5), 69.0, 66.2 (C-6, CH2Ph), 61.9 (OCH2), 54.3 (C-2),
40.3 (CH2NH), 29.4, 28.7, 25.7, 24.9 ((CH2)4), 22.9 (Ac),
20.4 (Ac).
1
ꢀ50.2 (c 1, DMF). Lit. [a]D ꢀ49.2 (c 1.5, DMF). H
NMR (400 MHz, CDCl3): d 7.51–7.31 (m, 10H, Ar),
5.55 (s, 1H, CHPh), 5.55 (br s, 1H, NHAc), 5.08 (br s,
2H, CH2Ph), 4.61 (d, 1H, J = 8.3 Hz, H-1 GlcNAc),
4.32 (dd, 1H, J = 4.9 Hz, J = 10.4 Hz, H-6), 3.92–3.37
(m, 7H, OCH2, H-2, H-3, H-4, H-5, H-6), 3.16 (m,
2H, CH2NH), 1.99 (s, 3H, Ac), 1.59–1.48 (m, 4H,
(CH2)2), 1.34 (m, 4H, (CH2)2). 13C NMR (CDCl3–
CD3OD 10:1): d 136.8, 128.9, 128.2, 127.9, 127.8,
127.6, (Ar), 101.5 (CHPh), 101.1 (C-1), 81.4, 70.9, 65.9
(C-3, C-4, C-5), 70.0 (C-6), 68.4 (CH2Ph), 66.3
(OCH2), 57.1 (C-2), 40.5 (CH2NH), 29.4, 28.6, 25.9,
22.5 (–(CH2)4–), 22.5 (CH3).
4.5. Ethyl 2-O-tert-Butyldimethylsilyl-3,4-O-isopropylid-
ene-1-thio-b-L-fucopyranoside (4)
A solution of ethyl 3,4-O-isopropylidene-1-thio-b-D-
fucopyranoside34 (1.71 g, 6.89 mmol), imidazole
(1.09 g,
16.18 mmol)
and
TBDMSCl
(1.62 g,
10.75 mmol) in DMF (10 mL) was stirred at room tem-
perature for 16 h and then evaporated. The residue was
dissolved in EtOAc (200 mL) and washed with aq satd
NaHCO3 and brine, dried (MgSO4), and evaporated.
The crude was purified by column chromatography
(Pet. Ether–EtOAc 30:1) to give 4 (2.24 g, 89%). Rf
(Pet. Ether–EtOAc 20:1): 0.55. [a]D +18.7 (c 1.1,
4.3. 6-Aminohexyl-2-acetamido-2-deoxy-b-D-glucopyr-
anoside (2)
Compound 1 (25 mg, 0.043 mmol) was treated with
NaOMe in MeOH (0.033 M, 3 mL) at rt for 15 h. The
mixture was then neutralized with Amberlite-Hꢁ, fil-
tered, and evaporated. The crude was dissolved in EtOH
90% in water (1.5 mL) and HCl 1 M (38 lL), Pd/C 10%
(36 mg) was added and the reaction mixture was stirred
overnight under H2 atmosphere. The reaction was fil-
tered over Hyfloꢂ and evaporated. The crude was puri-
fied by Gel permeation chromatography on a Superdex
Peptide PE 7.5/300 column (NH4HCO3 0.025 M in
water) to give 2 (11 mg, 73%). 1H NMR (600 MHz,
D2O): d 4.48 (d, J = 8.5 Hz, 1H, H-1 GlcNAc), 3.91–
3.86 (m, 2H, H-6, OCH2), 3.72–3.70 (m, 1H, H-2),
3.69 (dd, 1H, J = 5.2 Hz, J = 9.8 Hz, H-6 GlcNAc),
3.60 (t, 1H, J = 8.7 Hz, H-3), 3.55 (m, 1H, OCH2),
3.48 (t, 1H, J = 8.7 Hz, H-4), 3.42 (m, 1H, H-5 Glc-
NAc), 2.95 (t, 2H, J = 7.6 Hz, CH2NH), 1.99 (s, 3H,
Ac) 1.52 ( m, 2H, CH2), 1.45 (m, 2H, CH2), 1.30 (m,
4H, (CH2)2). 13C NMR (200 MHz, D2O): d 175.2
(CO), 102.6 (C-1 GlcNAc), 77.6, 75.7, 71.8 (C-3, C-4,
C-5 GlcNAc), 70.2 (CH2O), 62.5 (C-6 GlcNAc), 57.1
(C-2 GlcNAc), 40.4 (CH2NH), 29.9, 28.2, 26.8, 26.4
1
CH2Cl2). H NMR (200 MHz, CDCl3): d 4.27 (d, 1H,
J = 9.5 Hz, H-1), 4.02 (dd, 1H, J = 5.8 Hz, J = 2.2 Hz,
H-3), 3.93 (m, 1H, H-4), 3.80 (m, 1H, H-5), 3.54 (q,
1H, J = 9.5 Hz, J = 5.8 Hz, H-2), 2.64 (m, 2H, CH2SEt),
i
i
1.50 (s, 3H, CH3 Pr), 1.38–1.27 (m, 9H, CH3 Pr,
CH3Fuc, CH3SEt).13C NMR (50 MHz, CDCl3): d
109.1 (Cq Pr), 84.7 (C-1), 80.3, 76.3, 73.5, 72.01 (C-2,
i
i
C-3, C-4, C-5), 27.8, 26.2 (CH3 Pr), 25.7 (CH3 BuSi),
t
t
24.0 (CH2SEt), 17.9 (Cq BuSi), 16.6 (CH3Fuc), 14.6
(CH3SEt).
4.6. 6-[N-(Benzyloxycarbonylamino)hexyl]-2-acetamido-
4,6-O-benzylidene-2-deoxy-3-O-(2-O-tert-butyldimethyl-
silyl-3,4-O-isopropylidene-a-L-fucopyranosyl)-b-D-gluco-
pyranoside (5)
Acceptor 3 (334 mg, 0.63mmol) and donor 4 (280 mg,
0.77 mmol) were coevaporated together three times with
toluene, dissolved in DMF–CH2Cl2 (1:1, 8 mL) and stir-
red at room temperature in the presence of activated
((CH2)4), 22.8 (Ac). MALDI-TOF m/z: 321.
[M+H]+, 343.2 [M+Na]+.
3
˚
molecular sieves 4 A for 1 h. Bu4NBr (656 mg,
2.04 mmol) was added and 1 h later CuBr2 (456 mg,
2.04 mmol), and stirring was continued for 48 h at room
temperature. The reaction mixture was diluted with
CH2Cl2, filtered over Hyflo, washed three times with
aq satd NaHCO3 and brine, dried (MgSO4), filtered
and concentrated. The crude was purified by column
chromatrography (Pet. Ether–EtOAc 20:1!1:1) to give
5 (454 mg, 85 %). Rf (DCM–MeOH 40:1): 0.5. [a]D
4.4. 6-[N-(Benzyloxycarbonylamino)hexyl]-2-acetamido-
4,6-O-benzylidene-2-deoxy-b-D-glucopyranoside (3)
Compound 1 (6.53 g, 11.25 mmol) was treated with
NaOMe in MeOH (0.033 M, 300 mL) at rt for 15 h.
The mixture was then neutralized with Amberlite-Hꢁ,
filtered, and evaporated. The crude was dissolved in
CH3CN (8.5 mL) and benzaldehyde dimethyl acetal
(0.7 mL, 4.64 mmol) and p-TsOH (90 mg, 0.49 mmol)
1
ꢀ85.6 (c 0.53, CH2Cl2). H NMR (400 MHz, CDCl3):
d 7.49–7.33 (m, 10H, Ar), 5.93 (d, 1H, J = 7.5 Hz,

3560
A.-M. M. van Roon et al. / Bioorg. Med. Chem. 13 (2005) 3553–3564
NHAc), 5.50 (s, 1H, CHPh), 5.10 (s, 2H, CH2Ph), 4.98
(d, 1H, J = 2.9 Hz, H-1 Fuc), 4.86 (m, 1H, NHZ), 4.73
(d, 1H, J = 8.0 Hz, H-1 GlcNAc), 4.40 (m, 1H, H-5
Fuc), 4.32 (m, 2H, H-6 GlcNAc, H-3 GlcNAc), 4.08
(t, 1H, J = 6.5 Hz, H-3 Fuc), 3.91 (dd, 1H, J = 2.5 Hz,
J = 5.7 Hz, H-4 Fuc), 3.83 (m, 1H, OCH2), 3.73 (m,
2H, H-6 GlcNAc, H-2 Fuc), 3.59 (m, 2H, H-2 GlcNAc,
H-4 GlcNAc), 3.46 (m, 2H, OCH2, H-5 GlcNAc), 3.18
(m, 2H, CH2N), 1.98 (s, 3H, Ac), 1.49 (m, 4H,
(1.5 mL) and HCl 1 M (38 lL), Pd/C 10% (36 mg) was
added and the reaction mixture was stirred overnight
under H2 atmosphere. The reaction was filtered over
Hyfloꢂ and evaporated. The crude was purified by
HW-40 (NH4HCO3 0.15 M in water) to give 10
(14 mg, 68%). 1H NMR (600 MHz, D2O): d 4.96 (d,
J = 4.1 Hz, 1H, H-1 Fuc), 4.48 (d, J = 8.6 Hz, 1H, H-1
GlcNAc), 4.30 (m, 1H, H-5 Fuc), 3.91–3.86 (m, 2H,
H-6 GlcNAc, OCH2), 3.81–3.76 (m, 3H, H-3 Fuc, H-4
Fuc, H-2 GlcNAc), 3.72 (dd, 1H, J = 5.7 Hz, J =
12.2 Hz, H-6 GlcNAc), 3.67 (dd, 1H, J = 4.1 Hz, J =
10.4 Hz, H-2 GlcNAc), 3.60 (t, 1H, J = 8.7 Hz,, H-3
GlcNAc), 3.55 (m, 1H, OCH2), 3.48 (t, 1H, J = 8.7 Hz,
H-4 GlcNAc), 3.44 (m, 1H, H-5 GlcNAc), 2.64 (t, 2H,
J = 7.1 Hz, CH2NH), 1.99 (s, 3H, Ac) 1.52 (m, 2H,
CH2), 1.45 (m, 2H, CH2), 1.30 (m, 4H, (CH2)2), 1.13
(d, J = 6.6 Hz, 3H, CH3Fuc). 13C NMR (D2O): d
175.2 (CO), 101.7 (C-1 GlcNAc), 100.7 (C-1 Fuc),
81.2, 76.6, 72.6, 70.3, 69.4, 68.8, 67.7 (C-2, C-3, C-4,
C-5 Fuc, C-3, C-4, C-5 GlcNAc), 71.3 (CH2O), 61.6
(C-6 GlcNAc), 56.1 (C-2 GlcNAc), 41.7 (CH2NH),
29.3, 26.4, 25.6 ((CH2)4), 23.0 (Ac), 15.9 (CH3Fuc).
MALDI-TOF m/z: 467.25 [M+H]+, 489.26 [M+Na]+.
i
i
(CH2)2), 1.46 (s, 3H, CH3 Pr), 1.32 (s, 3H, CH3 Pr),
1.30 (s, 3H, CH3), 1.08 (d, 1H, J = 6.6 Hz, CH3Fuc),
t
0.92 (s, 9H, BuSi), 0.14 (s, 3H, CH3Si), 0.11 (s, 3H,
CH3Si). 13C NMR (CDCl3): d 170.1 (CO), 137.1,
128.7, 128.3, 127.9, 127.8, 125.8 (Ar), 108.4 (CqiPr),
101.1 (CHPh), 100.9 (C-1), 97.8 (C-10), 80.6, 76.1,
75.8, 74.3, 71.9, 65.9, 63.6, (C-3, C-4, C-5, C-20, C-30,
C-40, C-50) 57.1 (C-2), 69.4, 68.6 (CH2Ph, C-6), 66.2
(OCH2), 40.5 (CH2NH), 28.9 (CH2), 25.7 (tBuSi,
i
CH3 Pr), 25.1 (CH2), 19.5 (Ac), 17.9 (CqSi), 16.3
(CH3Fuc). HRMS m/z: calcd for C44H67N2O12Si
[M+H]+: 843.4463; found 843.4464.
4.7. 6-[N-(Benzyloxycarbonylamino)hexyl]-2-acetamido-
4,6-O-benzylidene-2-deoxy-3-O-(3,4-O-isopropylidene-a-
L-fucopyranosyl)-b-D-glucopyranoside (6)
4.9. 6-[N-(Benzyloxycarbonylamino)hexyl]-2-acetamido-
4,6-O-benzylidene-2-deoxy-3-O-[3,4-O-isopropylidene-2-
O-(2-O-tert-butyldimethylsilyl-3,4-O-isopropylidene-a-L-
fucopyranosyl)-a-L-fucopyranosyl]-b-D-glucopyranoside
(7)
Disaccharide 5 (372 mg, 0.44 mmol) was treated with
TBAF (1 M in THF, 0.9 mL) under N2 for 16 h at rt.
The reaction mixture was then evaporated, the residue
dissolved in EtOAc and washed with NaHCO3 and
brine, dried (MgSO4), filtrated, and evaporated. The
crude was purified by column chromatography
(EtOAc–MeOH 30:1!15:1) to give 6 (285 mg, 88%).
Rf (DCM–MeOH 25:1): 0.25. [a]D ꢀ105.6 (c 1.2,
CH2Cl2). 1H NMR (400 MHz, CDCl3): d 7.47–7.26
(m, 10H, Ar), 6.75 (d, 1H, J = 8.3 Hz, NHAc), 5.46 (s,
1H, CHPh), 5.11 (m, 2H, CH2Ph), 4.95 (br s, 1H,
NHZ), 4.87 (d, 1H, J = 3.3 Hz, H-1 Fuc), 4.68 (d, 1H,
J = 8.1 Hz, H-1 GlcNAc), 4.32 (m, 2H, H-5 Fuc, H-6
GlcNAc), 4.06 (m, 2H, H-3 Fuc, H-3 GlcNAc), 3.87–
3.73 (m, 4H, OCH2, H-2 GlcNAc, H-4 Fuc, H-6 Glc-
NAc), 3.57 (m, 2H, H-2 Fuc, H-4 GlcNAc), 3.48–3.42
(m, 2H, H-5 GlcNAc, OCH2), 3.19 (m, 2H, CH2NH),
1.98 (s, 3H, Ac), 1.50 (m, 4H, (CH2)2), 1.46 (s, 3H,
Acceptor 6 (430 mg, 0.59 mmol) and donor 4 (278 mg,
0.77 mmol) were coevaporated together three times with
toluene, dissolved in DMF–CH2Cl2 (1:1, 7.5 mL) and
stirred at room temperature in the presence of activated
˚
molecular sieves 4 A for 1 h. Bu4NBr (643 mg,
1.99 mmol) was added and 1 h later CuBr2 (444 mg,
1.99 mmol), and stirring was continued for 4 days at
room temperature. The reaction mixture was diluted
with CH2Cl2, filtered over Hyflo, washed three times
with aq satd NaHCO3 and brine, dried (MgSO4), filtered
and concentrated. The crude was purified by column
chromatrography (Pet. Ether–EtOAc 7:1!0:1) to give
a-7 (291 mg), b-7 (24 mg), yield 52%, a/b = 12:1. Rf
(DCM–MeOH 40:1): 0.45. [a]D ꢀ96.5 (c 1.5, CH2Cl2).
1H NMR (400 MHz, CDCl3): d 7.5–7.2 (m, 10H, Ar),
6.0 (br s, 1H, NHAc), 5.5 (s, 1H, CHPh), 5.09 (s, 2H,
CH2Ph), 5.03 (d, 1H, J = 2.7 Hz, H-1 Fuc), 4.94 (d,
1H, J = 3.3 Hz, H-1 Fuc), 4.74 (d, 1H, J = 8.3 Hz, H-1
GlcNAc), 4.50 (m, 1H, H-5 Fuc), 4.45 (m, 1H, H-5
Fuc), 4.33 (m, 1H, H-6 GlcNAc), 4.19–4.09 (m, 4H,
H-3 Fuc, H-3 GlcNAc, H-4 Fuc, F-4 Fuc), 3.95 (m,
1H, H-4 Fuc), 3.85–3.74 (m, 4H, OCH2, H-6, H-2
Fuc, H-2 Fuc), 3.64 (m, 1H, H-2 GlcNAc), 3.53 (m,
1H, H-4 GlcNAc), 3.46 (m, 2H, H-5 GlcNAc, OCH2),
3.18 (m, 2H, CH2NH), 2.01 (s, 3H, Ac), 1.49–1.30 (m,
i
i
CH3 Pr), 1.32 (m, 4H, (CH2)2), 1.29 (s, 3H, CH3 Pr),
0.79 (d, 1H, J = 6.6 Hz, CH3Fuc). 13C NMR (CDCl3):
d 170.6, 156.5 (CO), 137.0, 129.1, 128.4, 128.1, 127.9,
i
127.7, 126.2, 126.1 (Ar), 108.7 (Cq Pr), 101.9 (CHPh),
100.3 (C-1 GlcNAc), 99.7 (C-1 Fuc), 80.1, 77.4, 76.4,
75.9, 70.2, 66.1, 63.3 (C-3, C-5, C-5 GlcNAc, C-2, C-3,
C-4, C-5 Fuc), 69.0, 68.7 (C-6, CH2Ph), 66.5 (OCH2),
56.8 (C-2 GlcNAc), 40.4 (CH2NH), 29.6, 28.7, 25.6,
i
24.8 ((CH2)4), 28.1, 26.1 (CH3 Pr), 23.2 (Ac), 15.5
(CH3Fuc). HRMS m/z: calcd for C38H53N2O12
[M+H]+: 729.3598; found 729.3640.
i
15H, 4CH3 Pr, CH3Fuc), 1.08 (d, 3H, J = 6.6 Hz,
t
4.8. 6-Aminohexyl-2-acetamido-2-deoxy-3-O-(a-L-fuco-
pyranosyl)-b-D-glucopyranoside (10)
CH3Fuc), 0.89 (s, 3H, BuSi), 0.14 (s, 6H, CH3Si). 13C
NMR (50 MHz, CDCl3): d 170.6, 156.1 (CO), 137.1,
128.8, 128.4, 128.0, 127.9, 126.0 (Ar), 108.6, 108.4
i
(Cq Pr), 101.2 (CHPh, C-1 GlcNAc), 97.3 (C-1 Fuc),
Disaccharide 5 (25 mg, 0.044 mmol) was treated with
acetic acid 60% (10 mL) at 50 ꢁC for 6 h. The reaction
mixture was then concentrated coevaporating with tolu-
ene. The crude was dissolved in EtOH 90% in water
97.1 (C-1 Fuc), 81.0, 76.4, 75.9, 74.5, 72.1, 66.0, 63.7,
63.4 (C-3, C-4, C-5 GlcNAc, C-2, C-3, C-4, C-5 Fuc,
C-2, C-3, C-4, C-5 Fuc), 69.7, 68.6 (CHPh, C-6), 66.0

A.-M. M. van Roon et al. / Bioorg. Med. Chem. 13 (2005) 3553–3564
3561
(OCH2), 56.8 (C-2 GlcNAc), 40.7 (CH2NH), 29.6, 29.1,
25.3, ((CH2)2), 28.2 (CH3 Pr), 26.1 (CH3 Pr), 23.6 (Ac),
4H, (CH2)2), 1.17 (m, 6H, 2CH3Fuc). 13C NMR
(50 MHz, CD3OD): d 173.6 (CO), 102.3 (C-1 GlcNAc),
97.4 (C-1 Fuc), 97.3 (C-1 Fuc), 80.3, 77.5, 73.9, 73.6,
73.5, 71.6, 71.0, 70.1, 69.2, 68.1, 67.4, 62.4 (C-3, C-4,
C-5 GlcNAc, C-2, C-3, C-4, C-5 Fuc, C-2, C-3, C-4,
C-5 Fuc), 69.7 (C-6 GlcNAc), 62.4 (OCH2), 56.6 (C-2
GlcNAc), 40.5 (CH2NH), 29.8, 28.1, 26.7, 26.4
((CH2)2), 23.2 (Ac), 16.3 (CH3-Fuc). MALDI-TOF m/z
613.30 [M+H]+, 635.32 [M+Na]+.
i
i
t
18.4 (Cq BuSi), 16.1, 16.0 (CH3Fuc), ꢀ3.8 (CH3Si),
ꢀ4.2 (CH3Si). HRMS m/z: calcd for C53H84N3O16Si
[M+NH4]+: 1046.5620; found 1046.5668.
4.10. 6-[N-(Benzyloxycarbonylamino)hexyl]-2-acetamido-
4,6-O-benzylidene-2-deoxy-3-O-[3,4-O-isopropylidene-2-
O-(3,4-O-isopropylidene-a-L-fucopyranosyl)-a-L-fucopyr-
anosyl]-b-D-glucopyranoside (8)
4.12. Ethyl 3,4-O-isopropylidene-2-O-(2,3,4-tri-O-benzyl-
a-L-fucopyranosyl)-1-thio-b-L-fucopyranoside (18)
Trisaccharide 7 (52.8 mg, 0.05 mmol) was treated with
TBAF (1 M in THF, 100 ll) under N2 for 6 h at rt
The reaction mixture was then evaporated, the residue
dissolved in EtOAc and washed with NaHCO3 and
brine, dried (MgSO4), filtrated, and evaporated. The
crude was purified by column chromatography (Pet.
Ether–EtOAc 1:4) to give 8 (42 mg, 94%). Rf (DCM–
MeOH 20:1): 0.5. [a]D ꢀ128.9 (c 0.7, CH2Cl2). 1H
NMR (400 MHz, CDCl3): d 7.46–7.34 (m, 10H, Ar),
5.47 (s, 1H, CHPh), 5.10 (br s, 2H, CH2Ph), 5.03 (br
s, 1H, H-1 Fuc), 4.89 (s, 1H, H-1 Fuc), 4.58 (m, 1H,
H-5 Fuc), 4.43 (m, 2H, H-5 Fuc, H-1 GlcNAc), 4.33
(m, 1H, H-6 GlcNAc), 4.22 (m, 1H, H-3 Fuc), 4.12
(m, 3H, OCH2, H-3 Fuc, H-4 Fuc), 3.89–3.73 (m, 7H,
H-2 Fuc, H-2 Fuc, H-4 Fuc, H-3 GlcNAc, H-4 GlcNAc,
H-6 GlcNAc, OCH2), 3.52 (m, 2H, H-2 GlcNAc, H-5
GlcNAc), 3.20 (m, 2H, CH2NH), 1.95 (s, 3H, Ac),
Bromine (45 lL, 0.88 mmol) was added to a solution of
ethyl 2,3,4-tri-O-benzyl-1-thio-b-L-fucopyranosyl (382
mg, 0.8 mmol) in CH2Cl2 (7.7 mL) at 0 ꢁC. After
10 min, the mixture was evaporated, coevaporating
twice with CH2Cl2 to give crude bromide 16. Acceptor
17 was coevaporated with toluene (three times), dis-
solved in DMF (3.7 mL) under N2, and stirred in the
˚
presence of activated molecular sieves 3 A for 15 min
at rt. Bu4NBr (159 mg, 0.49 mmol) was added followed
by the crude bromide dissolved in CH2Cl2 (2 mL), and
stirring was continued overnight at rt. The mixture
was filtrated, and the filtrated washed with aq satd NaH-
CO3, water, and brine, dried (MgSO4), and evaporated.
The crude was purified by column chromatography Pet.
Ether–EtOAc 6:1!3:1 (1% Et3N), to give 18 (178
mg, 68%). [a]D ꢀ90 (c 1.1, CH2Cl2). 1H NMR (400
MHz, CDCl3): d 7.42–7.26 (m, 15H, Ar), 5.51 (d, 1H,
J = 3.6 Hz, H-10), 4.99–4.62 (m, 6H, 3 CH2Ph), 4.43
(d, 1H, J = 10.2 Hz, H-1), 4.24 (br q, 1H, J = 6.1 Hz,
J = 12.7 Hz, H-5), 4.15 (t, J = 5.7 Hz, H-3), 4.08 (dd,
1H, J = 3.7 Hz, J = 10.2 Hz, H-20), 4.01 (dd, 1H,
J = 1.9 Hz, J = 5.4 Hz, H-4), 3.96 (dd, 1H, J = 2.7 Hz,
J = 10.2 Hz, H-30), 3.81–3.70 (m, 3H, H-2, H-40, H-50),
i
1.49–1.25 (m, 23H, 4CH3 Pr, CH3-Fuc, (CH2)2), 0.91
(d, 3H, J = 5.1 Hz, CH3-Fuc). 13C NMR (50 MHz,
CDCl3): d 170.5, 156.5 (CO), 137.1, 128.9, 128.4,
128.1, 128.0, 127.9, 126.1 (Ar), 108.8, 108.4 (Cq Pr),
i
101.5 (CHPh), 99.9 (C-1 GlcNAc), 95.7 (C-1 Fuc),
95.3 (C-1 Fuc), 80.7, 76.4, 76.1, 75.9, 74.5, 74.1, 73.6,
71.0, 65.7, 62.7 (C-3, C-4, C-5 GlcNAc, C-2, C-3, C-4,
C-5 Fuc, C-2, C-3, C-4, C-5 Fuc), 69.7, 68.8 (CHPh,
C-6), 66.6 (OCH2), 58.7 (C-2 GlcNAc), 40.5 (CH2NH),
i
2.70 (m, 2H, CH2SEt), 1.54–1.11 (m, 15H, 2CH3 Pr,
i
29.5, 28.9, 25.7, 25.1 ((CH2)2), 28.3 (CH3 Pr), 26.3
(CH3 Pr), 23.3 (Ac), 15.9, 15.6 (CH3Fuc). HRMS m/z:
2CH3Fuc, CH3SEt). 13C NMR (CDCl3): d 138.9,
138.6, 138.4 (C-ipso), 128.2, 128.0, 127.3, 127.2 (Ar),
i
calcd for C47H67N2O16 [M+H]+: 915.4490; found
915.4450.
109.3 (Cq Pr), 96.7 (C-10), 83.5 (C-1), 79.1, 78.8, 77.7,
i
76.1, 74.1, 72.9, 72.1, 66.3 (C-2, C-3, C-4, C-5, C-20,
C-30, C-40, C-50), 74.6, 73.1 (CH2Ph), 29.5 (CH2SEt),
i
4.11. 6-Aminohexyl-2-acetamido-2-deoxy-3-O-[2-O-(a-L-
fucopyranosyl)-a-L-fucopyranosyl]-b-D-glucopyranoside
(11)
26.4, 24.1 (CH3 Pr), 16.7, 16.3 (CH3Fuc, CH3Fuc0),
14.5 (CH3SEt). HRMS m/z: calcd for C38H52NO8S
[M+NH4]+: 682.3413; found 682.3460.
Compound 8 (38 mg, 0.04 mmol) was treated with
AcOH 60% (2.4 mL) at 50 ꢁC for 8 h, and then evapo-
rated to dryness with the aid of toluene. The crude
was dissolved in EtOH 90% in water (2.7 mL), and
HCl 1 M (60 lL), Pd/C 10% was added and the reaction
mixture was stirred under H2 atmosphere for 4 h, fil-
trated over Hyflo, and evaporated. The residue was
purified by gel filtration chromatography HW-40
(NH4HCO3 0.15 M in H2O) to give 11 (11 mg,
45%). [a]D ꢀ126.4 (c 0.5, CH3OH). 1H NMR
(400 MHz, CD3OD): d 5.25 (d, 1H, J = 2.7 Hz, H-1
Fuc), 4.94 (d, 1H, J = 3.1 Hz, H-1 Fuc), 4.45 (d, 1H,
J = 8.3 Hz, H-1 GlcNAc), 4.32 (m 1H, H-5 Fuc), 4.27
(m, 1H, H-5 Fuc), 3.88–3.39 (m, 14H, H-2, H-3, H-4,
H-4, 2H-6 GlcNAc, H-2, H-3, H-4 Fuc, H-2, H-3, H-4
Fuc), 2.90 (t, 1H, J = 7.4 Hz, CH2NH), 2.00 (s, 3H,
Ac), 1.63 (m, 2H, CH2), 1.54 (m, 2H, CH2), 1.39 (m,
4.13. 6-[N-(Benzyloxycarbonylamino)hexyl]-2-acetamido-
4,6-O-benzylidene-2-deoxy-3-O-{3,4-O-isopropylidene-2-
O-[3,4-O-isopropylidene-2-O-(2,3,4-tri-O-benzyl-a-L-
fucopyranosyl)-a-L-fucopyranosyl]-a-L-fucopyranosyl}-
b-D-glucopyranoside (13)
Route A: Acceptor 8 (60 mg, 0.065 mmol) and donor 12
(47 mg mg, 0.097 mmol) were coevaporated together
three times with toluene, dissolved in DMF–CH2Cl2
(1:1, 1 mL) and stirred at room temperature in the pres-
˚
ence of activated molecular sieves 4 A for 1 h. Bu4NBr
(81.3 mg, 0.25 mmol) was added and 1 h later CuBr2
(58.8 mg, 0.25 mmol), and stirring was continued for
48 h at room temperature. The reaction mixture was di-
luted with CH2Cl2, filtered over Hyflo, washed three
times with aq satd NaHCO3 and brine, dried (MgSO4),
filtered and concentrated. The crude was purified by

3562
A.-M. M. van Roon et al. / Bioorg. Med. Chem. 13 (2005) 3553–3564
column chromatrography (Pet. Ether–EtOAc 2:1!1:1)
to give 13 (14 mg, 16%) and recovered acceptor 8 (75%).
129.1, 128.9, 128.5, 128.3 (Ar), 101.7 (C-1 GlcNAc),
96.4 (C-1 Fuc), 94.9 (C-1 Fuc), 94.0 (C-1 Fuc), 79.9,
79.1, 77.6, 76.8, 73.3, 73.0, 70.5, 69.6, 69.5, 68.1, 68.0
(C-3, C-4, C-5 GlcNAc, C-2, C-3, C-4, C-5 Fuc1, 2,
3), 75.9, 73.7, 70.5, 67.0 (CH2Ph, C-6), 62.7 (OCH2),
57.7 (C-2 GlcNAc), 41.5 (CH2NH), 30.6, 30.2, 27.2,
26.5 (CH2), 23.3 (Ac), 16.7, 16.2 (CH3Fuc). HRMS m/z:
calcd for C61H83N2O20 [M+H]+: 1163.5539; found
1163.5562.
Route B: Acceptor 6 (42 mg, 0.057 mmol) and donor 18
(50 mg, 0.075 mmol) were coevaporated together three
times with toluene, dissolved in DMF–CH2Cl2 (1:1,
710 lL) and stirred at room temperature in the presence
˚
of activated molecular sieves 4 A for 1 h. Bu4NBr
(62 mg, 0.19 mmol) was added and 1 h later CuBr2
(43 mg, 0.19 mmol), and stirring was continued for
5 days at room temperature. The reaction mixture was
diluted with CH2Cl2, filtered over Hyflo, washed three
times with aq satd NaHCO3 and brine, dried (MgSO4),
filtered, and concentrated. The crude was purified by
column chromatrography (Pet. Ether–EtOAc 3:1!1:1)
to give a-13 (27 mg, 36%), b-13 (12 mg, 16%), yield
4.15. 6-Aminohexyl-2-acetamido-2-deoxy-3-O-{2-O-[2-
O-(a-L-fucopyranosyl)-a-L-fucopyranosyl]-a-L-fucopyr-
anosyl}-b-D-glucopyranoside (15)
Compound 14 (8 mg, 0.007 mmol) was dissolved in
EtOH 90% in water (0.5 mL), and HCl 1 M (12 lL),
Pd/C 10% was added and the reaction mixture was stir-
red under H2 atmosphere for 4 h, filtrated over Hyflo,
and evaporated to give 15 (3.7 mg, 70%). 1H NMR
(600 MHz, D2O): d 5.50 (d, J = 3.6 Hz, 1H, H-1 Fuc),
5.18 (d, J = 3.8 Hz, 1H, H-1 Fuc), 5.14 (d, J = 3.4 Hz,
1H, H-1 Fuc), 4.40 (d, J = 8.4 Hz, 1H, H-1 GlcNAc),
4.30 (br q, 1H, H-5 Fuc), 4.25 (br q, 1H, H-5 Fuc),
4.06 (br q, 1H, H-5 Fuc), 4.00 (m, 2H, H-6 GlcNAc,
OCH2), 3.93–3.78 (m, 11H, C-2, C-3 GlcNAc, C-2, C-
3, C-4 Fuc 1, 2, 3), 3.58 (t, 1H, J = 9.1 Hz, H-4 Glc-
NAc), 3.51 (m, 1H, OCH2), 3.44 (m, 1H, H-5), 2.95 (t,
2H, J = 7.5 Hz, CH2NH), 2.00 (s, 3H, Ac), 1.61 (m,
2H, CH2), 1.51 (m, 2H, CH2), 1.31 (m, 4H, CH2), 1.19
(m, 9H, 3CH3Fuc). 13C NMR (D2O): d 174.9 (CO),
102.3 (C-1 GlcNAc), 96.9 (C-1 Fuc), 93.6 (C-1 Fuc),
92.4 (C-1 Fuc), 76.3, 75.8, 72.9, 72.7, 72.4, 71.1, 70.6,
68.9, 68.8, 68.6, 68.5, 67.8, 67.7, 67.5 (C-3, C-4, C-5 Glc-
NAc, C-2, C-3, C-4, C-5 Fuc1, 2, 3), 71.2.(C-6), 61.5
(OCH2), 56.2 (C-2 GlcNAc), 40.2 (CH2NH), 29.2,
27.5, 26.1, 25.5 (CH2), 23.2 (Ac), 16.1, 16.1, 16.0
(CH3Fuc). MALDI-TOF m/z 759.4 [M+H]+, 781.4
[M+Na]+.
1
52%, a/b = 2.25:1. [a]D ꢀ89.0 (c 0.2, CHCl3). H NMR
(600 MHz, CDCl3): d 7.35–7.16 (m, 25H, Ar), 6.64 (d,
1H, J = 7.2 Hz, NHAc), 5.38 (s, 1H, CHPh), 5.19 (br
s, 1H, H-1 Fuc), 5.11–5.07 (m, 4H, H-1 Fuc, H-1 Fuc,
CH2Ph), 4.93–4.61 (m, 8H, 3CH2Ph, H-1 GlcNAc,
NHZ), 4.56 (m, 1H, H-3 GlcNAc), 4.45–4.43 (m, 2H,
2H-5 Fuc), 3.20 (m, 1H, H-3 Fuc), 4.25 (dd, 1H,
J = 10.4 Hz, J = 4.6 Hz), 4.14 (m, 4H, H-2 Fuc, H-3
Fuc, H-4 Fuc, H-5 Fuc), 3.95 (dd, 1H, J = 10.1 Hz,
J = 2.7 Hz, H-3 Fuc), 3.83 (m, 1H, H-4 Fuc), 3.79–
3.64 (m, 5H, H-2 Fuc, H-2 Fuc, H-6 GlcNAc, H-4
Fuc, OCH), 3.48–3.43 (m, 2H, H-5 GlcNAc, H-4 Glc-
NAc), 3.33 (m, 2H, H-2 GlcNAc, OCH), 3.12 (m, 2H,
CH2NH), 1.97 (s, 3H, Ac), 1.60–1.21 (m, 23H, (CH2)4,
i
4CH3 Pr, CH3Fuc), 1.13 (d, 1H, J = 6.4 Hz, CH3 Fuc),
0.95 (d, 1H, J = 6.6 Hz, CH3Fuc). 13C NMR (CDCl3):
d 171.0 (CO), 138.6, 138.4, 138.3, 137.1, 128.9, 128.3,
128.2, 128.0, 127.6, 127.3, 127.0, 126.1 (Ar), 108.7,
i
108.4 (Cq Pr), 101.6 (CHPh), 100.1 (C-1 GlcNAc), 95.8
(C-1 Fuc), 95.9 (C-1 Fuc), 93.7 (C-1 Fuc), 81.1, 79.1,
75.9, 75.7, 75.2, 74.6, 74.2, 74.1, 66.7, 65.9, 62.2 (C-3,
C-4, C-5 GlcNAc, C-2, C-3, C-4, C-5 Fuc1, 2, 3), 72.5,
72.3, 69.2, 68.7 (CH2Ph, C-6), 60.2 (OCH2) 58.0 (C-2
GlcNAc), 40.7 (CH2NH), 29.5, 29.1, 26.1 (CH2), 28.3,
4.16. General procedure for the derivatization of glyco-
sides with diethyl squarate
i
28.1, 25.2 (CH3 Pr), 23.6 (Ac), 16.0, 15.7, 14.0
(CH3Fuc). HRMS m/z: calcd for C74H95N2O20
[M+H]+: 1331.6478; found 1331.6511.
Spacer containing 2, 10, 11, and 15 (1–7 mg) were dis-
solved a 1:1 solution of 0.1 M phosphate buffer pH 7.0
and MeOH (ꢂ250 lL total volume). Diethyl squarate
(1.1 equiv) was added and the reaction was allowed to
stand until TLC analysis (n-BuOH–MeOH–water–
HOAc 4:2:2:0.5) indicated complete conversion of the
glycoside into a faster moving product. After concentra-
tion, the crude residue was dissolved in water (0.5 mL)
and loaded onto a C-18 Sep-Pak cartridge. The column
was washed with water (3 · 0.5 mL), then the product
was eluted with water/MeOH (75/25!25/75 v/v). After
TLC analysis, the fractions containing the squarate ad-
duct were pooled, and evaporated.
4.14. 6-[N-(Benzyloxycarbonylamino)hexyl]-2-acetamido-
2-deoxy-3-O-{2-O-[2-O-(2,3,4-tri-O-benzyl-a-L-fucopyr-
anosyl)-a-L-fucopyranosyl]-a-L-fucopyranosyl}-b-D-
glucopyranoside (14)
Compound 13 (16 mg, 0.01 mmol) was treated with
AcOH 60% (700 lL) at 50 ꢁC for 8 h, and then evapo-
rated to dryness with the aid of toluene. The crude
was purified by column chromatography (CH2Cl2–
MeOH 25:1!15:1) to give 14 (8 mg, 57%). [a]D ꢀ92 (c
1
0.16, MeOH). H NMR (600 MHz, CD3OD): d 7.85–
7.22 (m, 20H, Ar), 5.42 (s, 1H, H-1 Fuc), 5.34 (s, 1H,
J = 3.5 Hz, H-1 Fuc), 5.31 (s, 3H, J = 3.1 Hz, H-1
Fuc), 5.06 (br s, 2H, CH2Ph), 4.81–4.52 (m, 7H,
3CH2Ph, H-1 GlcNAc), 4.41 (m, 1H, H-5 Fuc), 4.20
(m, 1H, H-5 Fuc), 4.09–3.35 (m, 17H, H-2, H-3, H-4,
H-5, 2H-6 GlcNAc, 3H-2, H-3, F-4 Fuc, OCH2), 3.07
(m, 2H, CH2NH), 1.91 (s, 3H, Ac), 1.68–0.94 (m, 17H,
(CH2)2, 3CH3-Fuc). 13C NMR (CD3OD): d 129.5,
4.17. General procedure for the preparation of the bovine
serum albumin (BSA) neoglycoconjugates
BSA was dissolved in 0.2 M carbonate buffer pH 9
(25 mg mLꢀ1) and stirred for 30 min. Then the diethyl
squarate adduct, dissolved in water (0.5 mg mLꢀ1),
was added to the BSA solution (20 mol equiv based on

A.-M. M. van Roon et al. / Bioorg. Med. Chem. 13 (2005) 3553–3564
3563
BSA) and the reaction was allowed to proceed for
5 days. The BSA neoglycoconjugates were purified using
gel permeation chromatography. The average degree of
incorporation of the glycosides onto BSA was deter-
mined by MALDI-TOF MS (Table 2). The LNFPIII-
HSA neoglycoconjugate was a kind gift of Dr. B. J.
Appelmelk (Amsterdam, The Netherlands). The neogly-
coconjugate was originally purchased from Isosep AB
(Tullinge, Sweden) and contained on average 22 mol
LNFPIII per mole HSA.
were observed with a Leica DM RBE fluorescence
microscope with the appropriate filter combination for
FITC fluorescence. A negative control consisted of incu-
bation with fresh culture medium followed by staining
with the FITC labeled conjugate. Fluorescence was
interpreted visually (Fig. 1).
Acknowledgements
The authors would like to thank D. Kornelis for per-
forming the immunofluorescence assays and M. Robijn
for helpful discussions.
4.18. Immobilization of BSA neoglycoconjugates on CM5
sensor chips
The Biacore 3000 instrument, CM5 sensor chips and an
amino coupling kit were purchased from Biacore AB
(Uppsala, Sweden). All buffers were filtered and de-
gassed under vacuum before use. Immobilization of
the neoglycoconjugates was performed using the stan-
dard amino coupling procedure according to the instruc-
References and notes
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1999, 85, 14–18.
3. Van de Vijver, K. K.; Hokke, C. H.; Van Remoortere, A.;
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ology 2000, 10, 601–609.
11. Van Remoortere, A.; Van Dam, G. J.; Hokke, C. H.; van
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tions of the manufacturer at a flow rate of 5 lL minꢀ1
.
In short, the carboxymethylated surface of the CM5 sen-
sor chip was activated by a 7 min pulse of a 1:1 mixture
of 0.1 M N-hydroxysuccinimide and 0.1 M N-Ethyl-
N0-(dimethylaminopropyl)carbodiimide, followed by
several injections of the neoglycoconjugate at a concen-
tration of 25 lg mLꢀ1 in 10 mM sodium acetate pH 4.5
until the desired level of immobilization was achieved.
The BSA neoglycoconjugates were immobilized to a le-
vel of approximately 10,000 response units (RU). After
immobilization unreacted groups were blocked by the
injection of 300 lL 1 M ethanolamine-HCl, pH 8.5.
The same protocol was followed for the LNFPIII-
HSA neoglycoconjugate. Unconjugated BSA was cou-
pled on one flow channel as a control.
4.19. Binding analysis of monoclonal antibodies to
fucosylated glycosides by SPR
Hybridoma culture supernatant (sup) containing the
Mab of interest was injected on the different CM5 sensor
chip channels containing immobilized neoglycoconju-
gates during 2 min with a flow of 20 lL sꢀ1. After a dis-
sociation period of 5 min following the end of the
injection, the response unit (RU) value was measured.
Subsequently, the surface was regenerated with 20 lL
20 mM HCl. At the start of each new cycle, a 3-min
equilibration period preceded injection of the sup, dur-
ing this period 5 lL HBS buffer was injected as a control
for the surface. After subtraction of the response units
of the BSA control channel, the binding pattern of each
Mab is represented in Table 3 as + (100–500 RU), ++
(500–1000 RU), or +++ (>1000 RU).
12. Van Remoortere, A.; Vermeer, H. J.; Van Roon, A. M.;
Langermans, J. A.; Thomas, A. W.; Wilson, R. A.; van
Die, I.; van den Eijnden, D. H.; Agoston, K.; Kerekgy-
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G. J.; Hokke, C. H.; Deelder, A. M. Exp. Parasitol. 2003,
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N. H.; Kapsenberg, M. L.; Deelder, A. M.; Tielens, A. G.;
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R.; Dell, A. Glycobiology 1997, 7, 653–661.
16. Robijn, M. L. M.; Wuhrer, M.; Kornelis, D.; Deelder, A.
M.; Geyer, R.; Hokke, C. H. Parasitology 2005, 130, 67–
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4.20. Immunofluorescence assay (IFA)
17. Agoston, K.; Kerekgyarto, J.; Hajko, J.; Batta, G.;
Lefeber, D. J.; Kamerling, J. P.; Vliegenthart, J. F. G.
Chem.-A Eur. J. 2002, 8, 151–161.
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ling, J. P.; Vliegenthart, J. F. G. J. Chem. Soc., Perkin
Trans. 1 2000, 2249–2263.
IFAs were carried out on 6 lm-thick cryostat liver sec-
tions of infected hamsters. These sections were incu-
bated with Mabs (Fig. 1), washed and then incubated
with an FITC labeled conjugate of RaM immunoglobu-
lin antibodies (Nordic Immunological Laboratories) di-
luted 1:50 in PBS (0.035 M phosphate, 0.15 M NaCl,
pH 7.8) containing 0.1 mg/mL EvanÕs blue. The slides
19. Nibbeling, H. A.; Kahama, A. I.; van Zeyl, R. J.; Deelder,
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