First demonstration of differential inhibition of lectin binding by synthetic tri- and tetravalent glycoclusters from cross-coupling of rigidified 2-propynyl lactoside.

Article abstract of DOI:10.1039/b307802g

The interplay of mammalian lectins such as galectins with cellular glycoconjugates is intimately involved in crucial reaction pathways including tumor cell adhesion, migration or growth regulation. These clinically relevant functions explain the interest

Full text of DOI:10.1039/b307802g

View Article Online / Journal Homepage / Table of Contents for this issue
First demonstration of differential inhibition of lectin binding by
synthetic tri- and tetravalent glycoclusters from cross-coupling of
rigidified 2-propynyl lactoside
Sabine André,a Bingcan Liu,b Hans-J. Gabiusa and René Roy*b
a Institut für Physiologische Chemie, Tierärztliche Fakultät,
Ludwig-Maximilians-Universität München, Veterinärstr. 13, 80539 München, Germany
b Department of Chemistry, Université du Québec à Montréal, P. O. Box 8888,
Succ. Centre-Ville, Montreal (Quebec), Canada H3C 3P8
Received 11th July 2003, Accepted 18th September 2003
First published as an Advance Article on the web 15th October 2003
The interplay of mammalian lectins such as galectins with cellular glycoconjugates is intimately involved in crucial
reaction pathways including tumor cell adhesion, migration or growth regulation. These clinically relevant functions
explain the interest in designing glycoclusters with potent activity to interfere with lectin binding. In view of the
perspective for medical applications the following objective arises: to correlate topological factors of ligand display
most favorably to reactivity against endogenous lectins. To date, plant agglutinins have commonly been used as
models. Properly addressing this issue we first prepared di- to tetravalent clusters from 2-propynyl lactoside under
mild oxidative homocoupling conditions and using the Sonogashira palladium-catalyzed cross-coupling reaction
with triiodobenzene or pentaerythritol cores. These products were tested for bioactivity in a competitive solid-phase
assay using different labeled sugar receptors as probes, i.e. the β-trefoil mistletoe lectin, the natural lactoside-binding
immunoglobulin G fraction from human serum and three mammalian galectins from two subgroups. The lactose
headgroups in the derivatives retained ligand properties. Differences in inhibitory capacity were marked between the
galectins. In contrast to homodimeric proto-type galectins-1 and -7 significant inhibition of galectin-3 binding with
a 7-fold increase in relative potency was observed for the trivalent compound. In comparison, the binding of the
β-trefoil mistletoe agglutinin was reduced best by tetravalent substances. The result for galectin-3 was independently
confirmed by haemagglutination and cytofluorometric cell binding assays. These data underline the feasibility of
galectin-type target selectivity by compound design despite using an identical headgroup (lactose) in synthesis.
density would automatically trigger a cluster effect.5 When thus
accomplishing a new synthetic approach to glycoclusters, it is
Introduction
The coining of terms such as sugar code or glycomics reflects
the growing realization of how well glycans of natural glyco-
conjugates are suited for biological information storage and
transfer. Their unrivalled potential for building isomers includ-
ing formation of branched structures, the versatile template-
free but intimately controlled production of glycan determin-
ants by a complex enzymatic machinery with regulation of
expression of distinct epitopes on different levels, and the
strategic positioning of the final products on cell surfaces read-
ily accessible for intermolecular interactions make a strong case
for carbohydrates as code words.1 That glycan diversity and
placement are matched by occurrence of various families of
lectins epitomizes the concept of information transfer by pro-
ductive protein–carbohydrate interactions.2 Naturally, insights
into lectin functionality will be the basis for devising medical
applications, for example to target drugs exploiting sugar
signals as postal code, to visualize lectin activity in relation
to disease, for example in tumor diagnosis, or to block lectin
binding, thereby interfering with glycan routing, unwanted cell
adhesion, tissue invasion or infections.3 With this aim in mind
synthetic efforts have been directed at defining scaffolds for
multivalent display of carbohydrate ligands.4
A major lesson taught by studies in this area is that multi-
valency will not necessarily lead to marked increases of bio-
logical activity of the carrier-immobilized lectin ligands. The
well-ordered positioning of binding sites in the membrane
engaged by endocytic C-type lectins,2a,2f,3a a blueprint for com-
plementarity design of neoglycoconjugates, is thus a particu-
larly favorable case. In fact, plant lectins such as concanavalin A
or Erythrina cristagalli agglutinin were instrumental in reveal-
ing the limits to naive expectations that increases in ligand
essential to thoroughly examine their potency in inhibition
assays. Given the medical perspective noted above, plant lectins
should still be regarded as models, with, eventually, endogenous
lectins involved in disease-associated processes entering the test
panel. Due to their involvement in tumor progression and
metastasis formation, as well as frequent correlation of expres-
sion to patients’ prognosis, the ligand-crosslinking members of
the galectin family of β-galactoside-specific lectins afford suit-
able test objects.6 Additionally, the different described modes of
presentation of the carbohydrate recognition domains may
even gain access to galectin-type selective reagents. Initial work
with starburst and wedge-like glycodendrimers has underscored
the potential of this concept.7 This study extends work along
this line.
Having recently developed a strategy to design tri- and
tetravalent glycoclusters using palladium cross-coupling and
Sonogashira reactions and triiodobenzene or pentaerythritol
cores,8 we herein describe the preparation of lactoside-bearing
glycotope bio-isosteres with assumed reactivity to lectins. Next,
we demonstrate the validity of this assumption. To figure out
whether and to what extent the glycoclusters can react with
carbohydrate-binding proteins we tested, following the reason-
ing given above, three galectins (the homodimeric proto-type
galectins-1 and -7 and the chimera-type galectin-3 monomeric
in solution and forming aggregates when surface immobil-
ized9). To add further modes of presentation of target sites to
testing the bioactive ligands we ran assays with the β-trefoil
mistletoe lectin (Viscum album agglutinin, VAA) and a natural
β-galactoside-specific immunoglobulin G fraction from human
serum in parallel. The capacity of the new bi- to tetravalent
lactose clusters to inhibit binding of these proteins to a matrix
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 9 – 3 9 1 6
3909

View Article Online
Scheme 1 Reactions and conditions: a) (Ph3P)2PdCl2, CuI, NEt3, DMF, rt, 3h (95%); b) NaOMe, MeOH, rt, 24 h, 95%; c) H2, 10% Pd–C, MeOH,
5 h, 97%.
Scheme 2 Reactions and conditions: a) 1,4-Diiodobenzene, (Ph3P)2PdCl2, CuI, NEt3, DMF, rt, 3 h (90%); b) NaOMe, MeOH, rt, 24 h, 95%; c) H2,
10% Pd–C, MeOH, 5 h, 95%.
established by surface-immobilized lactosylated neoglyco-
protein, a structural mimic of a cell surface, was comparatively
assessed. That glycoclusters effective in blocking galectin
binding to the model matrix were also able to interfere with
cell surface interactions was finally demonstrated in haemag-
glutination and cytofluorometric analysis using tumor cells. We
reveal a clear preference of cluster design for the plant lectin
and galectin-3.
and 14 (para) in 78 and 75% yields, respectively. Complete de-
O-acetylation of these clusters was uneventful, resulting in
freely water-soluble clusters 6,9,13 and 15 in more then 90%
yields. The solubility of these lactosylated clusters is in striking
contrast to those observed for the corresponding β--galacto-
side clusters.8a Thus, the extension of the sugar part improves
this physicochemical parameter and could also be exploited to
tailor lectin-specific headgroups, if bioactivity of the com-
pounds will be proven.
Tetramers 13 and 15 could not be hydrogenated due to the
benzylic nature of the core pentaerythritol moieties which
would be cleaved under such conditions. However, catalytic
hydrogenation of dimer 6 under standard conditions (H2, 10%
Pd–C, MeOH) gave dimer 7 in 95% yield which possesses a
slightly more flexible arm between the lactoside residues in
comparison to homodimer 6.
Having finished the synthesis of the test compounds and
their analysis, we next addressed the question as to whether the
derivatized sugar parts are still bioactive. For this purpose, we
used the experimental setting of a solid-phase lectin-binding
assay where these compounds were introduced as competitive
inhibitors.
Results and discussion
Synthesis
Starting with 2-propynyl lactoside 1 we prepared a series of di-,
tri, and tetravalent compounds (cpd) to initiate testing of their
bioactivity. In detail, 2-propynyl lactoside 1 was treated under
mild oxidative homocoupling conditions to provide dimer 2 in
95% yield using dichlorobis(triphenylphosphine)palladium()
((Ph3P)2PdCl2), copper() iodide (NEt3 : DMF, 1 : 1, v/v, 25 ЊC)
(Scheme 1). The choice of the catalysts was not critical since
other palladium catalysts such as tetrakis(triphenylphosphine)-
palladium(0) ((Ph3P)4Pd) or tris(dibenzylideneacetone)-
dipalladium (0) (Pd2(dba)3) were equally effective. Zemplén de-
O-acetylation of dimer 2 (NaOMe, MeOH) produced freely
water-soluble di-lactoside 3 in 95% yield. Hydrogenation of 3
with 10% palladium on charcoal gave dimer 4 in quantitative
yield.
All the next cross-coupling reactions were done using the
general protocol described above for 2 ((Ph3P)2PdCl2, DMF :
Et3N (1 : 1. v/v), CuI, rt, 3–5 h). The simultaneous cross-
coupling of 2-propynyl lactoside 1 to 1,4-diiodobenzene
(Scheme 2), 1,3,5-triiodobenzene (Scheme 3), and to penta-
erythritol tetrakis(m- and p-iodobenzyl)ether 10 and 11 (Scheme
4) afforded an efficacious entry into this family of carbohydrate
clusters 5 (dimer 90%), 8 (trimer 80%), and tetramers 12 (meta)
Biochemical and cell biological assays
The binding of the different sugar receptors to the matrix-
immobilized neoglycoprotein was consistently dependent on
ligand presentation on the surface and sensitive to the presence
of lactose. If bioactivity of the synthetic compounds’ head-
groups was maintained, they should thus also interfere with
protein–carbohydrate interaction responsible for signal gener-
ation in this assay. Indeed, this assumption was experimentally
verified. Therefore, it was possible to assess the concentration
which reduced the initial signal by 50% (IC50), as shown
exemplarily for the mistletoe lectin in Fig. 1. Stepwise increases
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 9 – 3 9 1 6
3910

View Article Online
Scheme 3 Reactions and conditions: a) 1,3,5-Triiodobenzene, (Ph3P)2PdCl2, CuI, NEt3, DMF, rt, 5 h (81%); b) NaOMe, MeOH, rt, 24 h, 92%.
Scheme 4 Reactions and conditions: a) (Ph3P)2PdCl2, CuI, NEt3, DMF, rt, 5 h (78% meta, 75% para); b) NaOMe, MeOH, rt, 24 h, (quant).
IC50-values. Only the tetravalent compound 15 for galectin-1
and compounds 4,9,13 and 15 for galectin-7 harbored inhibi-
tory capacity in the range of free lactose, establishing cases of
negative correlation between carrier-dependent presentation
of lactose and inhibitory efficiency. As seen in Table 1, the
chimera-type galectin-3, however, reacted with the compounds
rather well except for the divalent lactosides linked by an aro-
matic spacer. The strongest cluster effect was seen with the tri-
valent compound, a result shown for this murine, and also
obtained for human, galectin-3. Independently, we determined
the comparative inhibitory potency of these compounds in
haemagglutination. These assays confirmed that compound 9
was the strongest inhibitor, again clearly surpassing free lactose.
It should be noted that no evidence for formation of aggregates
by visible precipitation was obtained under either of the
experimental conditions.
Fig. 1 Inhibition curves of binding of biotinylated Viscum album L.
Compared to the galectins the tetravalent compounds were
more effective inhibitors in the case of the plant lectin VAA,
while the meta-derivative 13 was also a good inhibitor for the
natural immunoglobulin G fraction. The previous observation
that the topological aspects of ligand display (e.g. presentation
of bi-, tri, and tetraantennary N-glycans) modulate lectin affin-
ity and inhibitory capacity of glycoclusters7c,10 calls for a con-
firmation of the effectiveness of the trivalent compound to
interfere differentially with galectin binding. For this purpose,
we performed binding assays with tumor cells in vitro, a system
with increased clinical relevance relative to the solid-phase
assays. As shown in the upper panel of Fig. 2, differential activ-
ity of the trivalent compound 9 to impair galectin-3 but not
(VAA) agglutinin to surface-immobilized lactose maxiclusters using
galactose (᭺), lactose (ᮀ), cpd-3 (᭹), cpd-4 (᭢) and cpd-15 (ꢀ) in an
exemplary series; for compound structures, please see illustrations in
schemes.
of the inhibitor concentration reduced lectin binding to the
surface-presented ligands, and the different curves reflect that
glycoclusters can well surpass the inhibitory capacity of lactose.
Running these assays systematically for the different sugar
receptors led to the data compilation in Table 1. Of note, bind-
ing of the two homodimeric proto-type galectins-1 and -7 was
not influenced effectively by the presence of these di- to tetra-
valent compounds, precluding the determination of accurate
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 9 – 3 9 1 6
3911

View Article Online
Fig. 2 Cytofluorometric analysis of binding of galectins to tumor cell
surfaces (a, b: murine monocyte/macrophage line J774.A1; c, d: human
ovary adenocarcinoma line NIH-OVCAR 3) in an exemplary series
testing the effect of the absence or presence of glyco-inhibitors (the
molar concentration of lactose in the cell suspension was deliberately
kept constant regardless of the nature of the test substance in each
assay series). a: Level of background staining (—), staining with
galectin-1 (——, 25 µg mlϪ1) and staining with galectin-1 (25 µg mlϪ1) in
the presence of 0.5 mg mlϪ1 cpd-9 (grey——); b: level of background
staining (—), staining with galectin-3 (——, 20 µg mlϪ1) and staining
with galectin-3 (20 µg mlϪ1) in the presence of 0.5 mg mlϪ1 cpd-9
(grey——); c: level of background staining (—), staining with galectin-3
(——, 20 µg mlϪ1) and staining with galectin-3 in the presence of 1.25
mM lactose (grey——); d: level of background staining (—), staining
with galectin-3 (——, 20 µg mlϪ1) and staining with galectin-3 in the
presence of 0.5 mg mlϪ1 cpd-9 (1.25 mM in lactose content) (grey——).
galectin-1 binding to a tumor cell surface was recorded. Next,
we monitored the effects of this glycocluster relative to lactose
used at increased molarity to reach equal total ligand concen-
trations and observed strong effects of the inhibitors (Fig. 2,
bottom panel). Thus, the trivalent compound 9 (an illustration
of a low-energy conformation of compounds 9 and 15, respect-
ively, is presented in Fig. 3) is a potent inhibitor of galectin-3
binding to native cell surfaces with the same differential activity
against homodimeric galectins noted in the solid-phase assay.
Conclusion
Our study has proven that the lactoside headgroups in these
glycoclusters retained their ligand activity. In fact, the tetra-
valent products obtained from the m- and p-iodobenzyl pre-
cursors showed a remarkable enhancement of inhibitory
capacity on binding of the mistletoe lectin relative to free
lactose. That an extrapolation of these data for a plant lectin to
any mammalian lectin is not valid is clearly demonstrated by
the respective experimental series. The emerging prominent role
of galectins in tumor biology involved in cell growth, migration,
invasion and metastasis formation has guided the decision to
scrutinise members of this family. Also, different modes of
target-site presentation and reports on antagonistic activities of
galectins-1 and -3 give reasons for aiming at the development
of target-selective glycoclusters.9 Indeed, the trivalent design
brought about selectivity for the only known chimera-type
galectin under these experimental conditions. In view of the
documented effect of ligand topology on inhibitory activity of
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 9 – 3 9 1 6
3912

View Article Online
Fig. 3 Schematic representations of trimer 9 (upper panel: stick left, CPK right) and tetramer 15 (lower panel: stick left, CPK right) in one of their
low-energy (MM3) extended conformations. The lactose portion was adapted from the X-ray data on the galectin-7/lactose complex (PDB 4GAL).
Illustrations were rendered from SymApps software (BioRad) for the stick models and with CAChe 6.0 software from Fujitsu for the CPK models.
multivalent probes7 further studies with bi- to tetraantennary
N-glycans in addition to the tested maxiclusters are warranted.
At any rate, the reported analyses by haemagglutination and
cytofluorometry support the interpretation that the trivalent
compound has preference for blocking galectin-3 binding. Hav-
ing thus shown that this type of trivalency can distinguish
between proto- and chimera-type galectins, further activity and/
or selectivity enhancements might be feasible by tailoring the
ligand structure and conformation and by hydrophobic tag-
ging.11 In more general terms, this work – together with pre-
was purified by silica gel column chromatography using hexane
: ethyl acetate (2 : 3) to provide 3 as a white solid in 95% yield.
[α]D Ϫ22.1 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.31
(2H, bd, J = 3.4, H-4Ј), 5.19 (2H, t, J = 9.3, H-3), 5.07 (2H, dd,
J = 8.0, 10.3, H-2Ј), 4.91 (2H, dd, J = 3.4, 10.3, H-3Ј), 4.88 (2H,
dd, J = 8.0, 9.4, H-2), 4.66 (2H, d, J = 8.0, H-1), 4.49 (2H, dd,
J = 1.6, 12.0, H-6a), 4.45 (2H, d, J = 8.0 H-1Ј), 4.39 (2H, s,
H-1Љ), 4.02–4.11 (6H, m, H-6b and H-6Ј), 3.84 (2H, t, J = 7.0,
H-5Ј), 3.79 (2H, t, J = 9.5, H-4), 3.61–3.64 (2H, m, H-5), 2.12,
2.10, 2.03, 2.02, 2.01, 1.93 (42H, 6s, CH3CO); 13C NMR (125.7
MHz, CDCl3) δ 170.3, 170.1, 170.0, 169.7, 169.6 and 169.0
(CH3CO), 101.0 (C-1Ј), 98.2 (C-1), 76.0 (C-4), 74.2 (alkynyl),
72.8 (C-5), 72.6 (C-3), 71.2 (C-2), 70.9 (C-3Ј), 70.8 (alkynyl),
70.7 (C-5Ј), 69.0 (C-2Ј), 66.6 (C-4Ј), 61.7 (C-6), 60.8 (C-6Ј), 56.3
(C-1Љ), 20.8, 20.7, 20.6, 20.5 and 20.4 (CH3CO); ESI-MS calcd
for C58H74O36 ϩ (NH4ϩ): 1364.4; found: 1364.3.
vious studies on starburst and wedge-like glycodendrimers7
–
proves the potential of the concept to view glycoclusters as
effective blocking reagents against clinically interesting endo-
genous lectins. Further efforts to interfere with antibody bind-
ing, for example in xenotransplantation, appear to be likewise
rewarding.
De-O-acetylation of 3,6,9,13 and 15 under Zemplén condi-
tions. The fully protected homodimer 2 (68 mg, 0.05 mmol) was
suspended in methanol (15 mL), to which was added a catalytic
amount of sodium methoxide. The solution was stirred at room
temperature for 24 h. After neutralization of sodium methoxide
with Amberlite resin (120 Hϩ), the solution was filtered.
Removal of methanol under reduced pressure gave 3 (95%
yield). Compounds 6, 9, 13, and 15 were prepared in the same
manner in 95%, 92%, 90%, and 91% yields, respectively.
Experimental
Synthetic procedures
All the chemicals were purchased from Aldrich Chemicals and
used without further purification. Instrumentation description
follows that described previously.8a
1,6-Bis-[(2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl)-(1–4)-
2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyloxy]-2,4-hexadiyne
(2).
Prop-2-ynyl (2,3,4,6-tetra-O-acetyl-β--galactopyranosyl)-(1–
4)-2,3,6-tri-O-acetyl-β--glucopyranoside (1)12 (67.4 mg, 0.10
mmol) was dissolved in a solution of 10 mL of DMF and TEA
(1 : 1) to which were added Pd(PPh3)2Cl2 (3.6 mg, 5 mol%) and
CuI (3.8 mg, 20 mmol%). After stirring for 3 h at rt, the solvents
were removed under reduced pressure. The brownish residue
1,6-Bis-[(ꢀ-D-galactopyranosyl)-(1–4)-ꢀ-D-glucopyranosyl-
oxy]-2,4-hexadiyne (3). [α]D Ϫ20.8 (c 1.0, H2O); 1H NMR
(500 MHz, D2O) δ 4.70 (2H, J = 8.0, d, H-1), 4.63 (4H, s, H-1Љ),
4.49 (2H, d, J = 7.8, H-1Ј), 4.03 (2H, dd, J = 1.7, 11.8, H-6a),
3.98 (2H, d, J = 3.2, H-4Ј), 3.96 (2H, dd, J = 6.9, 12.0, dd,
H-1aЉ), 3.81–3.88 (8H, m, H-5Ј, H-6b, H-6,), 3.67–3.78 (8H,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 9 – 3 9 1 6
3913

View Article Online
m,H-3, H-3Ј, H-4, H-5), 3.59 (2H, dd, J = 7.8, 9.9, H-2Ј), 3.40
(2H, t, J = 8.3, H-2); 13C NMR (125.7 MHz, D2O) δ 102.5
(C-1Ј), 100.1 (C-1), 77.8, 74.9 (C-5Ј), 74.4 (C-5), 73.9, 72.4,
72.1, 72.0, 70.5 (C-2Ј), 69.9 (alkynyl), 68.1 (C-4Ј), 60.5 (C-6Ј),
59.5 (C-6), 56.6 (C-1Љ); ESI-MS calcd for C30H46O22 ϩ (Naϩ):
781.1; found: 781.2.
in 95% yield. [α]D Ϫ6.0 (c 1.0, H2O); 1H NMR (500 MHz, D2O)
δ 7.30 (4H, s, aromatic), 4.50 (4H, d, J = 7.8, H-1 and H-1Ј),
3.94–4.06 (6H, m, H-4Ј, H-1aЉ, H-6a), 3.58–3.86 (24H, m, H-2Ј,
H-3, H-3Ј, H-4, H-4Ј, H-5, H-5Ј, H-6b, H-6Ј, H-1Љ), 3.37 (2H, t,
J = 8.3, H-2), 2.74 (4H, t, J = 7.4, H-3Љ), 1.97 (4H, t, J = 6.8,
H-2Љ); 13C NMR (125.7 MHz, D2O) δ 139.3 and 128.2 (aro-
matic), 102.5 (C-1Ј), 102.4 (C-1), 78.0, 74.9, 74.3, 74.0, 72.4,
72.1, 70.5, 69.2 (C-1Љ), 68.1, 59.7 (C-6Ј), 59.4 (C-6), 30.3 and
30.1 (C-2Љ and C-3Љ); ESI-MS calcd for C36H58O22 ϩ (Naϩ):
865.3; found: 865.2.
1,6-Bis-[(ꢀ-D-galactopyranosyl)-(1–4)-ꢀ-D-glucopyranosyloxy]-
hexane (4). Homodimer 3 (39 mg, 0.05 mmol) was dissolved in
methanol (10 mL) to which was added a catalytic amount of 10%
Pd–C. The mixture was stirred at rt for 5 h. After filtration, the
filtrate was concentrated to dryness under reduced pressure to
1
Trimer 8. [α]D Ϫ22.0 (c 1.0, CHCl3); H NMR (500 MHz,
1
provide 4 (97% yield). [α]D Ϫ21.5 (c 1.0, H2O); H NMR (500
CDCl3) δ 7.42 (3H, s, aromatic), 5.31 (3H, dd, J = 0.9, 3.4,
H-4Ј), 5.20 (3H, t, J = 9.3, H-3), 5.07 (3H, dd, J = 7.9, 10.4,
H-2Ј), 4.91 (3H, dd, J = 3.4, 10.4, H-3Ј), 4.90 (3H, dd, J = 7.9,
9.4, H-2), 4.73 (3H, d, J = 7.9, H-1), 4.55 (3H, d, J = 16.1,
H-1aЉ), 4.50 (3H, d, J = 16.1, H-1bЉ), 4.44–4.48 (6H, m, H-6a
and H-1Ј), 4.02–4.11 (9H, m, H-6b and H-6Ј), 3.85 (3H, t,
J = 7.1, H-5Ј), 3.80 (3H, t, J = 9.2, H-4), 3.61–3.66 (3H, m, H-5),
2.11, 2.08, 2.03, 2.02, 2.01, 2.00, 1.99 and 1.93 (63H, 7s,
CH3CO); 13C NMR (125.7 MHz, CDCl3) δ 170.7, 170.5, 170.4,
170.1, 170.0, 169.4 (CH3CO), 135.1 and 123.5 (aromatic), 101.4
(C-1Ј), 98.6 (C-1), 85.5 and 85.3 (alkynyl), 76.4 (C-4), 73.1
(C-5), 73.0 (C-3), 71.7 (C-2), 71.3 (C-3Ј), 71.0 (C-5Ј), 69.4
(C-2Ј), 66.9 (C-4Ј), 62.1 (C-6), 61.1 (C-6Ј), 56.9 (C-1Љ), 21.2,
MHz, D2O) δ 4.53 (2H, J = 8.0, d, H-1), 4.50 (2H, J = 7.8, d, H-
1Ј), 4.02 (2H, d, J = 12.0, H-6a), 3.97 (2H, bd, J = 3.2, H-4Ј), 3.96
(2H, dd, J = 6.9, 12.0, H-1aЉ), 3.63–3.90 (18H, m, H-3, H-3Ј, H-4,
H-5, H-5Ј, H-6b, H-6, H-1bЉ), 3.59 (2H, dd, J = 7.8, 9.9, H-2Ј),
3.45 (2H, t, J = 8.3, H-2), 1.69 (4H, m, H-2Љ), 1.44 (4H, bs, H-3Љ);
13C NMR (125.7 MHz, D2O) δ 102.5 (C-1Ј), 101.5 (C-1), 78.0,
74.9, 74.3, 74.0, 72.4, 72.1, 71.3, 70.5, 70.3, 70.1 (C-2), 69.9, 68.1
(C-2Ј), 67.9 (C-1Љ), 60.5 (C-6Ј), 59.7 (C-6), 28.1 (C-2Љ), 24.3(C-3Љ);
ESI-MS calcd for C30H54O22 ϩ (NH4ϩ): 784.3; found: 784.2.
General procedure for the cross-coupling reactions
1,4-Diiodobenzene (16.6 mg, 0.05 mmol) was dissolved in 10
mL of DMF and TEA (1 : 1), to which were added Pd(PPh3)2-
Cl2 (3.6 mg, 5 mol%), lactoside 1 (80.9 mg, 0.12 mmol, 2.2 eq.)
and CuI (3.8 mg, 20 mmol%). The solution was stirred at rt for
5 h under a nitrogen atmosphere. The solvent and triethylamine
were removed under reduced pressure. The residue was purified
by silica gel column chromatography using hexane and ethyl
acetate (2 : 3) to give 5 (90% yield). The identical procedure was
used toward the synthesis of clusters 8,12 and 14 in yields of
81%, 78% and 75%, respectively.
21.1, 21.0 and 20.8 (CH3CO); ESI-MS calcd for C93H114O54
ϩ
(NH4ϩ): 2112.6; found: 2112.4.
Deprotected trimer 9. This compound was obtained as
1
described above for 3: [α]D Ϫ24.8 (c 1.0, H2O); H NMR (500
MHz, D2O) δ 7.62 (3H, s, aromatic), 4.73–4.78 (9H, m, H-1,
H-1Љ), 4.53 (3H, J = 7.8, d, H-1Ј), 4.03 (3H, bd, J = 11.2, H-6a),
4.00 (3H, bd, J = 3.2, H-4Ј), 3.73–3.92 (24H, m, H-3, H-3Ј, H-4,
H-4Ј, H-5Ј, H-6b, H-6Ј), 3.67 (3H, m, H-5), 3.63 (3H, dd,
J = 7.8 and 9.5, H-2Ј), 3.47 (3H, m, H-2); 13C NMR (125.7
MHz, D2O) δ 134.4 and 122.3 (aromatic), 102.5 (C-1Ј), 100.3
(C-1), 85.4 and 84.6 (alkynyl), 77.8, 74.9, 74.4, 73.9, 72.2 (C-2),
72.1, 70.5 (C-2Ј), 68.1 (C-4Ј), 60.1 (C-6Ј), 59.6 (C-6), 56.7
(C-1Љ); ESI-MS calcd for C51H72O33 ϩ (NH4ϩ): 1230.4; found:
1230.4.
1,4-Phenylenedi-2-propyne-3,1-diyl
bis[(2,3,4,6-tetra-O-
acetyl-ꢀ-D-galactopyranosyl)-(1–4)-2,3,6-tri-O-acetyl-ꢀ-D-gluco-
pyranoside] (5). [α]D Ϫ31.0 (c 1.0, CHCl3); 1H NMR (500
MHz, CDCl3) δ 7.35 (4H, s, aromatic), 5.31 (2H, bd, J = 2.9,
H-4Ј), 5.21 (2H, t, J = 9.2, H-3), 5.07 (2H, dd, J = 7.9, 10.3,
H-2Ј), 4.91 (2H, dd, J = 3.5, 10.3, H-3Ј), 4.90 (2H, dd, J = 7.9,
9.4, H-2), 4.76 (2H, d, J = 7.9, H-1), 4.54 (2H, d, J = 16.1,
H-1aЉ), 4.50 (2H, d, J = 16.1, H-1bЉ), 4.44–4.48 (4H, m,
H-6a and H-1Ј), 4.02–4.11 (6H, m, H-6b and H-6Ј), 3.84 (2H, t,
J = 6.8, H-5Ј), 3.80 (2H, t, J = 9.6, H-4), 3.61–3.64 (2H, m, H-5),
2.11, 2.08, 2.03, 2.02, 2.01, 2.00, 1.99 and 1.93 (42H, 7s,
CH3CO); 13C NMR (125.7 MHz, CDCl3) δ 170.3, 170.2, 170.1,
170.0, 169.7, 169.6 and 169.0 (CH3CO), 131.6 and 122.5
(aromatic), 101.0 (C-1Ј), 98.2 (C-1), 86.4 and 85.4 (alkynyl),
76.0 (C-4), 72.8 (C-5), 72.7 (C-3), 71.4 (C-2), 70.9 (C-3Ј), 70.7
(C-5Ј), 69.1 (C-2Ј), 66.6 (C-4Ј), 61.8 (C-6), 60.8 (C-6Ј), 56.7
(C-1Љ), 20.8, 20.7, 20.6, 20.5 and 20.4 (CH3CO); ESI-MS calcd
for C64H78O36 ϩ (Naϩ): 1445.3; found: 1445.2.
Tetramer (meta) 12. This compound was obtained by the
cross-coupling of 1 and tetraiodide 10: [α]D Ϫ24.5 (c 1.5,
1
CHCl3); H NMR (500 MHz, CDCl3) δ 7.17–7.41 (16H, m,
aromatic), 5.29 (4H, bd, J = 3.1, H-4Ј), 5.18 (4H, t, J = 9.3,
H-3), 5.05 (4H, dd, J = 7.9, 10.4, H-2Ј), 4.91 (4H, dd, J = 3.2,
10.3, H-3Ј), 4.90 (4H, dd, J = 7.9, 9.4, H-2), 4.73 (4H, d, J = 7.9,
H-1), 4.51 (4H, d, J = 16.0, H-1aЉ), 4.50 (4H, d, J = 16.0, H-1bЉ),
4.44–4.48 (8H, m, H-6a and H-1Ј), 4.40 (8H, s, benzyl), 4.02–
4.11 (12H, m, H-6b and H-6Ј), 3.83 (4H, t, J = 6.7, H-5Ј), 3.78
(4H, t, J = 9.6, H-4), 3.61–3.64 (4H, m, H-5), 3.49 (8H, s,
C(CH2OR)4), 2.10, 2.05, 2.00, 1.99, 1.98, 1.96 and 1.91 (84H,
7s, CH3CO); 13C NMR (125.7 MHz, CDCl3) δ 170.2, 170.0,
169.9, 169.7, 169.6 and 168.9 (CH3CO), 139.0, 130.7, 130.3,
128.3, 127.6 and 121.9 (aromatic), 100.9 (C-1Ј), 98.1 (C-1), 86.9
and 83.3 (alkynyl), 76.0 (C-4), 72.7 (C-5 and C-3), 72.6 (benzyl-
ic), 71.4 and 70.9 (C-3Ј and C-2), 70.6 (C-5Ј), 69.2 (C(CH2-
OR)4), 69.0 (C-2Ј), 66.5 (C-4Ј), 61.8 (C-6), 60.7 (C-6Ј), 56.7
(C-1Љ), 45.6 (C(CH2OR)4), 20.7, 20.6, 20.5 and 20.4 (CH3CO);
ESI-MS calcd for C149H180O76 ϩ (2NH4ϩ): 1610.5; found:
1610.5.
1,4-Phenylenedi-2-propyne-3,1-diyl bis[(ꢀ-D-galactopyrano-
syl)-(1–4)-ꢀ-D-glucopyranoside] (6). This compound was
obtained under the Zemplén conditions described above for 3:
[α]D Ϫ7.0 (c 1.0, DMSO); 1H NMR (500 MHz, DMSO) δ 7.30
(4H, s, aromatic), 4.64 (2H, d, J = 15.9, H-1aЉ), 4.53 (2H, d,
J = 15.9, H-1bЉ), 4.38 (2H, d, J = 7.9, H-1), 4.17 (2H, d, J = 7.1,
H-1Ј), 3.26–3.79 (22H, m, H-2Ј, H-3, H-3Ј, H-4, H-4Ј, H-5,
H-5Ј, H-6, H-6Ј), 3.03–3.08 (2H, t, J = 8.3, H-2); 13C NMR
(125.7 MHz, DMSO) δ 131.7 and 122.2 (aromatic), 103.8
(C-1Ј), 100.9 (C-1), 87.7 and 85.0 (alkynyl), 80.6, 75.5, 75.0,
74.9, 73.2, 73.0, 70.5, 68.1, 60.5 (C-6Ј), 60.3 (C-6), 55.8 (C-1Љ);
ESI-MS calcd for C36H50O22 ϩ (NH4ϩ): 852.3; found: 852.2.
Deprotected meta-tetramer 13. This compound was obtained
as described above for 3: [α]D Ϫ7.2 (c 1.0, H2O), 1H NMR (500
MHz, D2O at 60 ЊC) δ 7.47–7.78 (16H, m, aromatic), 4.98–5.10
(12H, m, H-1, H-1Љ), 4.92 (4H, d, J = 7.4, H-1Ј), 4.65 (8H, bs,
PhCH2), 4.44 (4H, bs, H-4Ј), 4.06–4.36 (36H, m, H-2Ј, H-3,
H-3Ј, H-4, H-5Ј, H-6, H-6Ј), 3.93 (4H, bs, H-5), 3.88 (4H, 8,
H-2), 3.47 (4H, m, H-2), 3.29 (8H, bs, C(CH2OR)4); 13C NMR
(125.7 MHz, D2O) δ 138.7, 131.0, 130.2, 128.5, 127.6 and 121.9
1,4-Bis-[[[(ꢀ-D-galactopyranosyl)-(1–4)-ꢀ-D-glucopyranosyl]-
oxy]propyl]benzene (7). Hydrogenation of homodimer 6 (42
mg, 0.05 mmol) was done as above for 4 to provide compound 7
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 9 – 3 9 1 6
3914

View Article Online
(aromatic), 103.1 (C-1Ј), 101.1 (C-1), 86.8 and 85.0 (alkynyl),
78.7, 75.3, 74.8 (C-5), 74.5, 72.8, 72.3 (PhCH2), 71.0, 68.8
(C(CH2OR)4), 68.6 (C-4Ј), 61.0 (C-6Ј), 60.4 (C-6), 57.2 (C-1Љ),
45.9 (C(CH2OR)4); ESI-MS calcd for C93H124O48 ϩ (Naϩ):
2031.7; found: 2031.4.
individual experimental series were always performed with
aliquots of the same cell batch on the same day (for further
experimental details, please see legend to Fig. 2).
References
Tetramer (para) 14. This compound was obtained by the
Sonogashira coupling of 1 and tetraiodide 11 as described
above: [α]D Ϫ22.7 (c 1.2, CHCl3); 1H NMR (500 MHz, CDCl3)
δ 7.32 (8H, d, J = 8.1, aromatic), 7.19 (8H, d, J = 8.1, aromatic),
5.30 (4H, bd, J = 2.9, H-4Ј), 5.19 (4H, t, J = 9.3, H-3), 5.06 (4H,
dd, J = 7.8, 10.4, H-2Ј), 4.91 (4H, dd, J = 3.4, 10.4, H-3Ј), 4.89
(4H, dd, J = 7.9, 9.4, H-2), 4.76 (4H, d, J = 7.9, H-1), 4.53 (4H,
d, J = 16.0, H-1aЉ), 4.50 (4H, d, J = 16.0, H-1bЉ), 4.48 (4H, m,
H-6a), 4.46 (4H, J = 7.8, d, H-1Ј), 4.40 (8H, s, benzyl), 4.02–4.11
(12H, m, H-6b and H-6Ј), 3.84 (4H, t, J = 6.7, H-5Ј), 3.80 (4H, t,
J = 9.6, H-4), 3.60–3.65 (4H, m, H-5), 3.52 (8H, s, C(CH2OR)4),
1 (a) P. J. Winterburn and C. F. Phelps, Nature, 1972, 236, 147–151;
(b) R. A. Laine, in Glycosciences: Status and Perspectives (Eds.: H.-J.
Gabius and S. Gabius), Chapman & Hall, London-Weinheim, 1997,
pp. 1–14; (c) G. Reuter and H.-J. Gabius, Cell. Mol. Life Sci., 1999,
55, 368–422; (d ) S. J. Williams and G. J. Davies, Trends Biotechnol.,
2001, 19, 356–362; (e) H.-J. Gabius, S. André, H. Kaltner and H.-C.
Siebert, Biochim. Biophys. Acta, 2002, 1572, 165–177.
2 (a) H.-J. Gabius, Eur. J. Biochem., 1997, 243, 543–576; (b) H. Kaltner
and B. Stierstorfer, Acta Anat., 1998, 161, 162–179; (c) T. Angata
and E. C. M. Brinkman-van der Linden, Biochim. Biophys. Acta,
2002, 1572, 294–316; (d ) N. M. Dahms and M. K. Hancock,
Biochim. Biophys. Acta, 2002, 1572, 317–340; (e) D. C. Kilpatrick,
Biochim. Biophys. Acta, 2002, 1572, 187–197; ( f ) P. H. Weigel and
J. H. N. Yik, Biochim. Biophys. Acta, 2002, 1572, 341–363.
3 (a) Y. C. Lee and R. T. Lee, J. Biomed. Sci., 1996, 3, 221–237;
(b) K. G. Rice, in Glycosciences: Status and Perspectives (Eds.: H.-J.
Gabius and S. Gabius), Chapman & Hall, London-Weinheim, 1997,
pp. 471–483; (c) N. Yamazaki, S. Kojima, N. V. Bovin, S. André,
S. Gabius and H.-J. Gabius, Adv. Drug Deliv. Rev., 2002, 43, 225–
244; (d ) H.-J. Gabius, Anat. Histol. Embryol., 2001, 30, 3–31;
(e) B. G. Davis and M. A. Robinson, Curr. Opin. Drug Discov.
Develop., 2002, 5, 279–288.
4 (a) Y. C. Lee and R. T. Lee (Eds.) Neoglycoconjugates: Preparation
and Applications . Academic Press, San Diego, 1994; (b) N. V. Bovin
and H.-J. Gabius, Chem. Soc. Rev., 1995, 24, 413–421; (c) R. Roy,
Trends Glycosci. Glycotechnol., 1996, 8, 79–99; (d ) B. T. Houseman
and M. Mrksich, Top. Curr. Chem., 2002, 218, 1–44; (e) R. Roy,
J. Carbohydr. Chem., 2002, 21, 769–798; ( f ) C.-L. Schengrund,
Biochem. Pharmacol., 2003, 65, 699–707.
5 (a) R. T. Lee and Y. C. Lee in Neoglycoconjugates: Preparation and
Applications (Eds.: Y. C. Lee and R. T. Lee), Academic Press, San
Diego, 1994, pp. 23–50; (b) D. Zanini and R. Roy, Bioconjugate
Chem., 1997, 8, 187–192; (c) P. R. Ashton, E. F. Hounsell,
N. Jayaraman, T. M. Nilsen, N. Spencer, J. F. Stoddart and
M. Young, J. Org. Chem., 1998, 63, 3429–3437; (d ) T. Furuike,
S. Aiba and S.-I. Nishimura, Tetrahedron, 2000, 56, 9909–9915.
6 (a) H.-J. Gabius, Cancer Invest., 1987, 5, 39–46; (b) D. W.
Ohannesian and R. Lotan in Glycosciences: Status and Perspectives
(Eds.: H.-J. Gabius and S. Gabius), Chapman & Hall, London-
Weinheim, 1997, pp. 459–469; (c) H. Lahm, S. André, A. Hoeflich,
J. R. Fischer, B. Sordat, H. Kaltner, E. Wolf and H.-J. Gabius,
J. Cancer Res. Clin. Oncol., 2001, 127, 375–386; (d ) C. F. Brewer,
Biochim. Biophys. Acta, 2002, 1572, 255–262; (e) A. Danguy,
I. Camby and R. Kiss, Biochim. Biophys. Acta, 2002, 1572, 285–293;
( f ) U. Wollina, T. Graefe, S. Feldrappe, S. André, K. Wasano,
H. Kaltner, Y. Zick and H.-J. Gabius, J. Cancer Res. Clin. Oncol.,
2002, 128, 103–110; (g) N. Nagy, H. Legendre, O. Engels, S. André,
H. Kaltner, K. Wasano, Y. Zick, J.-C. Pector, C. Decaestecker, H.-J.
Gabius, I. Salmon and R. Kiss, Cancer, 2003, 97, 1849–1858.
7 (a) S. André, P. J. C. Ortega, M. A. Perez, R. Roy and H.-J. Gabius,
Glycobiology, 1999, 9, 1253–1261; (b) S. André, B. Frisch,
H. Kaltner, D. L. Desouza, F. Schuber and H.-J. Gabius,
Pharmaceut. Res., 2000, 17, 985–990; (c) S. André, R. J. Pieters,
I. Vrasidas, H. Kaltner, I. Kuwabara, F.-T. Liu, R. M. J. Liskamp
and H.-J. Gabius, ChemBioChem, 2001, 2, 822–830; (d ) I. Vrasidas,
S. André, P. Valentini, C. Böck, M. Lensch, H. Kaltner, R. M. J.
Liskamp, H.-J. Gabius and R. J. Pieters, Org. Biomol. Chem., 2003,
1, 803–810.
2.10, 2.07, 2.01, 2.00, 1.99, 1.98 and 1.91 (84H, 7s, CH3CO); 13
C
NMR (125.7 MHz, CDCl3) δ 170.2, 170.0, 169.9, 169.7, 169.6
and 168.9 (CH3CO), 139.5, 131.6, 127.0 and 121.0 (aromatic),
100.9 (C-1Ј), 98.1 (C-1), 86.9 and 83.3 (alkynyl), 76.0 (C-4), 72.7
(C-5 and C-3), 72.6 (benzylic), 71.4 and 70.9 (C-3Ј and C-2),
70.6 (C-5Ј), 69.2 (C(CH2OR)4), 69.1 (C-2Ј), 66.6 (C-4Ј), 61.8
(C-6), 60.7 (C-6Ј), 56.8 (C-1Љ), 45.6 (C(CH2OR)4), 20.7, 20.6,
20.5 and 20.4 (CH3CO); ESI-MS calcd for C149H180O76
ϩ
(2NH4ϩ): 1610.5; found: 1610.7.
Deprotected para-tetramer 15. This compound was obtained
as described above for 3: [α]D 60.0 (c 0.5, H2O); 1H NMR (500
MHz, D2O) δ 7.31 (8H, bs, aromatic), 7.00 (8H, bs, aromatic),
4.62–4.78 (12H, m, H-1, H-1Љ), 4.52 (4H, J = 7.1, d, H-1Ј), 4.01
((8H, bs, PhCH2), 3.63–3.91 (44H, m, H-2Ј, H-3, H-3Ј, H-4,
H-4Ј, H-5, H-5Ј, H-6, H-6Ј), 3.47 (4H, m, H-2), 3.29 (8H, bs,
C(CH2OR)4); 13C NMR (125.7 MHz, D2O) δ 138.5, 131.3,
126.8 and 120.6 (aromatic), 102.5 (C-1Ј), 100.6 (C-1), 86.3 and
84.5 (alkynyl), 77.8, 74.9, 74.2, 74.0, 72.2, 71.7 (PhCH2), 70.5
(C-2Ј), 68.1, 65.5, 60.6 (C-6Ј), 59.7 (C-6), 56.7 (C-1Љ), 45.9
(C(CH2OR)4); ESI-MS calcd for C93H124O48 ϩ (Naϩ): 2031.7;
found: 2031.5.
Biochemical and cell biological procedures
The five different types of lactoside-binding proteins (human
galectins-1, -3, and -7, murine galectin-3, the IgG-fraction from
human serum and the mistletoe lectin) were purified under
optimized conditions with affinity chromatography on lactosyl-
ated Sepharose 4B, derived from divinyl sulfone activation,13 as
the crucial step, and purity was ascertained by gel filtration
and one- and two-dimensional gel electrophoretic analysis as
described.7 Following their biotinylation under activity-preserv-
ing conditions and rigorous controls to exclude any loss of
activity the solid-phase assay with bovine serum albumin expos-
ing 24–28 p-isothiocyanatophenyl lactoside moieties as ligand-
bearing matrix was performed as described.7 Experimental
conditions to obtain optimal responses (i.e. concentration of
neoglycoprotein in the coating step and of labeled lectin) were
defined in systematic experimental series. The individual
parameters are listed in Table 1 for each case. Inhibition of
haemagglutination and lectin binding to tumor cell surfaces
using the commercially available murine monocyte/macrophage
line J774A.1 and the human ovary adenocarcinoma line NIH-
OVCAR 3 (American Type Culture Collection, Rockville, MD,
USA) was measured as described.7c,9a,14 Trypsin-treated and
glutaraldehyde-fixed rabbit erythrocytes were tested in solution
containing 2-fold serial dilutions of standards with the com-
pounds and free lactose to determine the minimal inhibitory
concentration. Cytofluorometric analyses used at least three
separate cell batches independently. Identical molar concen-
trations of lactose (free or as constituent of the compounds)
were assayed in direct comparison. To allow valid comparison
8 (a) B. Liu and R. Roy, Tetrahedron, 2001, 57, 6909–6913; (b) B. Liu
and R. Roy, J. Chem. Soc., Perkin Trans. 1, 2001, 773–779; (c) B. Liu
and R. Roy, Chem. Commun., 2002, 6, 594–595.
9 (a) S. André, S. Kojima, N. Yamazaki, C. Fink, H. Kaltner,
K. Kayser and H.-J. Gabius, J. Cancer Res. Clin. Oncol., 1999, 125,
461–474; (b) J. Kopitz, C. von Reitzenstein, S. André, H. Kaltner,
J. Uhl, V. Ehemann, M. Cantz and H.-J. Gabius, J. Biol. Chem.,
2001, 276, 35917–35923; (c) J. Kopitz, S. André, C. von Reitzenstein,
K. Versluis, H. Kaltner, R. J. Pieters, K. Wasano, I. Kuwabara, F.-T.
Liu, M. Cantz, A. J. R. Heck and H.-J. Gabius, Oncogene, 2003, 22,
6277–6288; (d ) A. V. Timoshenko, I. V. Gorudko, O. V. Maslakova,
S. André, I. Kuwabara, F.-T. Liu, H. Kaltner and H.-J. Gabius, Mol.
Cell. Biochem., 2003, 250, 139–149.
10 (a) S. André, C. Unverzagt, S. Kojima, X. Dong, C. Fink, K. Kayser
and H.-J. Gabius, Bioconjugate Chem., 1997, 8, 845–855;
(b) C. Unverzagt, S. André, J. Seifert, C. Fink, G. Srikrishnan, H. H.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 9 – 3 9 1 6
3915

View Article Online
Freeze, K. Kayser and H.-J. Gabius, J. Med. Chem., 2002, 45, 478–
491.
( f ) N. Ahmad, H.-J. Gabius, H. Kaltner, S. André, I. Kuwabara,
F.-T. Liu, S. Oscarson, T. Norberg and C. F. Brewer, Can. J. Chem.,
2002, 80, 1096–1104; (g) P. Sörme, Y. Qian, P.-G. Nyholm, H. Leffler
and U. J. Nilsson, ChemBioChem, 2002, 3, 183–189; (h) A. M. Wu,
J. H. Wu, M.-S. Tsai, J.-H. Liu, S. André, K. Wasano, H. Kaltner
and H.-J. Gabius, Biochem. J., 2002, 367, 653–664.
11 (a) H.-C. Siebert, M. Gilleron, H. Kaltner, C.-W. von der Lieth,
T. Kozár, N. V. Bovin, E. Y. Korchagina, J. F. G. Vliegenthart and
H.-J. Gabius, Biochem. Biophys. Res. Commun., 1996, 219, 205–212;
(b) H.-J. Gabius, Pharmaceut. Res., 1998, 15, 23–30; (c) J. L. Asensio,
J. F. Espinosa, H. Dietrich, F. J. Cañada, R. R. Schmidt, M. Martín-
Lomas, S. André, H.-J. Gabius and J. Jiménez-Barbero, J. Am.
Chem. Soc., 1999, 121, 8995–9000; (d ) H. Rüdiger, H.-C. Siebert,
D. Solís, J. Jiménez-Barbero, A. Romero, C.-W. von der Lieth,
T. Díaz-Mauriño and H.-J. Gabius, Curr. Med. Chem., 2000, 7, 389–
416; (e) J. M. Alonso-Plaza, M. A. Canales, M. Jiménez, J. L.
Roldán, A. García-Herrero, L. Iturrino, J. L. Asensio, F. J. Cañada,
A. Romero, H.-C. Siebert, S. André, D. Solís, H.-J. Gabius and
J. Jiménez-Barbero, Biochim. Biophys. Acta, 2001, 1568, 225–236;
12 (a) H. B. Mereyala and S. R. Gurrala, Carbohydr. Res., 1998, 307,
351–354; (b) R. Roy, S. K. Das, F. Santoyo-González, F. Hernández-
Mateo, T. K. Dam and C. F. Brewer, Chem. Eur. J., 2000, 6, 1757–
1762.
13 H.-J. Gabius, Anal. Biochem., 1990, 189, 91–94.
14 (a) H.-J. Gabius, R. Engelhardt, S. Rehm and F. Cramer, J. Natl.
Cancer Inst., 1984, 73, 1349–1357; (b) S. Kojima, S. André, E. Y.
Korchagina, N. V. Bovin and H.-J. Gabius, Pharmaceut. Res., 1997,
14, 879–886.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 9 – 3 9 1 6
3916