Synthesis and galectin-binding activities of mercaptododecyl glycosides containing a terminal β-galactosyl group

Article abstract of DOI:10.1016/j.bmcl.2010.12.049

Mercaptododecyl glycosides containing a terminal β-galactosyl group were prepared from d-galactose or from d-lactose via hexa-O-acetyl-lactal (10) as a key intermediate. Interactions of these glycolipids (5 kinds) and galectins (β-galactoside binding lectins, 6 species) were evaluated by surface plasmon resonance (SPR) method. High binding responses were observed for the lactoside, 2-deoxy-lactoside, and lactosaminide with some galectins (Gal-3, -4, -8), whereas the galactoside and 2,3-dideoxy-lactoside showed low binding activities.

Full text of DOI:10.1016/j.bmcl.2010.12.049

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1265–1269
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and galectin-binding activities of mercaptododecyl glycosides
containing a terminal b-galactosyl group
Teiichi Murakami a, , Kyoko Yoshioka b, Yukari Sato b, Mutsuo Tanaka a, Osamu Niwa b, Soichi Yabuki b
⇑
a Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan
b Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Mercaptododecyl glycosides containing a terminal b-galactosyl group were prepared from D-galactose or
Received 24 August 2010
Revised 6 December 2010
Accepted 13 December 2010
Available online 16 December 2010
from
D-lactose via hexa-O-acetyl-lactal (10) as a key intermediate. Interactions of these glycolipids
(5 kinds) and galectins (b-galactoside binding lectins, 6 species) were evaluated by surface plasmon res-
onance (SPR) method. High binding responses were observed for the lactoside, 2-deoxy-lactoside, and
lactosaminide with some galectins (Gal-3, -4, -8), whereas the galactoside and 2,3-dideoxy-lactoside
showed low binding activities.
Keywords:
Glycolipids
Galectins
Lactal
Ó 2010 Elsevier Ltd. All rights reserved.
SAM
SPR
Recognition
Lectins are a large group of carbohydrate binding proteins
widely found in nature including plants, animals, and lower organ-
isms. The galectins,1 a subfamily of lectins, are defined by shared
conserved amino-acid sequence and affinity for b-galactosides.
To date, 14 members of the family have been identified in mam-
mals, 10 of which have been detected in human organs and tissues
such as lung, liver, and small intestine. Galectins are soluble cyto-
solic proteins with molecular weight of 14–36 kDa and having only
a carbohydrate recognition domain (CRD) and no other functional
domains. They appear to be involved in a broad range of biological
events such as cell growth and adhesion,2 inflammation and im-
mune response,3 apoptosis,4 and cancer.5 Galectins display an
intriguing combination of intracellular and extracellular activities.
However, much work remains to clearly understand the mecha-
nism by which they exert these functions.
Recent biomedical studies have demonstrated the relationships
between the galectin species and diseases. Among the family,
galectin-1 and -3 have been most extensively studied, and they
can be considered as promising tumor markers since their expres-
sion has been correlated with tumor progression6 and metastasis.7
Galectin-2 was found to be associated with increased risk for myo-
cardial infarction.8 Strong expression of galectin-4 is induced with
a proceeded malignancy of breast and liver cancer,9 and galectin-8
has been reported on its relevance to prostate and lung cancer.10
Therefore, highly sensitive and selective detection of each galectin
would be desirable in clinical and biomedical area.
In recent years various detection methods of galectins have
been developed,5c,11 for example, enzyme-linked immunosorbent
assay (ELISA),12 western-blotting, fluorescence measurements,13,14
isothermal titration microcalorimetry,15 and surface plasmon reso-
nance (SPR) measurement.16 We focused on the SPR measurement
to evaluate galectin–carbohydrate interactions. The advantages of
the SPR method are high sensitivity, the elimination of the need
for labeling of the analytes, a larger reduction of measurement
time, and a real-time monitoring.
Non-specific adsorption of biological species such as protein on
solid surfaces is a ubiquitous problem in the measurement. Re-
cently we found that, in the specific interaction of concanavalin
A with maltosyl-dodecanethiolate on gold surface, triethylene gly-
col (TEG)-terminated short-alkane (C6, C8)-thiols effectively sup-
press the non-specific adsorption of various proteins.17
In a preceding paper we have reported a highly sensitive detec-
tion (nanomolar level) of galectin-4 and -8 with lactoside-protu-
berant hybrid monolayer surface.18 In the present report, to
examine the carbohydrate specificity of each galectin, we designed
and prepared four kinds of 12-thiododecyl disaccharides composed
of a terminal b-galactoside and a spacer sugar (Fig. 1) including
non-natural analogues. After construction of their self-assembled
monolayers mixed with TEG-hexanethiol on gold chips, their inter-
actions with six members of galectins (-1, -2, -3, -4, -7, and -8)
were investigated by the SPR method.
⇑
Corresponding author. Tel.: +81 29 861 9394; fax: +81 29 861 4549.
E-mail address: t-murakami@aist.go.jp (T. Murakami).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.049

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T. Murakami et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1265–1269
in 55% yield. Separable 2-deoxy-b-lactoside 12b and unsaturated
11 were also obtained in 8% and 20% yield, respectively. Attempts
to suppress the formation of 11 were unsuccessful. Deacetylation
of 11 and 12a with sodium methoxide in MeOH afforded the thiol-
lipids 13 and 14, respectively. It is noteworthy that the glycolipids
13 and 14 were more soluble in aqueous EtOH than the lactoside 7.
We turned our attention to the synthesis of lactosamine deriv-
atives because galectins strongly bind to N-acetyllactosamine.1
Such compounds would be prepared27 from the lactal via azido-
nitration developed by Lemieux et al.28 As shown in Scheme 3,
lactal 10 was treated with cerium(IV) ammonium nitrate and
sodium azide in acetonitrile to give the 2-azide-1-O-nitrates as a
Figure 1. b-Galactoside-terminated dodecanethiols.
Glycosylation of alcohols (O-glycosylation) is an essential pro-
cess for the synthesis of oligosaccharides and glycoconjugates.
We have recently reported an efficient synthesis of mercaptoalkyl
1,2-trans-glycosides from sugar peracetates using ZnCl2 as a pro-
moter of the glycosylation.19 According to this approach, 12-mer-
captododecyl b-galactoside 6 and b-lactoside 7 were readily
prepared as shown in Scheme 1. Prior to the glycosylation, 12-acet-
ylthio-1-dodecanol 3 was prepared from 12-bromododecanol with
potassium thioacetate in DMF in nearly quantitative yield.
Treatment of 3 with b-galactose pentaacetate 1 or b-lactose octaac-
etate 2 in the presence of ZnCl2 in toluene afforded the correspond-
ing b-glycosides 4 or 5. Deacetylation of 4 and 5 with sodium
methoxide in MeOH afforded the thiol-lipids 6 and 7, respectively.
For the syntheses of other 12-thiododecyl glycosides containing
a terminal b-galactosyl group, hexa-O-acetyl-lactal was employed
stereoisomeric mixture:
b-gluco 15b and -manno 16 isomers (60% combined yield). Since
the latter two isomers were inseparable, the mixture of the
azido-nitrates was treated with LiBr in CH3CN29 to give the
-glu-
co-bromide 17 and -manno-bromide 18. Although these bromides
a-gluco-15a (14% yield), and less polar
a
a
a
were separable by chromatography, partial decomposition was ob-
served. Thus the mixture of the bromides after extractive work-up
was treated with acetylthiododecanol 3 and silver perchlorate as a
promoter to give the coupling products. The products were sepa-
rated by silica-gel chromatography to give the
a-gluco-isomer 19
and the -manno-isomer 20 in 42% and 28% yields, respectively.
a
The azide group in 19 was reduced with zinc in acetic acid and
acetic anhydride to give the acetamide 21 in high yield. Finally
deacetylation with sodium methoxide in MeOH afforded the
lactosamine-type thiol-lipid 22.
as a key intermediate. D-Lactose 8 was conveniently converted to
To understand the functions of galectins and search for their nat-
ural ligands, it is necessary to know their carbohydrate specificity.
All members of the galectin family can bind to b-galactoside termi-
nated sugar chains, but their oligosaccharide specificities seem to be
different.1c,30 We designed and synthesized several mercaptodode-
cyl glycosides as described above. The galactoside 6, lactoside
(Lac) 7, and N-acetyllactosaminide (LacNAc) 22 are natural carbohy-
drates, while 2-deoxy-lactoside (Deoxy) 14 and 2,3-unsaturated
glycoside (En) 13 are non-natural disaccharides. Binding inhibition
studies have suggested31 that 4-OH and 6-OH of the galactopyrano-
syl ring and 3-OH of the glucopyranoside ring in lactose and
lactosaminoglycans are primarily responsible for interactions with
galectin-1 and -3. The X-ray crystal structures of Gal-132a and -
332b in complex with lactose or N-acetyllactosamine have confirmed
the above interactions via hydrogen bonds with the specific amino
acid residues in the carbohydrate binding pocket of galectins.
Deoxygenation of the hydroxyl group(s) would affect directly to
the binding.
hexa-O-acetyl-lactal 10 in three steps according to a literature pro-
cedure20 with slight modification as shown in Scheme 2. Thus, lac-
tose 8 was treated with acetic anhydride in the presence of a
catalytic Cu(OTf)2 (solvent-free per-acetylation)21 to give the octa-
acetate 2a (a/b = 9), which was treated in the same flask with HBr
(25% in AcOH) to give the anomeric bromide 9. The reaction was
quenched by the addition of sodium acetate, and the mixture
was poured into a suspension of zinc in AcOH in the presence of
CuSO4 to afford the lactal 10 in high overall yield.
With the lactal in hand, we examined the coupling with acetyl-
thiododecanol 3. In the acid-catalyzed reactions of O-protected
glycals with alcohols, two major reaction pathways are possible:
addition of the alcohol to the double bond to form 2-deoxy-O-gly-
cosides22 and SN20-type reaction to form 2,3-dideoxy- and unsatu-
rated glycosides (Ferrier reaction).23 When O-acetyl-protected
glycals are employed with a variety of acidic catalysts,24 2,3-unsat-
urated glycosides are generally predominated owing to the pres-
ence of reactive allylic acetate group. Indeed, reaction of the
With the glycolipids in hand, we constructed their self-assem-
bled monolayers (SAM) mixed with TEG-hexanethiol on gold
chips,33 and investigated their binding activities with human
galectins (Gal-1, -2, -3, -4, -7, and -8)34 by the SPR method using
25
lactal 10 and dodecanol 3 in the presence of BF3ꢀOEt2 or I2 gave
acetylthiododecyl 2-deoxy-a-lactoside 11 (a/b = 6) in high yield
with no 2-deoxy-lactoside (The Table in Scheme 2).
We then explored a direct preparation of 2-deoxy-lactoside from
the lactal 10. Falck reported a selective synthesis of 2-deoxy-gluco-
pyranosides from tri-O-acetyl-glucal using triphenylphophine
hydrobromide (TPHB) as a catalyst.26 We tried the TPHB-catalyzed
a BIAcore T100 instrument. The galectin (0.5 or 1.0
lM in HBS-
EP35) was injected simultaneously to the mixed SAM and the refer-
ence monolayer, the latter of which was formed by applying 10 lM
TEG-C6SH solution. In our preceding report,18 the maximum
reaction and obtained 2-deoxy-a-lactoside 12a as a major product
adsorption of Gal-4 was observed for the 4% Lac 7 in TEG-C6SH,
Scheme 1. Reagents and conditions: (a) ZnCl2, toluene, 55–65 °C, 1 h, 52–65%; (b) NaOMe, MeOH, CH2Cl2, 5 °C, 75–82%.

T. Murakami et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1265–1269
1267
Scheme 2. Reagents and conditions: (a) Ac2O, Cu(OTf)2, rt, 12 h; (b) HBr, AcOH, 5 °C; (c) Zn, CuSO4, AcOH, NaOAc, H2O, 5 °C, 90% from 8; (d) NaOMe, MeOH, CH2Cl2, 5 °C, 68–
77%.
Scheme 3. Reagents and conditions: (a) Ce(NH4)2(NO3)6, NaN3, CH3CN, ꢁ20 °C, 10 h, 74%; (b) LiBr, CH3CN, rt, 10 h; (c) AgClO4, MS4A, CH2Cl2, rt, 2 h, 70%; (d) Zn, AcOH, Ac2O,
THF, 5 °C, 2 h, 82%; (e) NaOMe, MeOH, CH2Cl2, 5 °C, 72%.
whereas the response was very low with 100% Lac 7. Thus the
binding activities were measured using the mixed SAM containing
4–6% glycolipid.36
Figure 2 shows the SPR sensorgrams of galectin binding to the
LacNAc 22 presenting surface. The ‘Diff. Response (RU)’ curve
was obtained after subtraction of the reference curve (100%

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T. Murakami et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1265–1269
Table 2
Dissociation constants (KD) of galectins for interaction with the glycolipid surfacesa
The point of measurement
for galectin binding (at 623 s)
Gal-3
Gal-4
Heterogeneous
ligand
Gal-8
Heterogeneous
ligand
Binding model 1:1 Binding
500
400
300
200
100
0
(1)
(2)
(1)
(2)
7 (Lac)
14 (Deoxy)
22 (LacNAc)
1.06
1.43
2.20
0.14
0.30
1.26
0.085
0.058
0.78
0.0016
0.037
0.00023
0.025
0.031
0.049
Gal-1
Gal-2
Gal-3
Gal-4
Gal-7
Gal-8
a
KD = kd/ka (lM).
binding models by using the evaluation software. The results were
similar; the Gal-3 curves fitted to the 1:1 binding model, and the
Gal-4 and -8 curves fitted to the heterogeneous ligand model.
These results as well as the binding activities shown in Table 1
may be correlated to the structures of the galectins: Gal-1, -2,
and -7 are simple proto-type dimers; Gal-3 is chimera-type having
a single CRD; Gal-4 and -8 are tandem-repeat type having two het-
erogeneous CRDs.
-100
-100
0
100 200 300 400 500 600 700 800 900 1000
Time (sec)
Figure 2. Differential SPR sensorgrams of galectin (Gal-1, -2, -3, -4, -7 and -8;
0.2 M) binding to the LacNAc 22 presenting surface. (The sensorgrams of Gal-1, -2,
-7 have overlapped.)
l
The kinetic parameters [association rate constant (ka (1/M s)),
dissociation rate constant (kd (1/s)), and dissociation constant (KD
(M))] were calculated from the best-fitted model curves, which
were obtained from the measurements at lower concentrations
(1–1000 nM) of the galectins (see Supplementary Tables S1–S3).
The dissociation constants (KD) are shown in Table 2, in which
interactions of Gal-4 (-8) with the glycosides consist of two com-
ponents: (1) and (2). The component (1) is in slow association
and slow dissociation, and the other (2) is in faster association
and dissociation. In the complexes of Gal-8, the slow component
(1) appears to contribute largely to the stability. These kinetic data
indicate the order of the affinities to the glycoside surfaces: Gal-
8 > -4 > -3.
TEG-C6SH). Specific binding to LacNAc 22 was observed for Gal-3,
Gal-4, and Gal-8. We defined the point for determining the amount
of bound galectin to be 23 s after the change to dissociation phase
(arrow in Fig. 2). The amounts of bound Gal-4, -8, and -3 were 274
RU, 85.5 RU, and 61.2 RU, respectively. Other galectins (Gal-1, -2,
-7) showed little or no differential response.
The binding studies were carried out with each glycolipid, and
the bound amounts of the galectins are summarized in Table 1.
The galactoside 6 showed much lower affinities to most galectins
than the Lac 7. The presence of the glucose residue in lactose would
be advantageous for binding to the CRD pocket by formation of
multiple hydrogen bondings. Gal-3, -4, and -8 bound to the Lac 7
and LacNAc 22 surfaces in much larger amounts than other galec-
tins (Gal-1, -2, -7) did. Especially, the bound amount of Gal-4 to 7
was five times larger than that of Gal-3, and that of Gal-4 to 22 was
three times larger than that of Gal-3 or Gal-8. The results also indi-
cate the feasibility of the Lac 7 and LacNAc 22 surfaces for the spe-
cific and sensitive detection of Gal-4. Gal-3, -4, and -8 showed good
affinities to the Deoxy 14, but they showed negligible specific bind-
ing to the En 13. The result suggests that the 3-OH in the adjacent
sugar would be significant for the galectin recognition, being con-
sistent with the previous reports.30
In summary, 12-mercaptododecyl b-D-galactoside and four kinds
of mercaptododecyl disaccharides composed of a terminal b-galac-
toside and a spacer sugar were prepared. Through construction of
their self-assembled monolayers mixed with TEG-C6SH on gold
chips, their binding activities with the galectins, promising tumor
markers, were evaluated by the SPR method. High binding responses
were observed for the lactoside, 2-deoxy-lactoside, and N-acetyl-
lactosaminide with Gal-3, -4, and -8. These findings would lead to
the rational design of novel potent ligands with selectivity for the
galectin species toward cancer diagnosis.
In the precedent paper,18 we fitted the SPR response curves to
the binding models by using the BIAcore T100 evaluation software
to estimate the most appropriate binding model for Gal-3, -4 and -
8 with the lactoside 7. It was indicated that the Gal-3 curve fitted
to the simple 1:1 binding model, whereas the Gal-4 and -8 fitted to
the heterogeneous ligand model, which has two or more binding
sites with different affinities. Herein we fitted the SPR response
curves of Gal-3, -4, -8 with the Deoxy 14 and LacNAc 22 to the
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.049.
References and notes
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Table 1
The bound amounts of galectins to the glycolipid surfacesa
Glycolipid
Gal-1
Gal-2
Gal-3
Gal-4
Gal-7
Gal-8
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Nbb
82.3
5.1
135.1
166.9
Nsbc
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4.2
34.1
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9.5
25.1
17.8
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2.0
13.4
11.0
Nbb
7 (Lac)
457.0
368.2
Nsbc
74.5
13 (En)
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22 (LacNAc)
Nsbc
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a
The values are the RUs (resonance units) of galectin binding to the carbohy-
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Nb: No binding detected in both the carbohydrate and the reference surface.
Nsb: Non-specific binding to the reference surface was too large to detect
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l
l
b
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33. The galactoside presenting monolayers were formed by applying the glycolipid
solution (1
ethanol) to the bare gold surface of the sensor chip (BIAcore SIA Kit Au, GE
Healthcare) for a brief time (20 s at 10
L/min ꢂ 1–3 times) and following with
the TEG-C6SH solution (10 M, in PBS added with 20% ethanol) for 3600 s at
L/min. The amounts of the glycolipid adsorbed on Au were controlled by
lM, in phosphate buffered saline (PBS; pH 7.2) added with 20%
l
l
1
l
changing the application time. The chip surface was washed sequentially with
18. Yoshioka, K.; Sato, Y.; Murakami, T.; Tanaka, M.; Niwa, O. Anal. Chem. 2010, 82,
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PBS and HBS-EP35 before the galectin binding measurement.
34. Human galectins (Gal-1, -2, -3, -4, -7 and -8) were obtained from R&D Systems,
Inc. (Minneapolis, USA).
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22. For a review, see: Marzabadi, C. H.; Franck, R. W. Tetrahedron 2000, 56, 8385.
23. For reviews, see: (a) Ferrier, R. J.; Zubkov, O. A. Org. React. 2003, 62, 569; (b)
Ferrier, R. J.; Hoberg, J. O. Adv. Carbohydr. Chem. Biochem. 2003, 58, 55.
24. Procopio, A.; Dalpozzo, R.; De Nino, A.; Maiuolo, L.; Nardi, M.; Oliverio, M.;
Russo, B. Carbohydr. Res. 2007, 342, 2125. and references cited therein.
35. HBS-EP: 10 mM HEPES buffer, 0.15 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH
7.4. HBS-EP was used as the running buffer for all SPR measurements.
36. The glycolipid solution was applied repeatedly (1–3 times) until the total
amount of the adsorbed thiol reached to around 100 Resonance Unit
(1 RU = 1 pg/mm2) by monitoring the SPR angle shift change. By this
procedure, a mixed monolayer containing 4–6% carbohydrate ligand filled
with TEG-C6SH was obtained.