Tuning the preference of thiodigalactoside- and lactosamine-based ligands to galectin-3 over galectin-1

Article abstract of DOI:10.1021/jm301677r

Inhibitors for galectin-1 and -3 were synthesized from thiodigalactoside and lactosamine by derivatization of the galactose C3. Introduction of 4-phenyl-1H-1,2,3-triazol-1-yl substituents at the thiodigalactoside C3 by CuAAC, targeting arginine-arene interactions, increased the affinity to 13 nM but yielded little selectivity. The bulkier 4-(4-phenoxyphenyl)-1H-1,2,3- triazol-1-yl substituent, however, increased the preference for galectin-3 over galectin-1 to more than 200-fold. Modeling showed more arginine-arene interactions for galectin-3 than for galectin-1. Introducing 4-phenoxyaryl groups on lactosamine had a similar effect.

Full text of DOI:10.1021/jm301677r

Brief Article
pubs.acs.org/jmc
Tuning the Preference of Thiodigalactoside- and Lactosamine-Based
Ligands to Galectin‑3 over Galectin‑1
Hilde van Hattum,† Hilbert M. Branderhorst,† Ed E. Moret,† Ulf J. Nilsson,‡ Hakon Leffler,§
and Roland J. Pieters*,†
†Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O.
Box 80082, 3508 TB Utrecht, The Netherlands
‡Centre for Analysis and Synthesis, Lund University, POB 124, 22100 Lund, Sweden
§Section MIG, Department of Laboratory Medicine, Lund University, Solvegatan 23, 22362 Lund, Sweden
̈
S
* Supporting Information
ABSTRACT: Inhibitors for galectin-1 and -3 were synthesized from thiodigalactoside and lactosamine by derivatization of the
galactose C3. Introduction of 4-phenyl-1H-1,2,3-triazol-1-yl substituents at the thiodigalactoside C3 by CuAAC, targeting
arginine−arene interactions, increased the affinity to 13 nM but yielded little selectivity. The bulkier 4-(4-phenoxyphenyl)-1H-
1,2,3-triazol-1-yl substituent, however, increased the preference for galectin-3 over galectin-1 to more than 200-fold. Modeling
showed more arginine−arene interactions for galectin-3 than for galectin-1. Introducing 4-phenoxyaryl groups on lactosamine
had a similar effect.
phenoxyphenyl, were coupled to a TDG azide by copper
assisted alkyne−azide cycloaddition (CuAAC).26 First, the
affinity of galectin-1 and -3 was strongly increased to the
nanomolar range. Modeling studies indicated that the
established arginine−arene interactions could be responsible
for this phenomenon. Moreover, galectin-3 bound 230 times
stronger to TDG with larger aromatic groups than galectin-1,
resulting in a selective inhibitor for galectin-3. In a parallel
approach, lactosamine (LacNAc), for which galectin-3 has an
affinity of 69 μM,27 was also derivatized to investigate whether a
similar effect could be achieved. LacNAc is hydrolytically less
stable than TDG, but the carbohydrate is easier to derivatize
with fluorescent labels or radiolabels for biological studies via its
reducing end. X-ray studies indicated that the two carbohy-
drates bind similarly to galectin-1,28 and therefore, LacNAc was
derivatized on the Gal-C3 position with 4-phenoxyaryl via an
ether or a triazole linkage and an aromatic phthaloyl or benzoyl
group was attached to the N2 to mimic the symmetric nature of
TDG. Additionally, the LacNAc derivatives contained an
azidopropyl spacer to enable their conjugation for biological
studies. Several LacNAc derivatives showed similar selectivity
for galectin-3 as their TDG counterparts; however, the affinities
were significantly lower to around 1 μM in the best cases.
INTRODUCTION
■
The protein family of galectins is defined by their affinity for β-
galactosides and their sequence homology.1 They function
intracellularly and extracellularly, and they are involved in many
biological processes such as cell signaling2 and adhesion.3
Moreover, they play a role in various pathological pathways like
metastasis,4 tumor cell survival,5 apoptosis,6 and inflammatory
response.7,8 However, the exact biological role of the various
galectins remains to be elucidated. Selective inhibitors for
different galectins would therefore be very beneficial to
deciphering the modes of action of these proteins, considering
the reported opposite roles of galectins-1 and -3.5 Furthermore,
selective inhibitors are important for the possible development
of therapeutics.
Previous (selective) inhibitor studies focused on peptide and
carbohydrate ligands.8−12 The peptidic structures include
pentapeptides13 and the antiangiogenic peptide anginex,14
which was recently found to enhance the binding affinity of
galectin-1 for glycoproteins.15 The carbohydrate-based ligands
have been used in a multivalent format to gain affinity.16−18
Besides this, optimization of monovalent carbohydrates yielded
nanomolar inhibitors, as shown by Nilsson et al., due to the
introduction of aromatic groups on the galactose C3 position
establishing favorable cation−π interactions with arginine
residues.19−24 Moreover, a triazole linkage between the
carbohydrate and the aromatic moiety instead of an ether,
ester, or amide linkage increased the affinity even further.25
Previous studies with proteomics probes carrying bulky
galactose C3 substituents had indicated that galectin-3
selectivity might be achievable using this strategy.25 These
foundations led to our aim to synthesize selective galectin-3
inhibitors based on thiodigalactoside (TDG) for which the
protein has an affinity of 43 μM.19 Three aromatic groups
differing in size, namely, phenyl, 3-hydroxyphenyl and 4-
CHEMISTRY
■
Synthesis of Thiodigalactoside Derivatives. The
thiodigalactoside-based inhibitors were synthesized from a
common thiodigalactoside intermediate with azide moieties on
the C3 and C3′ positions to allow the introduction of alkyne
containing aromatic groups through CuAAC (Scheme 1). To
this end, 3-3′-azidothiodigalactoside 1 was synthesized as
Received: November 14, 2012
Published: January 3, 2013
© 2013 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
a
Scheme 1
Scheme 2
a
Reaction conditions: (a) phenylacetylene, 3-hydroxyphenylacetylene,
or 1-ethynyl-4-phenoxybenzene, CuSO4, sodium ascorbate, DMF/
H2O, 80 °C, microwave (86−100%); (b) NaOMe, MeOH (86−
100%).
previously described25 in two steps by bromination of the
anomeric center of 3-azidogalactoside29 followed by glyco-
sylation with the thiourea treated 3-azidogalactoside acceptor.
Several aromatic groups varying in size and hydrophilicity were
subsequently coupled to 1 using CuAAC under microwave
irradiation to give the phenyl-, 3-hydroxylphenyl-, and 4-
phenoxyphenyl derivatives 2, 3, and 4 in good to excellent
yields. Finally, deacetylation of the hydroxyl groups yielded the
free thiodigalactosides 5, 6, and 7.
Synthesis of 4-(4-Phenoxybenzyl) Ether Linked
Lactosamine. Larger aromatic groups on TDG resulted in
higher selectivity of galectin-3 vs galectin-1. Therefore,
phenoxyaryl groups were introduced on the lactosamine
derivatives. The LacNAc derivatives with an ether-linked
aromatic moiety were obtained by the glycosylation of
derivatized galactoside and glucosamine (Scheme 2). First,
the known thiomethylgalactoside donor 830 was treated with
dibutyltin oxide to form the C3 to C4 stannylideneacetal,31
which was then functionalized with 4-phenoxybenzene bromide
on C3 and acetylated to give compound 9. Acceptor 11 was
synthesized from the fully protected glucosamine 10,32 which
was glycosylated with 3-bromopropanol using BF3·OEt2
followed by bromide displacement with sodium azide,
deacetylation, and selective low temperature acetylation of C6.
Target 12 was obtained by glycosylation of the galactoside
donor 9 and glucosamine acceptor 11 and careful deacetylation
without removal of the phthaloyl protection group. Lactos-
amine derivative 13 was obtained by removal of the phthaloyl
group of 12 with ethylenediamine followed by benzoylation.
Synthesis of 4-(4-Phenoxyphenyl)triazole-Linked Lac-
tosamine. For the triazole-linked lactosamine derivatives the
known 3-azidogalactoside 1429 and the bromide 16 were the
starting point (Scheme 3). Compound 14 was reacted with
(methylthio)trimethylsilane to give the desired methylthio
donor 15. The acceptor 17 was synthesized from 16 by low-
temperature acetylation.
In this route, the bromide was not displaced by an azide
immediately to avoid the formation of a diazidolactosamine
after glycosylation. Glycosylation of 15 and 17 was performed
as before and yielded the desired 1,4-linked disaccharide as the
major product and the 1,3-isomer as a side product. This
mixture was coupled with 1-ethynyl-4-phenoxybenzene by
CuAAC followed by azide introduction and careful deacetyla-
tion without removal of the phthaloyl protection group.
Preparative HPLC yielded isomerically pure 18. The benzoyl
analogue 19 was prepared by removal of the phthaloyl group
and subsequent benzoylation of the nitrogen.
a
Reaction conditions: (a) (i) Bu2SnO, MeOH; (ii) 4-phenoxybenzene
bromide, NBu4Br, benzene; (iii) Ac2O, py, 22%; (b) 3-bromo-1-
propanol, BF3·Et2O, CH2Cl2, 81%; (c) NaN3, DMF, 91%; (d)
NaOMe, MeOH, quant; (e) AcCl, 2,4,6-trimethylpyridine, CH2Cl2,
quant, −60 °C → −20 °C; (f) TfOH, NIS, CH2Cl2, 80%; (g) NaOMe,
MeOH, 73%; (h) (i) ethylene diamine, n-BuOH; (ii) Bz2O, DiPEA,
MeCN, 95%.
AFFINITY STUDIES
■
The binding affinities of galectin-1 and -3 for 5−7, 12, 13, 18,
and 19 and reference compounds thiodigalactoside and
LacNAc-β-OMe were evaluated by a fluorescent polarization
assay (Table 1).33 The symmetrical thiodigalactoside deriva-
tives 5−7 strongly benefited from the introduction of aromatic
substituents in comparison to the parent compound. The Kd of
galectin-3 for TDG was 43 μM and decreased 1000−2000
times to 44 and 22 nM, respectively, for 5 and 6. For galectin-1
the enhancement was between 500 and 1850 times for the
same compounds. Interestingly, the bulkier 7 showed different
behavior. Its Kd for galectin-3 was still submicromolar with 0.36
μM, but for galectin-1 the affinity was even worse than the free
TDG with a Kd of 84 μM. The result was that 7 has a 230-fold
preference for galectin-3. For the LacNAc derivatives (12, 13,
18, 19) that all contained the bulky phenoxyaryl group, several
effects are apparent. First, three of the four compounds showed
similar galectin-3 affinities of around 1−2 μM. The ether-linked
phenoxybenzyl group of 12 and 13 strongly reduced galectin-1
binding with Kd of 280 and 180 μM, respectively. This resulted
in a selectivity factor of 230 for 12. The triazole-linked
phenoxyphenyl group (18 and 19) reduced the galectin-1
affinity less than in the TDG series. Additional observations
include the affinity of galectin-1 benefiting more from a
triazole-linked than an ether-linked phenoxyaryl and at least in
one case (12) galectin-3 binding benefiting from a phthaloyl on
the N2 over the benzoyl group.
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Journal of Medicinal Chemistry
Brief Article
a
cation−π interactions with the phenyl and triazole groups that
could explain its enhanced affinities for both galectins in
comparison to TDG. Interestingly, galectin-3 was modeled to
have three arginine−arene interactions with 7 while for
galectin-1 there was only one, which might account for the
large observed differences in affinity (Figure 1). Additionally,
the larger structures could cause steric hindrance and hence
lower the affinity of galectin-1; however, this is not apparent
from the computational data.
Scheme 3
a
Reaction conditions: (a) MeSSiMe3, TMSOTf, DCE, 71%; (b) AcCl,
2,4,6-trimethylpyridine, CH2Cl2, 79%; (c) TfOH, NIS, CH2Cl2, 64%;
(d) 1-ethynyl-4-phenoxybenzene, CuSO4, sodium ascorbate, DMF/
H2O, 70%; (e) NaN3, DMF, 83%; (f) NaOMe, MeOH, 84%; (g) (i)
ethylenediamine, n-BuOH; (ii) Bz2O, DiPEA, MeCN, 73%.
Table 1. Kd Values of Galectin-1 and -3 for the
Thiodigalactoside and Lactosamine Derivatives As Measured
by a Fluorescence Polarization Assay
Figure 1. PoseView of 7 positioned in galectin-3 via molecular
modeling in which Arg168, Arg186, and Arg144 interact with the
aromatic substituents.
galectin-1
Kd (μM)
galectin-3
Kd (μM) rel aff ratio
a
compd
TDG
rel aff
CONCLUSIONS
■
24
1
43
1
0.6
1
Aromatic substitution at C3 of TDG with 4-aryltriazol groups
results in potent galectin-3 inhibitors. Moreover, larger
aromatic groups provide selective inhibitors for galectin-3 vs
-1, presumably because of a larger number of favorable aryl−
arginine interactions for galectin-3 and possible steric impedi-
ments for galectin-1. Furthermore, substitution of LacNAc on
the C3 position and the N2 similarly resulted in selectivity
especially for 12. These results indicate that the newly
synthesized TDG and LacNAc inhibitors can be very useful
for the biological investigations of galectin-3 or possibly for
therapeutics development. Attempts in the former direction
have already been made with the conjugation of TDG and
LacNAc derivatives to DOTA chelators for radiolabeling. These
results will be presented in due course.
5
6
7
0.049 0.012
0.013 0.004
84 46
490
1850
0.3
1
0.044 0.005
0.022 0.007
0.36 0.09
69
980
1950
120
1
0.6
230
1
LacNAc-
β-OMe
70
12
13
18
19
280
8
0.3
0.4
2.5
10
1.2 0.13
8.1 0.92
2.2 0.29
2.8 0.69
58
8.5
31
25
230
22
180 25
28 7.6
6.7 1.0
13
2.4
a
Galectin-3 preference, Kd(gal-1)/Kd(gal-3).
To better understand the experimental observations,
computer simulations of the ligands were undertaken following
a rigid docking protocol (see Supporting Information). The
lowest energy structures obtained for reference compounds
TDG and LacNAc in galectin-1 and -3 by rigid docking
mimicked the known X-ray structures of Gal-X-TDH and Gal-
Y-LacNAc-OX with rmsd of ∼1.1 Å (Gal-1 PDB code 3t2t,
Gal-3 PDB code 1kjr), and the calculated affinities accurately
resembled the experimentally measured data.27,34 The calcu-
lated free energy of binding for the newly synthesized
compounds showed a similar trend as the experimental Kd;
however, the trends were less pronounced. Modeling of 5
showed similar interactions with the two galectins. Arg73 for
galectin-1 and Arg186 and Arg144 for galectin-3 showed
EXPERIMENTAL SECTION
■
General 1,3-Cycloaddition of 3-Azidothiodigalactoside. 3-
Azidothiodigalactoside 1 (75 mg, 114 μmol), alkyne (58 mg pf
phenylacetylene, 67 mg of 3-hydroxyphenylacetylene, or 111 mg of 1-
ethynyl-4-phenoxybenzene, 570 μmol), CuSO4·5H2O (27 mg, 108
μmol), and sodium ascorbate (23 mg, 116 μmol) were dissolved in
DMF/H2O (5 mL, 4:1). The 1,3-cycloaddition reaction was
performed under microwave irradiation at 80 °C for 40 min.
Subsequently, the solvent was evaporated and the residue was
redissolved in CH2Cl2 (100 mL), washed with H2O (50 mL) and
saturated aqueous NaCl (50 mL), dried over Na2SO4, and filtered. The
residue was purified by silica chromatography (CH2Cl2/MeOH 49:1),
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Journal of Medicinal Chemistry
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and products 2−4 were obtained as white solids. Bis-{2,4,6-tri-O-
acetyl-3-deoxy-3-[4-(phenyl)-1H-1,2,3-triazol-1-yl]-β-D-
galactopyranosyl}sulfane 2: yield 100 mg, quant, Rf = 0.6 (CH2Cl2/
MeOH 19:1). Bis-{2,4,6-tri-O-acetyl-3-deoxy-3-[4-(3-hydroxyphenyl)-
1H-1,2,3-triazol-1-yl]-β-D-galactopyranosyl}sulfane 3: yield 97 mg,
quant, Rf = 0.29 (CH2Cl2/MeOH 19:1). Bis-{2,4,6-tri-O-acetyl-3-
deoxy-3-[4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl]-β-D-
galactopyranosyl}sulfane 4: yield 102 mg, 86%, Rf = 0.29 (CH2Cl2/
MeOH 19:1).
Deacetylation of 3-Triazolylthiodigalactoside. Compounds
2−4 (15 μmol) were separately dissolved in MeOH (5 mL) and
treated with NaOMe (100 μL, 30% w/v in MeOH) for 2.5 h. The
mixture was neutralized with DOWEX H+ resin, filtered, and
concentrated in vacuo. Products 5−7 were obtained after preparative
HPLC (elution gradient of 5% MeCN and 0.1% TFA in H2O to 5%
H2O and 0.1% TFA in MeCN) as white solids. Bis-{3-deoxy-3-[4-
(phenyl)-1H-1,2,3-triazol-1-yl]-β-D-galactopyranosyl}sulfane 5: Yield 9
mg, 98%. HRMS (m/z): calcd for C28H32N6O8S [M + H]+ 613.2080;
found 613.2045. Bis-{3-deoxy-3-[4-(3-hydoxyphenyl)-1H-1,2,3-triazol-
1-yl]-β-D-galactopyranosyl}sulfane 6: yield 7.5 mg, 86%. HRMS (m/
z): calcd C28H32N6O10S [M + H]+ 645.1979; found 645.1877. Bis-{3-
deoxy-3-[4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl]-β-D-
galactopyranosyl}sulfane 7: yield 12 mg, quant. HRMS (m/z): calcd
C40H40N6O10S [M + H]+ 797.2605; found 797.2514.
[3-O-(4-Phenoxybenzyl)-β-D-galactopyranosyl]-β-(1,4)-2-
deoxy-2-N-phthalimido-3-azidopropyl-β-D-glucopyranoside
12. Donor 9 (295 mg, 0.57 mmol) and acceptor 11 (206 mg, 0.47
mmol) were azeotropically dried by triple coevaporation with toluene.
The reagents and NIS (160 mg, 1.5 mmol) were dissolved in dry
CH2Cl2 (15 mL), and the mixture was stirred under N2 flow in the
presence of molecular sieves (4 Å, 2.5 g) at −50 °C. After 15 min triflic
acid (8.3 μL, 94 μmol) was added and the mixture was stirred for 1 h.
The mixture was diluted with CH2Cl2 (100 mL) and washed with
saturated aqueous Na2S2O5 (50 mL) and the organic layer dried over
Na2SO4, filtered, and evaporated. Silica chromatography (Hex/EtOAc
2:1 to 1:2) yielded the disaccharide 12I as white foam (80%, 342 mg).
Rf = 0.41 (Hex/EtOAc 1:1). Subsequently, NaOMe (17 μL, 30% w/v
in MeOH) was added to a solution of the dissaccharide (35 mg, 39
μmol) in MeOH (14 mL) and the mixture was stirred for 6 h at rt.
The solution was neutralized with DOWEX-H+ resin, filtered, and
evaporated. 12 was obtained as a white solid after preparative HPLC
(73%, 21 mg). HRMS (m/z): calcd C36H40N4O13 [M + Na]+
759.2490; found 759.2418.
μmol) were dissolved in DMF/H2O (19:1) and reacted at 80 °C
under microwave irradiation for 40 min. The solvents were evaporated.
The residue was redissolved in CH2Cl2 (50 mL) and washed with H2O
(25 mL) and aqueous NaCl (25 mL, saturated). The organic layer was
dried over Na2SO4, filtered, and concentrated. Compound 18II was
obtained after silica chromatography (Hex/EtOAc 1:1) as a solid (62
mg, 70%). Rf = 0.63 (Hex/EtOAc 1:3). Compound 18II (50 mg, 51
μmol) and NaN3 (16 mg, 73 μmol) were dissolved in dry DMF (1.4
mL) and stirred at 100 °C for 18 h. The organic solvent was
evaporated, and the residue was redissolved in EtOAc (50 mL) and
washed with H2O (50 mL) and saturated aqueous NaCl (50 mL). The
organic layer was dried over Na2SO4, filtered, and evaporated. The
crude product was subjected to silica chromatography (Hex/EtOAc
1:1) to give 18III as a clear oil (83%, 40 mg). Rf = 0.51 (Hex/EtOAc
1:3). NaOMe (12.5 μL, 30% w/v in MeOH) was added to a solution
of 18III (43 mg, 39 μmol) in MeOH (17 mL). The mixture was
stirred for 1.5 h at rt. The solution was neutralized with DOWEX-H+
resin, filtered, and evaporated. Compound 18 was obtained as a white
solid after preparative HPLC (84%, 29 mg). HRMS (m/z): calcd for
C37H39N7O12 [M + H]+ 774.2735; found 774.2654.
3-[4-(4-Phenoxyphenyl)-1H-1,2,3-triazol-1-yl]-3-deoxy-β-D-
galactopyranosyl)-β(1,4)-2-deoxy-2-N-benzamido-3-azido-
propyl-β-D-glucopyranoside 19. A solution of 18 (10 mg, 13
μmol) in n-butanol (2.5 mL) and ethylenediamine (0.5 mL) was
heated to 80 °C for 20 h. Then the solvent was evaporated and the
compound was purified by column chromatography (CH2Cl2/MeOH
4:1). The free N2 was immediately protected with benzoic anhydride
(12.8 mg, 54 μmol) in acetonitrile (2 mL) and DiPEA (17 μL) for 1 h.
After preparative HPLC 19 was obtained as a white solid (73%, 7.3
mg). HRMS (m/z): calcd for C36H41N7O11 [M + H]+ 748.2942; found
748.2892.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, characterization data, and modeling
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +31-6-202 933 87. Email: r.j.pieters@uu.nl.
[3-O-(4-Phenoxybenzyl)-β-D-galactopyranosyl]-β-(1,4)-2-
deoxy-2-N-benzamido-3-azidopropyl-β-D-glucopyranoside 13.
A solution of 12 (4 mg, 5 μmol) in n-butanol (2.5 mL) and
ethylenediamine (0.5 mL) was heated to 80 °C for 20 h. Subsequently,
the solvent was evaporated and the compound was purified by column
chromatography (CH2Cl2/MeOH 4:1). The nitrogen was immediately
protected with benzoic anhydride (9.4 mg, 41 μmol) in acetonitrile (2
mL) and DiPEA (12 μL) for 1 h. Column chromatography (CH2Cl2/
MeOH 9:1) yielded 13 as a white solid (95%, 3.4 mg). HRMS (m/z):
calcd C35H42N4O12 [M + H]+ 711.2877; found 711.2869.
3-[4-(4-Phenoxyphenyl)-1H-1,2,3-triazol-1-yl]-3-deoxy-β-D-
galactospyranosyl)-β(1,4)-2-deoxy-2-N-phthalimido-3-azido-
propyl-β-D-glucopyranoside 18. Donor 15 (620 mg, 1.7 mmol)
and acceptor 17 (675 mg, 1.43 mmol) were azeotropically dried by
coevaporation with toluene. Then the reagents were dissolved in dry
CH2Cl2 (30 mL) and combined with NIS (482 mg, 2.1 mmol). The
mixture was stirred under N2 flow with activated molecular sieves (4 Å,
6 g) at −50 °C. After 15 min triflic acid (32 μL, 0.36 mmol) was added
and the mixture was stirred for 1 h. The mixture was diluted with
CH2Cl2 (100 mL) and filtered to remove the molecular sieves. The
mixture was washed with saturated aqueous Na2S2O5 (50 mL) and the
organic layer dried over Na2SO4, filtered, and evaporated. Silica
chromatography (Hex/EtOAc 1:1) yielded the disaccharide 18I as a
white foam (1 g, 64%) with galactospyranosyl-β(1,3)-glucopyranoside
as side product. Rf = 0.15 (Hex/EtOAc 1:1). Disaccharide 18I (75 mg,
90 μmol), 1-ethynyl-4-phenoxybenzene (36 mg, 180 mmol),
CuSO4·5H2O (12 mg, 45 μmol), and sodium ascorbate (9 mg, 45
Notes
The authors declare the following competing financial
interest(s): U.J.N. and H.L. own shares in Galacto Biotech AB.
ACKNOWLEDGMENTS
■
The authors thank Barbro Kahl-Knutson for galectin
production and binding analysis, Mirjam Damen for HRMS
analysis, and Johan Kemmink for HSQC NMR. This research is
supported by the Dutch Technology Foundation STW, Applied
Science Division of NWO, and the Technology Program of the
Ministry of Economic Affairs, the Swedish Research Council
(Grants 621-2009-5656 to H.L. and 621-2003-4265 to U.J.N.),
and European Community’s Seventh Framework Programme
[FP7-2007-2013] under Grant Agreement HEALTH-F2-2011-
256986 (PANACREAS).
REFERENCES
■
(1) Di Lella, S.; Sundblad, V.; Cerliani, J. P.; Guardia, C. M.; Estrin,
D. A.; Vasta, G. R.; Rabinovich, G. A. When galectins recognize
glycans: from biochemistry to physiology and back again. Biochemistry
2011, 50, 7842−7857.
(2) Levy, R.; Biran, A.; Poirier, F.; Raz, A.; Kloog, Y. Galectin-3
mediates cross-talk between K-Ras and Let-7c tumor suppressor
microRNA. PLoS One 2011, 11, e27490.
1353
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Journal of Medicinal Chemistry
Brief Article
(3) Elola, M. T.; Wolfenstein-Todel, C.; Troncoso, M. F.; Vasta, G.
R.; Rabinovich, G. A. Galectins: matricellular glycan-binding proteins
linking cell adhesion, migration, and survival. Cell. Mol. Life Sci. 2007,
13, 1679−1700.
(20) Cumpstey, I.; Salomonsson, E.; Sundin, A.; Leffler, H.; Nilsson,
U. J. Double affinity amplification of galectin−ligand interactions
through arginine−arene interactions: synthetic, thermodynamic, and
computational studies with aromatic diamido thiodigalactosides.
Chem.Eur. J. 2008, 14, 4233−4245.
(4) Carlsson, M. C.; Cederfur, C.; Schaar, V.; Balog, C. I.; Lepur, A.;
(21) Delaine, T.; Cumpstey, I.; Ingrassia, L.; Le Mercier, M.;
Okechukwu, P.; Leffler, H.; Kiss, R.; Nilsson, U. J. Galectin-inhibitory
thiodigalactoside ester derivatives have antimigratory effects in
cultured lung and prostate cancer cells. J. Med. Chem. 2008, 51,
8109−8114.
Touret, F.; Salomonsson, E.; Deelder, A. M.; Ferno, M.; Olsson, H.;
̈
Wuhrer, M.; Leffler, H. Galectin-1-binding glycoforms of haptoglobin
with altered intracellular trafficking, and increase in metastatic breast
cancer patients. PLoS One 2011, 6, e26560.
(5) Liu, F. T.; Rabinovich, G. A. Galectins as modulators of tumour
(22) Salameh, B. A.; Cumpstey, I.; Sundin, A.; Leffler, H.; Nilsson, U.
J. 1H-1,2,3-Triazol-1-yl thiodigalactoside derivatives as high affinity
galectin-3 inhibitors. Bioorg. Med. Chem. 2010, 18, 5367−5378.
(23) Mackinnon, A. C.; Gibbons, M. A.; Farnworth, S. L.; Leffler, H.;
Nilsson, U. J.; Delaine, T.; Simpson, A. J.; Forbes, S. J.; Hirani, N.;
Gauldie, J.; Sethi, T. Regulation of transforming growth factor-β1-
driven lung fibrosis by galectin-3. Am. J. Respir. Crit. Care Med. 2012,
185, 537−546.
(24) Volarevic, V.; Milovanovic, M.; Ljujic, B.; Pejnovic, N.;
Arsenijevic, N.; Nilsson, U.; Leffler, H.; Lukic, M. L. Galectin-3
deficiency prevents concanavalin A-induced hepatitis in mice.
Hepatology 2012, 55, 1954−1964.
progression. Nat. Rev. Cancer 2005, 5, 29−41.
(6) Nakahara, S.; Oka, N.; Raz, A. On the role of galectin-3 in cancer
apoptosis. Apoptosis 2005, 10, 267−275.
(7) Rabinovich, G. A.; Liu, F. T.; Hirashima, M.; Anderson, A. An
emerging role for galectins in tuning the immune response: lessons
from experimental models of inflammatory disease, autoimmunity and
cancer. Scand. J. Immunol. 2007, 66, 143−158.
(8) Pieters, R. J. Inhibition and detection of galectins. ChemBioChem
2006, 7, 721−728.
(9) Ingrassia, L.; Camby, I.; Lefranc, F.; Mathieu, V.;
Nshimyumukiza, P.; Darro, F.; Kiss, R. Anti-galectin compounds as
potential anti-cancer drugs. Curr. Med. Chem. 2006, 13, 3513−3527.
(25) van Scherpenzeel, M.; Moret, E. E.; Ballell, L.; Liskamp, R. M. J.;
Nilsson, U. J.; Leffler, H.; Pieters, R. J. Synthesis and evaluation of new
thiodigalactoside-based chemical probes to label galectin-3. Chem-
BioChem. 2009, 10, 1724−1733.
̀
(10) Giguere, D.; Sato, S.; St-Pierre, C.; Sirois, S.; Roy, R. Aryl O-
and S-galactosides and lactosides as specific inhibitors of human
galectins-1 and -3: role of electrostatic potential at O-3. Bioorg. Med.
Chem. Lett. 2006, 16, 1668−1672.
(26) Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on
solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-
dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem.
2002, 67, 3057−3064.
́
(11) Andre, S.; Jarikote, D. V.; Yan, D.; Vincenz, L.; Wang, G. N.;
Kaltner, H.; Murphy, P. V.; Gabius, H. J. Synthesis of bivalent
lactosides and their activity as sensors for differences between lectins
in inter- and intrafamily comparisons. Bioorg. Med. Chem. Lett. 2012,
22, 313−318.
(27) Sorme, P.; Arnoux, P.; Kahl-Knutsson, B.; Leffler, H.; Rini, J. M.;
̈
Nilsson, U. J. Structural and thermodynamic studies on cation−π
interactions in lectin−ligand complexes: high-affinity galectin-3
inhibitors through fine-tuning of an arginine−arene interaction. J.
Am. Chem. Soc. 2005, 127, 1737−1743.
́
(12) Andre, S.; Maljaars, C. E.; Halkes, K. M.; Gabius, H. J.;
Kamerling, J. P. Discovery of galectin ligands in fully randomized
combinatorial one-bead-one-compound (glyco)peptide libraries. Bio-
org. Med. Chem. Lett. 2007, 17, 793−798.
(28) Bianchet, M. A.; Ahmed, H.; Vasta, G. R.; Amzel, L. M. Soluble
beta-galactosyl-binding lectin (galectin) from toad ovary: crystallo-
graphic studies of two protein−sugar complexes. Proteins 2000, 15,
378−388.
́
(13) Arnusch, C. J.; Andre, S.; Valentinia, P.; Lensch, M.;
Russwurmb, R.; Siebert, H.-C.; Fischer, M. J. E.; Gabius, H.-J.;
Pieters, R. J. Interference of the galactose-dependent binding of lectins
by novel pentapeptide ligands. Bioorg. Med. Chem. Lett. 2004, 14,
1437−1440.
(29) Lowary, T. L.; Hindsgaul, O. Recognition of synthetic O-methyl,
epimeric, and amino analogues of the acceptor α-L-Fucp-(1→2)-β-D-
Galp-OR glycosyltransferases. Carbohydr. Res. 1994, 251, 33−67.
(30) Koike, K.; Sugimoto, M.; Sato, S.; Ito, Y.; Nakahara, Y.; Ogawa,
T. Total synthesis of globotriaosyl-E and Z-ceramides and
isoglobotriaosyl-E-ceramide. Carbohydr. Res. 1987, 163, 189−208.
(31) Ballell, L.; Joosten, J. A. F.; Ait el Maate, F.; Liskamp., R. M. J.;
Pieters, R. J. Microwave-assisted, tin-mediated, regioselective 3-O-
alkylation of galactosides. Tetrahedron Lett. 2004, 45, 6685−6687.
(32) Zhang, G.; Guan, Z.; Zhang, L.; Min, J.; Zhang, L. Synthesis of
2-amino-2-deoxy-β-glycosyl-(1→5)-nucleosides and the interaction
with RNA. Bioorg. Med. Chem. 2003, 11, 3273−3278.
(14) Thijssen, V. L.; Postel, R.; Brandwijk, R. J.; Dings, R. P.;
Nesmelova, I.; Satijn, S.; Verhofstad, N.; Nakabeppu, Y.; Baum, L. G.;
Bakkers, J.; Mayo, K. H.; Poirier, F.; Griffioen, A. W. Galectin-1 is
essential in tumor angiogenesis and is a target for antiangiogenesis
therapy. Proc. Natl. Acad. Sci. U.S.A. 2006, 43, 15975−15980.
(15) Salomonsson, E.; Thijssen, V. L.; Griffioen, A. W.; Nilsson, U. J.;
Leffler, H. The anti-angiogenic peptide anginex greatly enhances
galectin-1 binding affinity for glycoproteins. J. Biol. Chem. 2011, 286,
13801−13804.
́
(16) Andre, S.; Pieters, R. J.; Vrasidas, I.; Kaltner, H.; Kuwabara, I.;
Liu, F. T.; Liskamp, R. M. J.; Gabius, H. J. Wedgelike glycodendrimers
as inhibitors of binding of mammalian galectins to glycoproteins,
lactose maxiclusters, and cell surface glycoconjugates. ChemBioChem
2001, 2, 822−830.
(33) Sorme, P.; Kahl-Knutsson, B.; Huflejt, M.; Nilsson, U. J.; Leffler,
̈
H. Fluorescence polarization as an analytical tool to evaluate galectin−
ligand interactions. Anal. Biochem. 2004, 334, 36−47.
̈
(34) Collins, P. M.; Oberg, C. T.; Leffler, H.; Nilsson, U. J.;
́
(17) Andre, S.; Ortega, P. J.; Perez, M. A.; Roy, R.; Gabius, H. J.
Blanchard, H. Taloside inhibitors of galectin-1 and galectin-3. Chem.
Biol. Drug Des. 2012, 79, 339−346.
Lactose-containing starburst dendrimers: influence of dendrimer
generation and binding-site orientation of receptors (plant/animal
lectins and immunoglobulins) on binding properties. Glycobiology
1999, 9, 1253−1261.
́
(18) Vrasidas, I.; Andre, S.; Valentini, P.; Bock, C.; Lensch, M.;
̈
Kaltner, H.; Liskamp, R. M.; Gabius, H. J.; Pieters, R. J. Rigidified
multivalent lactose molecules and their interactions with mammalian
galectins: a route to selective inhibitors. Org. Biomol. Chem. 2003, 5,
803−810.
(19) Cumpstey, I.; Sundin, A.; Leffler, H.; Nilsson, U. J. C2-
symmetrical thiodigalactoside bis-benzamido derivatives as high-
affinity inhibitors of galectin-3: efficient lectin inhibition through
double arginine−arene interactions. Angew. Chem. 2005, 117, 5240−
5242.
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