Convenient assembly of trimeric Lex determinants using carbosilane scaffolds by means of Huisgen cycloaddition

Article abstract of DOI:10.1016/j.tetlet.2012.10.004

The Le^X glycoside having an N₃ moiety at the ω position, which was prepared by using living cells from GlcNAcO(CH ₂)₁₂N₃ as a starting material, was efficiently introduced into a trivalent-type carbos

Full text of DOI:10.1016/j.tetlet.2012.10.004

Tetrahedron Letters 53 (2012) 6793–6796
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Convenient assembly of trimeric Lex determinants using carbosilane scaffolds
by means of Huisgen cycloaddition q
Koji Matsuoka a, , Hiroki Yamaguchi a, Tatsuya Kohzu a, Jun-Ichi Sakamoto a, Tetsuo Koyama a,
⇑
Ken Hatano a, Shigeto Yamamoto b, Tsutomu Mori b, Kenichi Hatanaka c
a Area for Molecular Function, Division of Material Science, Graduate School of Science and Engineering, Saitama University, Sakura, Saitama 338-8570, Japan
b R&D Center, Hayashibara Co., Ltd, Fujisaki, Okayama 702-8006, Japan
c Institute of Industrial Science, The University of Tokyo, Meguro, Tokyo 153-8505, Japan
a r t i c l e i n f o
a b s t r a c t
The LeX glycoside having an N3 moiety at the
x position, which was prepared by using living cells from
Article history:
Received 29 August 2012
Revised 26 September 2012
Accepted 1 October 2012
Available online 11 October 2012
GlcNAcO(CH2)12N3 as a starting material, was efficiently introduced into a trivalent-type carbosilane core
scaffold by means of Huisgen cycloaddition reaction to yield the corresponding glycocluster having three
LeX moieties at each end. Structural elucidation of the product was performed by a combination of NMR
and mass spectroscopic analyses, and the results of the analyses supported the structure of the glycoclus-
ter. Evaluation of the glycocluster was carried out using Lotus lectin as a model lectin. Environmental
changes of tryptophan residues located at or near the binding sites of Lotus lectin caused fluorescence
intensity change.
Keywords:
Lewis X
Glycosides
Huisgen cycloaddition
Glycodendrimers
Glycoclusters
Carbosilanes
Ó 2012 Elsevier Ltd. All rights reserved.
Oligosaccharide chains in glycoconjugates, such as glycopro-
teins and glycolipids, play important roles in biological events.
Among the various oligosaccharides, Lewis X determinant [LeX;
introduced a series of glycodendrimers using carbosilanes as piv-
otal supporting tools for bioactive carbohydrate moieties.8 In this
Letter, we describe the efficient construction of a trivalent-type
carbosilane having LeX trisaccharidic determinants 2 as shown in
Figure1 and the biochemical evaluation of the glycocluster for Lotus
tetragonolobus agglutinin (LTA) by means of fluorospectroscopic
analysis.
Galb1 ? 4(Fuca1 ? 3)GlcNAc] is known as an extremely valuable
core structure of glycoconjugates.1 Recently, biocombinatorial syn-
thesis of oligosaccharides by means of animal cells in culture has
been developed, and oligosaccharide libraries of various biologi-
cally important sugars are now under investigation.2 The LeX deriv-
We have reported efficient coupling procedures in order to
build up glycodendrimers using carbosilanes through sulfide link-
ages by means of a simple SN2 reaction between an alkyl halide 4
and a thiolate 6 (Scheme 1).9 Although the sulfide linkage in 7 is
suitable and stable for biochemical and biomedical uses, an alter-
native procedure for effective coupling between a carbohydrate
moiety and a multivalent-type compound was explored. Huisgen
cycloaddition reaction10 of an azido functionalized compound
and an alkynylated compound reminded us that the reaction can
be applicable for the formation of a cluster-type compound, be-
cause of the high yield and high specificity for the coupling reac-
tion.11 Thus, an alkynylated carbosilane compound was needed
as a core scaffold. Scheme 1 summarizes an elongation reaction
of a known triol 312 compound of a carbosilane by means of the
Williamson ether synthesis. It was noted that the reaction required
3 equiv molar excess of propargyl bromide against an alcoholic
function since usual ether synthesis required slightly excess alkyl
bromide. Therefore, triol 3 was treated with 9 equiv molar of
ative 13 having an azide moiety at the
x-position of the aglycon
was isolated from the residues of cell culture using HL-60 (Human
promyelocytic leukemia cells) in the presence of an N-acetyl glu-
cosaminyl primer (GlcNAcO(CH2)12N3).4 Since synthetic assembly
of the LeX unit was paid much attention, this approach to obtain
the LeX trisaccharidic component is of great interest for biochem-
ists and medicinal scientists.5 On the other hand, a cluster-type tri-
saccharide is also very attractive from the point of view of
biochemical as well as biomedical applications.6 In our previous
study,7 a convergent chemical synthesis of the polymerizable Lex
trisaccharidic glycomonomer via Gabriel amine synthesis and the
chemical conversion into a highly clustered glycopolymer were
accomplished. In addition to linear-type glycoclusters, we have
q
Glyco-Silicon Functional Materials. Part 14. For Part 13, see Ref. 8a.
⇑
Corresponding author. Tel./fax: +81 48 858 3099.
E-mail address: koji@fms.saitama-u.ac.jp (K. Matsuoka).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.10.004

6794
K. Matsuoka et al. / Tetrahedron Letters 53 (2012) 6793–6796
OH
O
OH
O
HO
HO
N
O
OH
O
3
O
O
HL-60
NHAc
12
OH
HO
HO
O
N
3
CH3
O
NHAc
GlcNAcO(CH )
OH
OH
N
2 12
HO
3
1
OH
HO
HO
Huisgen Cycloaddition
OH
O
O
O
O
OH
N
N
N
O
H3C
N
O
NHAc
O
OH
HO
OH
OH
O
OH
O
N
N
O
O
HO
HO
SiPh
N
12
O
O
NHAc
OH
CH3
O
OH
O
N
N
OH
HO
OH
O
O
OH
O
O
NHAc
O
HO
HO
OH
OH
O
2
H3C
HO
OH
Figure 1. Synthetic assembly of trimeric Lex derivatives.
Previous study
Na
R S
6
Si
R SBn
SR
liq NH
3
Si
Br
3
5
7
3
OH
OH
4
Si
This study
(i)
OH
OH
OH
N
N
3
O
O
O
HO
HO
SiPh
(ii)
O
N
O
O
Si
O
NHAc
OH
12
CH3
O
3
OH
8
OH
HO
3
2
Scheme 1. Reagents and conditions: (i) BrCH2C„CH (9 equiv molar), NaH, DMF, 0 °C, 5 h, 66.9%, (ii) compound 1 (3.75 equiv molar), 0.1 M aq CuSO4, 1 M aq Na ascorbate,
MeOH, 50 °C, 1 d, 57.9%.
propargyl bromide in the presence of NaH in DMF and the reaction
proceeded smoothly to afford trialkynylated carbosilane 813 in
66.9% yield after chromatographic purification on silica gel with
10/1 (v/v; n-hexane–EtOAc).
glycocluster. In our ongoing synthetic study of glycoclusters, preli-
minary investigations of the glycoclusters against lectins have
been carried out by using fluorescence measurement.7,14 The spec-
tral change of the lectin due to tryptophan residues located around
the binding site of target sugars was monitored when the titrimet-
ric sugars were added to the measuring solution.15 Thus, Lotus
tetragonolobus agglutinin (LTA) was selected as a suitable lectin
Since the scaffold for supporting the carbohydrate moieties was
prepared, chemical introduction of LeX derivative 1 into the carbos-
ilane 8 was attempted under slightly modified Huisgen cycloaddi-
tion conditions. Thus, a methanolic solution of an alkyne 8 and an
azide 1 was treated with 0.1 M aq CuSO4 and 1 M aq sodium ascor-
bate at 50 °C for 24 h. Silica gel chromatography of the reaction
mixture followed by gel filtration using LH-20 gave the corre-
sponding carbosilane 2 having three LeX moieties at each end in
57.9% yield, Rf 0.47 [3:3:1 (v/v/v) CHCl3–MeOH–H2O]; 1H NMR
(CD3OD) d 7.94 [s, 3 H, 3 N–CH@ (triazole)], 5.04 [d, 3 H,
for binding to L
-fucose residue.16 The structure of LTA was recently
estimated to be a homotetramer.16c Figure2 shows fluorescence
emission of LTA and of its complexes with various concentrations
of trimeric LeX 2. When the protein was saturated with the glyco-
cluster 2, the maximum fluorescence intensity was enhanced by
7.2% and the emission maximum was not downfield-shifted, when
a monomeric LeX glycoside 10 and
L-fucose 9 slightly shifted
J1 ,2 = 3.7 Hz, 3 H-100 (Fuc)], 4.66 (s, 6H, 3@C–CH2O), 4.45 [d, 3 H,
(ꢀ2 nm). The results suggested that the environment of trypto-
phan residues located at or near the binding sites of LTA is altered
from hydrophilic to relatively more hydrophobic upon interaction
00 00
J1 ,2 = 7.7 Hz, 3H-10 (Gal)], 4.40 (t, 6H, J = 6.8 Hz, 3 CH2N), 1.97 (s,
0
0
9H, 3 NAc), 1.19 (d, 9 H, J5 ,6 = 6.3 Hz, 3 H-600 (Fuc)], MALDI-TOF
00 00
MS calcd for [M+Na+]: 2635.374; Found m/z 2635.451.
with the glycocluster 2. In a plot of DF/F0 versus [S] based on sugar
Given the success of preparation of trimetric LeX derivative 2,
our attention was turned toward the biological properties of the
unit concentration followed by analyses using the Hill equation,17
association constant Ka was estimated to be 1.2 ꢁ 106 (Mꢀ1). The

K. Matsuoka et al. / Tetrahedron Letters 53 (2012) 6793–6796
6795
0.16
(A)
(B)
(C)
0.12
0.08
0.04
0
0
0.5
1
1.5
2
[S] (μM)
1
0.5
0
-0.5
-1
-1.5
-6.8
-6.6
-6.4
-6.2
-6
-5.8
310
330
350
370
390
410
430
450
Wavelength (nm)
log [S]
Figure 2. Fluorescence intensity change of LTA by addition of
trimeric LeX 2 (34.5
M in 0.1 mM CaCl—0.05 M phosphate buffer) at 5 °C, pH 6.8. The excitation wavelength (kex) was used at 295 nm. (B) Plots of
is the change in the intensity at 335 nm of LTA with various concentrations of 2, F0 is the intensity of LTA alone, and [S] is the total ligand concentration. (C) Hill plots of log
F/( FMAX F)] versus log [S].
L-fucose. (A) Changes in fluorescence emission spectra of LTA (0.57
lM) upon addition of 20-
lL aliquots of
l
DF/F0 versus [S], where DF
[D
D
-D
Table 1
Results of binding assay for LTA on the basis of fluorescence measurements
F0a (kJ/mol)
Compounds k (nm)
D
D
F/F0 (%)
Ka (Mꢀ1
)
Hill coefficient
2.0
Relative potency
1.1
D
OH
OH
CH3
HO
O
ꢀ2
ꢀ2
6.1
8.9
22.1
21.8
1.4 ꢁ 104a
OH
9
OH
O
OH
O
OH
O
HO
HO
N
3
O
O
NHAc
5
1.3 ꢁ 104
1.7
1
CH3
O
OH
10
OH
HO
OH
O
OH
N
N
N
O
HO
HO
O
SiPh
O
O
O
OH
NHAc
12
0
7.2
—
32.4
—
1.2 ꢁ 106
1.8
—
92
—
CH3
O
OH
OH
HO
3
2
OH
O
HO
HO
b
O
—
—
NHAc
11
a
Also see Ref. 16b,18 (0.9-1.4 ꢁ 104 Mꢀ1).
b
Not determined due to no binding.
results, summarized in Table 1, clearly indicated that the lectin
Acknowledgments
showed ca. 100-fold higher affinity for the trimeric LeX 2 than that
for L
-fucose 918 and monomeric LeX glycoside 10, where a sugar-
This work was supported by a Grant for ‘Development of Novel
Diagnostic and Medical Applications through Elucidation of Sugar
Chain Functions’ from New Energy and Industrial Technology
Development Organization (NEDO).
clustering effect19 was obviously observed.
In summary, we have successfully described the preparation of
a carbosilane having trimeric Lex units by means of Huisgen cyclo-
addition and biological evaluation of the glycocluster for LTA by
using a fluorometric assay. The results clearly indicated that the
trimeric compound has the sugar-clustering effect. This methodol-
ogy includes simplicity, rapidity, and precision of construction of
glycoclusters having biologically active carbohydrate moieties of
glycoconjugates by means of Huisgen cycloaddition. In addition
to the synthesis, an appropriate combination of carbohydrate res-
idues and lectins provides a wide variety of applications based
on fluorescence measurement of lectins from the viewpoint of bio-
chemical, medicinal, and other fields.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.10.004.
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