Synthesis of multivalent N-acetyl lactosamine modified quantum dots for the study of carbohydrate and galectin-3 interactions

Article abstract of DOI:10.1016/j.tet.2012.06.035

Ternary core/shell CdSeS/ZnS-QDs coated with N-acetyl lactosamine was prepared as a fluorescent probe to study the interactions of N-acetyl lactosamine and galectin-3. The synthesis of N-acetyl lactosamine was achieved through the 'azidoiodoglycosylation'

Full text of DOI:10.1016/j.tet.2012.06.035

Tetrahedron 68 (2012) 7148e7154
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of multivalent N-acetyl lactosamine modified quantum dots
for the study of carbohydrate and galectin-3 interactions
,
Yang Yang a, Xiao-Chao Xue a, Xiao-Feng Jin a, Li-Jun Wang b, Yin-Lin Sha b, Zhong-Jun Li a
*
a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China
b Single-molecule & Nanobiology Laboratory, Department of Biophysics, School of Basic Medical Sciences and Biomed-X Center, Peking University, 38 Xueyuan Road, Beijing 100191,
China
a r t i c l e i n f o
a b s t r a c t
Article history:
Ternary core/shell CdSeS/ZnS-QDs coated with N-acetyl lactosamine was prepared as a fluorescent probe
to study the interactions of N-acetyl lactosamine and galectin-3. The synthesis of N-acetyl lactosamine
was achieved through the ‘azidoiodoglycosylation’ method. The amount of ligand coated on QDs was
determined by 1H NMR and ICP-OES. The interactions between carbohydrates and galectin-3 were
measured using SPR. The results revealed that the affinity of galectin-3 with di- and multivalent N-acetyl
lactosamine increased 20 and 184-fold, respectively. The prepared glyco-QDs could be used as an effi-
cient fluorescent probe to study carbohydrates and galectin-3 interactions.
Received 26 February 2012
Received in revised form 30 May 2012
Accepted 8 June 2012
Available online 15 June 2012
Keywords:
Glyco-quantum dots
NMR
Ó 2012 Elsevier Ltd. All rights reserved.
SPR
Galectin-3
N-Acetyl lactosamine
Cluster effect
1. Introduction
approach is the preparation of multivalent compounds, which were
designed to interact with more than one of the lectin’s subunits.
Galectin-3 is a member of the
b
-galactose-binding protein family
Pieters et al.6 synthesized two, four, and eight valent lactose clusters
using 3,5-di-(2-aminoethoxy) benzoic acid as the linking unit. The
inhibition assays of protein and cell levels showed that galectin-3
was markedly sensitive to increased sugar valency. Compared to
carrier-free lactose, the inhibitory potency of each lactose unit
reached a maximum value of 144-fold. However, the valence of
multivalent carbohydratesis determined bythe limited branchingof
the linking scaffold. Moreover, fluorescence labeling is usually
needed for the bioassay and organic dyes FITC7a and Rhodamine7b
are often conjugated to the carbohydrates as the fluorophores.
Recently, the use of quantum dots (QDs)8 to construct the gly-
coside cluster and as a fluorescent probe opens new opportunities
for studying the carbohydrateeprotein interactions.9 Our previous
work has demonstrated that binding of sugar molecules conjugated
QDs to their target proteins can be dramatically enhanced.10 It is
easy to attach more than one hundred sugar ligands on the surface
of the QDs due to its high surface/volume ratio. In addition, a new
strategy with Nuclear Magnetic Resonance (NMR) and Inductively
Coupled Plasma-Optical Emission Spectrometry (ICP-OES) has been
used to analyze the coated polysaccharide molecules and the inner
structure of the core and shell of the glyco-QDs.10,11
and plays a key role in initiating the adhesion of human breast and
prostate cancer cells to the endothelium by specifically interacting
with the cancer associated carbohydrate, T antigen.1 Moreover, it
was shown that galectin-3 possesses anti-apoptotic activity2,3 in
severaltumorcell types. The mechanism ofaction ofgalectin-3 is not
clear yet. And there is a high interest to discover high affinity in-
hibitors that could be used as tools for probing galectin-3 functions
and as lead compounds in the development of therapeutics.4c
Lactose and N-acetyl lactosamine (LacNAc) are the natural li-
gands of galectin-3, but they have weak dissociation constants in the
mM range. Two main approaches have been used to enhance their
binding affinity. One is the structural modification of LacNAc or
lactose. Nilsson et al. have reported that the 3-OH of galactoside is
located in the extended groove of the protein and chemical modi-
fications at this position can enhance the affinity greatly.4 Pieters
reported that thiodigalactoside also has strong affinity to galectin-
3.5 However, the synthesis of the high affinity free LacNAc or thio-
digalactoside is tedious, which contain complicated protection,
deprotection of the hydroxyl and conversion of the amine. The other
Herein we used the ternary core/shell CdSeS/ZnS-QDs to con-
struct multivalent N-acetyl lactosamine clusters, which has
* Corresponding author. Fax: þ86 01 8280 5496; e-mail addresses: zjli@
bjmu.edu.cn, zjli@mail.bjmu.edu.cn (Z.-J. Li).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.06.035

Y. Yang et al. / Tetrahedron 68 (2012) 7148e7154
7149
stronger binding affinity with galectin-3 than lactose. An efficient
azidoiodo-glycosylation reaction was used to synthesize LacNAc
ligand. We expect the high affinity fluorescent probe will be useful
in the study of carbohydrates and galectin-3 interactions.
needed. Under the condition d (Scheme1), two configurations4 and 5
-gluco configuration) were obtained. Unfortunately they were
(b-D
inseparable bycolumn chromatography, even after they were reacted
with the acceptor. Finally, another intermediate 3 was synthesized
first. Iodoacetoxylation of acetylated lactal 2 was achieved by reaction
of iodine in the presence of cupric acetate monohydrate in HOAc at
80 ꢀC. This reaction showed high stereoselectivity, only 3 can be
obtained by recrystallization with ethanol or purification by column
chromatography after the reaction. Treatment of 3 with trimethylsilyl
azide (TMSN3) and trimethylsilyl trifluoromethanesulfonate
(TMSOTf) afforded the donor 4 in high yield (Scheme 1). The config-
uration and structure of 4 were confirmed by 1H NMR (J1,2¼2.7 Hz),
2. Results and discussion
2.1. Synthesis of mono and bivalent N-acetyl lactosamine
It is difficult to synthesize oligosaccharide fragments containing
hexosamine because of the low activities and high steric hindrance
induced by amine protecting groups. The yield and stereoselectivity
of the glycosylation using 4-hydroxy group of N-acetyl glucosamine
derivatives as acceptors are not satisfactory.12 Therefore, lactal is
often used as the starting material in the synthesis of LacNAc. We
have reported a synthesis of divalent N-acetyl lactosamine using
lactal as the starting material, which needs much more changing of
13C NMR ( 90.71, C-1), and IR (nN3 2119 cmꢁ1).
d
After the synthesis of the donor 4, azidoiodo-glycosylation re-
action was used to synthesize the fully protected mono 9 and bi-
valent 7 LacNAc (Scheme 2). Typically, 4 and the alcohol were
dissolved in CH2Cl2 and triphenylphosphine in CH2Cl2 was added
dropwise. The reaction mixture was stirred overnight at room
temperature. Exchange with anion resin (Dowex 2X8 (OHꢁ)) and
treatment with catalytic amount of sodium methoxide in methanol
afforded the amino compound. Acetylation under classical condi-
tions (Ac2O/Py) yielded the fully protected compound. Compared
with other strategies, this method achieved glycosylation, azido
reduction, and amine acetylation in one pot and no purification was
needed. Moreover, this glycosylation reaction has high stereo-
the amino protecting groups and the a,b selectivity was influenced
by the strength of the promotor.13 In order to improve the selec-
tivity and synthesis route, Lafont et al.14 reported a convenient
method to synthesize the
b-glycosides of N-acetyl lactosamine
through an azidoiodo-glycosylation reaction. In this method 2-iodo
lactosyl azide 4 was used as the donor and PPh3 as Staudinger re-
ducer and Lewis acid. This methodology is easier to carry out and
more efficient. Herein we used this method to prepare ligands
containing N-acetyl lactosamine.
selectivity and only
b product was obtained. Deacetylation of 7 and
In order to obtain
2-deoxy-2-iodo lactosyl azides with
b
glycosylation product, intermediate 1,2-trans-
9 with sodium methoxide in methanol afforded mono 10 and bi-
valent LacNAc 8, respectively.
a-D-manno configuration 4 was
1
Lactose
a
Method d, 4 and 5 were formed
OAc
OAc
O
OAc
O
AcO
O
AcO
AcO
Method b, only 3 was formed
2
d
b
OAc
OAc
O
AcO
I
Method b and c only 4 was formed
O
O
RO
AcO
O
I+
AcO
AcO
OAc
3
d
c
OAc
OAc
O
I+
AcO
I
O
O
RO
RO
AcO
O
AcO
AcO
N3
-
4
N3
OAc
OAc
O
AcO
O
O
I
N3
AcO
O
AcO
I+
AcO
5
Scheme 1. Synthesis of the donor. (a) (i) CH3COBr/MeOH, 95%; (ii) Zn/50% HOAc, 71%; (b) I2, CuAc2$H2O, HOAc, 80 ꢀC, overnight, 77.2%; (c) (CH3)3SiN3, TMSOTf, CH2Cl2, 4 A MS, Ar, rt
ꢀ
52 h, 87.5%; (d) NaN3, ICl, CH3CN, 0 ꢀC, 30 min, 76%.

7150
Y. Yang et al. / Tetrahedron 68 (2012) 7148e7154
Scheme 2. Synthesis of the glyco-QDs and mono and bivalent LacNAc. (a) (i) CH2Cl2; (ii) Dowex OH-; (iii) CH3ONa/CH3OH; (iv) Ac2O/Py; (b) CH3ONa/CH3OH; (c) (i) H2, Pd(OH)2/C;
(ii) MsCl/Py; (iii) KSAc/butanone.
2.2. Synthesis of QDs
QDs (lem,
2.4. Determination of the glyco-QDs formula
¼534 nm) were prepared according to previous
To quantitatively determine the number of sugar molecules on
QDs surface, we have developed a facile strategy using NMR, which
can provide more structural information and does not destroy the
glyco-QDs.10,11 In our previous works methanol and acetonitrile
were used as internal standards (IS) to quantitatively determine the
amount of the ligands on QDs and glyco-QDs, respectively. In this
study, due to the NMR peak of methyl group in acetonitrile (singlet,
max
reports with minor modifications.8d,e The prepared CdSeS/ZnS-QDs
displayed a photoluminescence emission (lem, max¼534 nm) with
the full width at the half maximum (FWHM) of less than 30 nm. The
diameter of the QDs is 4.5ꢂ0.5 nm confirmed by Transmission
electron microscopy (TEM) (see Supplementary data).
d
near 2.0) overlap with the N-acetyl group (singlet,
d
near 2.0), we
near 1.05)
used ethanol as IS, in which the methyl group (triplet,
d
2.3. Synthesis of LacNAc-QDs
displays NMR signal different from 13 (Fig. 1).
When the thiol compound 13 attached on the surface of QDs, the
NMR peak of the eCH2SH in the PEG linker is almost missing.
In order to conjugate the sugar onto the QDs surface, a thiol
group was attached through a PEG linker. Mono benzyl protected
PEG (n¼3) 6 was prepared first, which can be achieved in the
presence of freshly prepared silver (I) oxide and benzyl bromide in
excellent yield (94%).15 Benzyl-protected PEGylated N-acetyl lac-
tosamine 11 can also be synthesized through azidoiodo-
glycosylation reaction as described above. Introduction of a thiol
group to 11 was achieved in four steps (Scheme 2), and the ligand
13 for the preparation of glyco-QDs was finally synthesized.
We have reported a facile method to fabricate glyco-QDs using
two phase reaction system.16 Typically, the CdSeS/ZnS-QDs
ꢁ
Similar observation has been reported by Penades, S. et al. on lac-
tose and Lex capped gold-nanoparticles.17 In this experiment, the
(lem¼534 nm) (13 mg) was first dispersed in chloroform (10 mL),
followed by the addition of a solution of LacNAcPEGSH 13 (65 mg)
in 50 mM phosphate buffered saline (PBS 30 mL, pH 8.0). Unlike 1-
thiol sugar used in our previous reports,10,11 13 is very sensitive to
the air and easily oxidized to form disulfide, which cannot be
coated on QDs. Therefore, the coating of QDs must be operated
under argon. After vigorously stirred for 2 h at room temperature,
the lower layer became colorless and the upper became green,
indicating the reaction was finished. The upper aqueous layer was
subsequently collected and purified with Sephdex G-70, followed
by lyophilization to afford a yellow powder.
Fig. 1. 1H NMR of LacNAcPEG-QDs with ethanol as IS.

Y. Yang et al. / Tetrahedron 68 (2012) 7148e7154
7151
accurate integration of the anomeric or the N-acetyl could be used
to calculate the amount of 13 on the QDs surface.
Based on the NMR data, we calculated the amount of the sugar
ligand on QDs was 44.7% of total weight. Together with ICP-OES
data and the size of the sugar and QDs, the formula of the glyco-
QDs was determined to be [Cd171Se12S356-(ZnS)9]@(LacNAc-
PEGS)108. The number of the sugar molecules on the QDs surface
was in agreement with previous reports for the carbohydrate-
encapsulated QDs with 5 nm diameter,18 6 nm gold nano-
particles19 and our 4.5 nm lactose and galactose coated QDs.11
2.5. Affinity study of glyco-QDs with galectin-3
The binding of galectin-3 to glyco-QDs was measured using
a BIACORE 3000 instrument (Biacore AB, Uppsala). Two N-acetyl
lactosamine derivatives, LacNAcOEt 10 and (LacNAc)2PEG 8, were
tested as mono and bivalent ligands, respectively. Although Surface
Plasmon Resonance (SPR) has been used for the investigation of
galectinecarbohydrate binding, lectin was often immobilized to the
sensor surface rather than the glycan moiety,20 because of the low
sensitivity induced by the low molecular weight of the glycan. In
order to compare the binding of different carbohydrates containing
N-acetyl lactosamine to galectin-3, we immobilized the lectin to the
sensor chip.
Fig. 2 showed the kinetic binding curves of the three sugar
conjugates 8, 10, and 14 with galectin-3. All three compounds
displayed binding to galectin-3. Fig. 2a showed that the bound
amount ‘Diff. Response (RU)’ of 10 was small even at high con-
centration of 50 mM, because of weak binding. However, the
binding of bivalent carbohydrate 8 was approximately five times
higher than that of 10 at the same concentration (Fig. 2b), which
can be explained by the cluster effect.21 This result also showed that
the galectin-3 was sensitive to the sugar valency.6 Indeed, the
largest amount of sugar binding on the sensor was observed for the
glyco-QDs, even at lower concentration (<0.1 mM). Moreover, the
glyco-QDs cannot be removed unless using the regeneration buffer
(0.05 M NaOH), indicating a strong binding. Furthermore, the data
indicated that N-acetyl lactosamine retained its specific binding to
galectin-3 after conjugating onto the QDs surface.
Fig. 2. SPR sensorgrams of three glycoconjugates binding to galectin-3 presenting
In order to further quantitatively study the interactions of gly-
coconjugates and galectin-3, the KD of each ligand binding to
galectin-3 were calculated (Table 1). The sensorgrams were ana-
lyzed with the software provided by the instrument manufacturer
(BIAEVAL 4.1). It was found N-acetyl lactosamine 10 having
surface.
method measure different carbohydrateeprotein interaction phe-
nomena, the enhancement of the cluster glycoside effect can also be
different.21 Our data showed the binding affinity of N-acetyl lactos-
amine to galectin-3 can be dramatically enhanced by cluster effect.
KD¼5.26
m
M, a value consistent with that obtained by Murakami
m
et al. (2.20
M).22 It should be noted that they immobilized the
sugar on the chip, and the galectin-3 was flown over the sensor.
Due to the high molecular weight and the aggregation behavior23 of
the galectin-3, a higher RU could be observed. Nilsson et al.4c used
isothermal titration microcalorimetry (ITC) to investigate the in-
teraction between galectin-3 and LacNAc. The Kd they reported was
2.6. Fluorescent properties of the glyco-QDs
QDs have attractive optical properties, such as precisely tunable
and sharp photoluminescence (PL), broad absorption cross section,
high quantum yield (QY), and good photostability.26 As shown in
Fig. 3, the fluorescence emission peak of QDs was obtained at
534 nm. After being coated with carbohydrates, the maximum
wavelength of the fluorescence emission exhibited small red shift.
The full width at half maximum (FWHM) of the QDs and the glyco-
QDs is less than 30 nm, which is much narrower than the organic
dyes.
82 mM. It should be emphasized that the ITC method measures the
solutionesolution carbohydrate and protein interactions, which is
different from the solution-solid interactions measured by SPR.24
The different phase interactions may affect the kinetic and dy-
namic properties of the carbohydrateeprotein interactions.
Because the precise number of the ligand in target interactions
in SPR experiment is unknown, an enhancement factor
b (Kmono/
Kmultivalent),25 which defines the relative affinity of a multivalent
interaction is introduced. The bivalent (LacNAc)2PEG 8 showed 20-
fold higher affinity than the mono LacNAc 10, and glyco-QDs 14
showed 184-fold higher affinity than the mono LacNAc 10.
Table 1
Dissociation constants (KDa) of three sugar ligands
Entry
KD
(
m
M)
b
(Kfree/Kmultivalent)
LacNAcOEt
(LacNAc)2PEG
LacNAcPEG-QDs
5.26
0.25
1
20
It should be also noticed that, it is very hard to determinate the
formula of the glyco-QDs. Although we have given an approximate
formula, it is only estimations. Therefore, the molar concentration
could not be accurately calculated. On the other hand, different
5.7ꢃ10ꢁ2
184
a
KD¼kd/ka.

7152
Y. Yang et al. / Tetrahedron 68 (2012) 7148e7154
in 50% AcOH/H2O (25 mL), zinc dust (2.04 g, 31.4 mmol) was added
at 0 ꢀC. The reaction mixture was stirred for 1.5 h at 0 ꢀC when TLC
analysis showed the reaction was complete. The mixture was fil-
tered and extracted with CH2Cl2. The combined organic extracts
were washed successively with saturated aqueous NaHCO3 (50 mL)
and brine (50 mL), dried (Na2SO4), filtered, and concentrated. Pu-
rification by column chromatography (petroleum ether/EtOAc
2.5:1, v/v) afforded hexa-O-acetyl-
D
-lactal (2) (0.74 g, 71%) as
1.99 (s, 3H), 2.06 (s, 3H),
a colorless oil. 1H NMR (300 MHz, CDCl3):
d
2.07 (s, 3H), 2.09 (s, 3H), 2.16 (s, 3H), 2.12 (s, 3H), 3.91 (ddd, 1H,
J¼10.0, 4.2, 2.0 Hz, H-50), 4.00 (dd, 1H, J¼5.4, 7.2 Hz, H-5), 4.08 (dd,
1H, J¼2.0, 12.4 Hz, H-6b0), 4.15 (dd, J¼12.4, 4.2 Hz, H-6a0), 4.18 (dd,
1H, J¼2.4, 11.6 Hz, H-6b), 4.21 (dd, 1H, J¼6.0, 5.4 Hz, H-4), 4.44 (dd,
1H, J¼2.4, 11.4 Hz, H-6a), 4.66 (d, 1H, J¼8.0 Hz, H-10), 4.84 (dd, 1H,
J¼6.4, 3.2 Hz, H-2), 5.01 (dd, 1H, J¼3.6, 10.4 Hz, H-30), 5.19 (dd, 1H,
J¼10.4, 8.0 Hz, H-20), 5.37 (dd, 1H, J¼9.6, 9.2 Hz, H-40), 5.41 (dd, H,
J¼4.0, 4.4 Hz, H-3), 6.42 (d, 1H, J¼6.0 Hz, H-1); 13C NMR (75 MHz,
Fig. 3. The fluorescence spectrum of QDs and glyco-QDs.
CDCl3):
d
20.4, 20.5, 20.6, 20.8, 20.9 (6ꢃ COCH3), 60.9, 61.8, 66.7,
3. Conclusion
68.8, 70.7, 74.1, 74.7, 99.0, 101.0 (C-10), 145.4 (C-1), 169.2, 169.9,
170.0, 170.1, 170.3, 170.4 (6ꢃ COCH3).
We have prepared N-acetyl lactosamine, bivalent N-acetyl lac-
tosamine, and N-acetyl lactosamine coated QDs. The bioassay using
SPR showed that coating of N-acetyl lactosamine on QDs surface
can dramatically enhance its binding to galectin-3 through cluster
effect. The N-acetyl lactosamine coated QDs could be used as an
efficient fluorescent probe to assess the carbohydrateeprotein in-
teractions and possibly to image the glycoproteins and their cellular
activities.
4.2.2. 1,3,6-Tri-O-acetyl-4-O-(20,30,40,60-tetra-O-acetyl-
anosyl)-2-deoxy-2-iodo- -manno-pyranose (3). Hexa-O-acetyl-lac-
b-D-galactopyr-
a-D
tal 2 (1.0 g,1.78 mmol), cupricacetate hydrate (0.39 g,1.95 mmol), and
iodine (0.55 g, 2.17 mmol) were successively added to AcOH (10 mL).
The mixture was stirred overnight at 80 ꢀC, and cooled to room tem-
perature. CH2Cl2 (20 mL) was added to the mixture and the organic
layer was washed with saturated aqueous NaHCO3 until neutral, sat-
uratedaqueousNa2S2O3 (15 mL),andwater(10 mL).Theclearsolution
was dried with Na2SO4 and evaporated. The crude product was puri-
fied by recrystallization from ethanol as a yellow syrup (1.03 g, yield:
4. Experimental section
4.1. General information
77.2%).1H NMR (300 MHz, CDCl3):
d
6.34(d,1H, J¼2.1 Hz, H-1), 5.38 (d,
1H, J¼3.0 Hz, H-40), 5.16 (dd, 1H, J¼8.1, 10.5 Hz, H-20), 5.00 (dd, 1H,
J¼3.6, 10.5 Hz, H-30), 4.68 (dd, 1H, J¼4.2, 7.2 Hz, H-3), 4.62 (d, 1H,
J¼8.1 Hz, H-10), 4.52 (dd,1H, J¼2.1, 3.9 Hz, H-2), 4.44 (d,1H, J¼13.5 Hz,
H-6a), 4.21e4.03 (m, 5H, H-6a0,4,5,6b,6b0), 3.95 (m, 1H, H-50),
All solvents were dried prior to use. When dry conditions were
required, the reactions were performed under an argon atmo-
sphere. Thin-layer chromatography (TLC) was purchased from
Liangchen Chemical Engineering Co. Ltd. (Anhui Province). All
compounds were stained with 5% H2SO4 in ethanol followed by
heating. Detection with UV light was employed when possible.
Flash column chromatography was performed on silica gel
200e300 mesh. The boiling range of petroleum ether used as fluent
in column chromatography is 60e90 ꢀC. NMR spectra were recor-
ded on a JEOL-300 (300 MHz) or Bruker AMX-400 (400 MHz) in-
2.17e1.98 (7s, 21H, CH3CO); 13C NMR (75 MHz, CDCl3):
d 170.3, 170.0,
169.9,169.4,169.30,168.4 (6ꢃ COCH3),101.3 (C-10), 94.3 (C-1), 75.3 (C-
4), 71.6 (C-5), 70.8 (C-30), 70.6 (C-50), 69.2 (C-20), 69.0 (C-3), 66.6 (C-40),
61.6, 61.0 (C-6,60), 27.3 (C-2), 20.9, 20.8, 20.7, 20.6, 20.5, 20.4 (6ꢃ
COCH3).
4.2.3. 3,6-Di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-
b-D-galactopyr-
struments. Chemical shifts (d) were reported in parts per million
anosyl)-2-deoxy-2-iodo- -manno-pyranosylazide (4). To a solution
a-D
downfield from TMS, the internal standard; J values were given in
Hertz. Mass spectra were recorded on a Bruker Apex IV FTMS in-
strument. Inductively Coupled Plasma-Optical Emission Spec-
trometer (ICP-OES) was recorded on Thermo-iCAP 6000. Surface
Plasmon Resonance (SPR) measurements were carried out with
BIAcore 3000 instrument using carboxymethylated sensor chips
(CM5, BIAcore). Galectin-3 was purchased from SigmaeAldrich
(G5170).
of 3 (1.0 g, 1.34 mmol) in anhydrous CH2Cl2 (10 mL) were added
TMSN3 (0.33 mL, 2.50 mmol) and TMSOTf (0.06 mL, 0.22 mmol) un-
derargon. Stirringwascontinued until completedisappearanceofthe
starting material onTLC. The solutionwas then neutralized by careful
addition of saturated aqueous NaHCO3 and the organic layer was
dried with Na2SO4, concentrated, and purified by column chroma-
tography (petroleum ether/EtOAc 2:1, v/v) to give 4 (0.86 g, yield:
87.5%) as syrup.1H NMR (400 MHz, CDCl3):
d
5.64 (d,1H, J¼2.7 Hz, H-
1), 5.38 (d, 1H, J¼3.9 Hz, H-40), 5.16 (dd, 1H, J¼9.9, 9.2 Hz, H-20), 5.00
(dd,1H, J¼2.4,10.5 Hz, H-30), 4.68 (dd,1H, J¼3.6, 6.9 Hz, H-3), 4.59 (d,
1H, J¼8.0 Hz, H-10), 4.45 (m, 2H, H-6a, 2), 4.22e4.06 (m, 4H, H-
5,6b,60a,60b), 3.95 (m, 2H, H-50,4), 2.17e1.98 (6s, 18H, 6ꢃ COCH3); 13C
4.2. Synthesis of mono, bivalent LacNAc and other sugar
ligand
4.2.1. 3,6,20,30,40,60-Hexa-O-acetyl-
D-lactal
(2). Acetyl
bromide
NMR (100 MHz, CDCl3): d
170.4,170.1, 170.0,169.3, 169.2, 101.3 (C-10),
(0.92 mL, 12.3 mmol) and CH3OH (0.5 mL, 12.30 mmol) were added
to AcOH (10 mL) and stirred at room temperature (protected from
light) for 15 min, followed by addition of lactose (1.0 g, 3.08 mmol)
and Ac2O (3.4 mL, 35.97 mmol). The reaction mixture was further
stirred overnight at room temperature and the solution was diluted
with chloroform, washed with saturated solution of NaHCO3, and
dried with Na2SO4. The solvent was removed, and cold ether was
90.7 (C-1), 75.5 (C-4), 71.5 (C-5), 70.8 (C-50), 70.7 (C-30), 69.5 (C-20),
68.9 (C-3), 66.7 (C-40), 61.6(C-6), 61.1 (C-60), 27.5(C-2), 20.9, 20.8, 20.7,
20.6, 20.5; IR (film KBr): nN3 2119 cmꢁ1
.
4.2.4. 2-{2-[2-(Benzyloxy)ethoxy]ethoxy}ethan-1-ol (6). Ag2O (6.9 g,
30 mmol) was added to a solution of triethylene glycol (3.0 g,
20 mmol) in dry CH2Cl2. BnBr (2.6 mL, 22 mmol) was then added
dropwise over 5 min, and the mixture was stirred for 2 h. After this
time, the suspension was filtered through a pad of Celite, which was
added to give 2,3,6,20,30,40,60-sept-O-acetyl-
(2.15 g, yield: 95%) as a white solid. Then the product was dissolved
a-D-lactosyl bromide

Y. Yang et al. / Tetrahedron 68 (2012) 7148e7154
7153
thoroughly washed with CH2Cl2. The combined filtrate was con-
centrated under reduced pressure, and the residue was purified by
column chromatography to yield a yellow oil (4.46 g, 92.9%). 1H
69.6, 68.9, 68.5, 61.0, 60.0, 55.0, 22.2; HRMS m/z calcd for
C9H16N2O4Na [MþNa]þ: 239.1002. Found: 239.0997.
NMR (300 MHz, CDCl3):
d
7.26e7.35 (m, 5H, Ph), 4.56 (s, 2H,
4.2.8. Ethyl b-D-galactopyranosyl-(1/4)-2-acetamido-2-deoxy-b-D-
eCH2Ph), 3.73e3.58 (m, 12H, eOCH2CH2Oe), 2.80 (br, 1H, eOH);
glucopyranoside (10). The title compound was obtained as a white
13C NMR (75 MHz, CDCl3):
70.5, 70.4, 70.2, 69.2, 61.5.
d
138.0, 128.2, 127.6, 127.5, 73.1, 72.4,
solid in 95% yield using the general procedure as described above. 1H
NMR (400 MHz, D2O):
d
4.42 (d, 1H, J¼7.6 Hz, H-1), 4.33 (d, 1H,
J¼7.6 Hz, H-10), 3.86e3.38 (m, 14H, H-2,3,4,5,6a,6b, H-
4.2.5. 3,6-Dioxaoct-1,8-diyl-bis[2,3,4,6-tetra-O-acetyl-2-acetamido-2-
deoxy-4-O-(2,3,4,6-tetra-O- -galactopyranosyl)- -glucoside]dimer
20,30,40,50,6a0,6b0, eOCH2CH3), 1.90 (s, 3H, eCH3CO), 1.04 (t, 3H,
b-D
b-D
J¼4.0 Hz, eOCH2CH3); 13C NMR (100 MHz, D2O):
d 174.6 (NHCO),
(7). Under argon atmosphere, compound 4 (466 mg, 0.639 mmol)
102.9 (C-10), 100.6 (C-1), 78.5, 75.3, 74.8, 72.5, 70.9, 68.5, 66.2, 61.0,
60.1, 55.1, 22.1, 14.2.
and polyethylene glycol (32 mg, 0.213 mmol) was dissolved in dry
ꢀ
CH2Cl2 (10 mL), then 4 A MS was added. The mixture was stirred at
0
ꢀC for 30 min. PPh3 (201 mg, 0.767 mmol) was dissolved in dry
4.2.9. 8-Phenylmethyloxy-3,6-dioxaoctyl 2,3,4,6-tetra-O-acetyl-
b
-
-
D
D
-
-
CH2Cl2 and added dropwise to the mixture above at 0 ꢀC. The mixture
was allowed to warm to room temperature and then stirred over-
galactopyranosyl (1/4)-3,6-di-O-acetyl-2-acetamido-2-deoxy-
b
glucopyranoside (11). The title compound was obtained as oil in
56.2% yield from the reaction of 3 and 5 using the general procedure
ꢀ
night. 4 A MS was removed by filtration, and the filtrate was con-
centrated to dryness. The residue was dissolved in ethanol. Dowex
OHꢁ/EtOH exchange resin was added and the mixture was stirred at
room temperature for 6 h before filtration. The filtrate was concen-
trated to dryness and the residue was dissolved in methanol. A cat-
alytic amount of sodium was added to the solution and the mixture
was stirred overnight at room temperature. The mixture was con-
centrated to dryness and Ac2O/Py (10 mL, 1:2 v/v) was added. The
mixture was stirred overnight at room temperature, followed by
cooling to 0 ꢀC. Methanol was added dropwise to terminate the re-
action. The mixture was concentrated to dryness and then diluted
with CH2Cl2. The organic solution was washed successively with 1 M
HCl, saturated aqueous NaHCO3 and water, dried over Na2SO4, fil-
trated, and concentrated. The crude was purified by silica gel column
chromatography (35:1 CH2Cl2/methanol) to give 4 (227 mg, 81%
as described above. 1H NMR (400 MHz, CDCl3):
d 7.34 (m, 5H, Ph),
6.67 (d,1H, J¼9.6 Hz, NHCO-), 5.34 (d,1H, J¼4.2 Hz, H-40), 5.14e4.94
(m, 3H, H-20,30,3), 4.69 (d, 1H, J¼8.1 Hz, H-1), 4.57 (s, 2H, PhCH2e),
4.47 (d, 1H, J¼8.0 Hz, H-10), 4.37 (d, 1H, J¼11.7 Hz, H-2), 4.10 (m, 4H,
H-6a,6b,60a,60b), 3.60e3.85 (m, 15H, H-4,5,50, CH2O),1.91e2.13 (7s,
21H, 7ꢃ COCH3); 13C NMR (100 MHz, CDCl3):
d 170.6 (NHCOe),
170.4e169.2 (6C, CH3CO), 138.2, 128.4, 127.7, 127.6 (Phe), 101.9 (C-
1), 101.2 (C-10), 73.7 (C-4), 72.9 (CH2O), 72.4 (C-3), 71.7 (CH2O), 71.0
(C-30), 70.7 (C-50), 70.6 (CH2O), 70.5 (CH2O), 70.4 (C-20), 69.4 (C-5),
69.1 (CH2O), 68.7 (C-40), 66.6 (C-6), 63.2 (CH2O), 62.4 (C-60), 61.6 (C-
2), 60.7 (PhCH2e), 23.0 (CH3CONH), 20.5e21.0 (6C, CH3CO).
4.2.10. (8-Acetylthio-3,6-dioxaoctyl) 2,3,4,6-tetra-O-acetyl-
b-D-gal-
actopyranosyl (1/4)-3,6-di-O-acetyl-2-acet-amido-2-deoxy-
b-D-
yield) as a pale yellow syrup. 1H NMR (400 MHz, CDCl3):
d
6.65 (d, 2H,
glucopyranoside (12). Pd(OH)2/C was added to a solution of 11 in
methanol. The reaction mixture was stirred under hydrogen at-
mosphere (4 atm) for 4 h when TLC analysis showed the reaction
was complete. The mixture was filtered over Celite and concen-
trated to afford the alcohol, which was directly used in the next
step. To a solution of alcohol (0.4 g, 0.52 mmol) in pyridine (5 mL)
was added methanesulfonyl chloride (0.1 mL, 0.148 g, 1.3 mmol).
The resulting solution was stirred at room temperature for 1 h
when TLC indicated complete conversion of the starting alcohol
into the methanesulfonate, CH2Cl2 (25 mL) was added and the or-
ganic solution was washed sequentially with 1 M hydrochloric acid
(100 mL), NaHCO3 (aq) (50 mL), and water (50 mL) and then dried
with Na2SO4. The solvent was evaporated to afford yellow oil
(0.42 g, 85%), which was used directly in the next step. A mixture of
the methanesulfonate (0.38 g, 0.63 mmol) and potassium thio-
acetate (0.144 g, 1.26 mmol) in butanone (25 mL) was heated under
reflux for 2 h, when TLC showed that the starting material had been
replaced with the thioacetate. The solvent was removed under
reduced pressure, and the residue was distributed between CH2Cl2
(25 mL) and water. The separated organic layer was dried and
concentrated, and the product was subjected to column chroma-
tography (EtOAc/Petrol ether, 1:1) to give thioacetate 12 (0.26 g,
eNH), 5.35 (d, J¼2.8 Hz, 2H, H-40), 5.08 (m, 2H), 5.04e4.96 (m, 6H),
4.64 (dd, 2H), 4.53e4.45 (m, 2H), 4.21e4.04 (m, 8H), 3.93e3.90 (m,
4H), 3.81e3.77 (m, 4H), 3.68e3.61 (m, 10H), 2.18e1.95 (7s, 42H, 6ꢃ
OAc, CH3CONHe); 13C NMR (100 MHz, CDCl3):
d 170.4, 170.4, 170.3,
170.3, 170.2, 170.0, 169.9, 169.2, 101.4 (C-10), 101.0 (C-1), 76.2, 73.4,
72.4, 70.8, 70.5, 69.0, 68.7, 66.5, 62.3, 60.7, 53.3, 30.8, 22.9, 22.8, 20.7,
20.5, 20.5, 20.4; HRMS m/z calcd for C9H16N2O4Na [MþNa]þ:
239.1002. Found 239.0997.
4.2.6. Ethyl 2,3,4,6-tetra-O-acetyl- -galactopyranosyl (1/4)-3,6-
b
-
D
di-O-acetyl-2-acetamido-2-deoxy-b-D-gluco-pyranoside (9). The ti-
tle compound was obtained as syrup in 73.9% yield from the re-
action of 4 with ethanol using the general procedure as described
above. 1H NMR (400 MHz, CDCl3):
d 6.72 (d, 1H, NHCO), 5.85 (dd,
1H, H-40), 5.34 (dd, 1H, H-20), 5.08 (m, 2H, H-3, 30), 4.95 (d, 1H,
J¼7.9 Hz, H-1), 4.50 (d, 1H, J¼7.9 Hz, H-10), 4.48, 4.15e4.11 (m, 4H,
H-6a,6b, H-6a0,6b0), 4.08 (dd, 1H, H-2), 3.99 (dd, 2H, eOCH2CH3),
3.76 (m, 1H, H-5), 3.62 (m, 1H, H-50), 3.50 (m, 1H, H-4), 2.15e1.97
(7s, 21H, 6ꢃ CH3CO, eCH3CONH); 1.16 (t, 3H, eOCH2CH3); 13C NMR
(100 MHz, CDCl3):
d
170.6e169.3 (7C, 6ꢃ COCH3, CONH), 100.9 (C-
1), 100.5 (C-10), 77.4, 77.1, 76.7, 75.8, 72.5, 72.4, 70.8, 70.7, 69.1, 66.6,
65.0, 62.4, 60.8, 53.2, 23.2, 20.9, 20.6, 20.5, 15.0.
71%). 1H NMR (400 MHz, CDCl3):
d
1.97e2.34 (m, 24H, 8ꢃ OAc);
3.09 (t, 2H, J¼6.8 Hz, eCH2 in eCH2SAc); 3.57e3.64 (m, 8H, eOCH2
in PEG); 3.69e3.74 (m, 1H); 3.79 (t, 1H, J¼9.9, 9.2 Hz); 3.86e3.92
(m, 2H); 4.05e4.15 (m, 3H); 4.48e4.58 (m, 3H); 4.89 (dd, 1H, J¼8,
9.2 Hz); 4.94 (dd, 1H, J¼3.2, 10.4 Hz); 5.10 (dd, 1H, J¼8, 10.4 Hz);
5.19 (t, 1H, J¼9.2, 9.2 Hz); 5.10 (d, 1H, J¼2.8 Hz, H-4); 13C NMR
4.2.7. 3,6-Dioxaoct-1,8-diyl bis[
b-D-galactopyranosyl-(1-4)-2-acet-
amido-2-deoxy- -glucopyranoside] (8). A catalytic amount of so-
b-D
dium was added to a solution of compound 7 (100 mg) in methanol
(5 mL). The mixture was stirred at room temperature for 1 h, and
then neutralized with Hþ cation-exchange resin. The solution was
filtered and concentrated and the residue was dissolved in 10 mL
water and freeze-dried to give 5 as a white solid (61 mg, 96%). 1H
(100 MHz, CDCl3): d 20.4, 20.5, 20.6, 20.7, 20.7, 28.7, 29.6, 30.4, 60.7,
61.9, 66.5, 69.0, 69.0, 69.7, 70.2, 70.5, 70.6, 70.9, 71.6, 72.5, 72.7, 76.2,
100.5, 101.0, 168.9, 169.5, 169.6, 169.9, 170.0, 170.2, 170.2, 195.4.
NMR (400 MHz, D2O):
d
4.50 (d, 2H, J¼7.6 Hz, H-1), 4.38 (d, 2H,
J¼7.6 Hz, H-10), 3.91e3.89 (m, 4H), 3.84e3.83 (m, 2H), 3.76e3.56 (m,
4.2.11. (8-Thiol-3,6-dioxaoctyl)-
amido-2-deoxy- -glucopyranoside (13). A solution of 13 (100 mg)
in dry MeOH (25 mL) was treated with 0.05 M methanolic solution
b-D-galactopyranosyl (1/4)-2-acet-
26H), 3.50e3.43 (m, 4H), 1.95 (s, 6H, 2ꢃ OAc); 13C NMR (100 MHz,
b-D
D2O): d
174.4, 102.8 (C-10), 100.9 (C-1), 78.4, 75.3, 74.7, 72.4, 70.9, 69.7,

7154
Y. Yang et al. / Tetrahedron 68 (2012) 7148e7154
of NaOMe. The reaction was stirred under an argon atmosphere.
After 1 h, the solution was acidified with DOWEX 50WX4-100 ion-
exchange resin (SigmaeAldrich) to pH 7.0. Evaporation of the solu-
tion under reduced pressure produced a syrup. Because of the in-
stability of the intermediate, it was immediately used in the
following step without further characterization 1H NMR (400 MHz,
References and notes
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This work was financially supported by the Natural Science
Foundation of China (Grant Nos. 90713004, 20732001), the State
New Drug Innovation (Grant Nos. 2009ZX09103-044,
2009ZX09501-011), the National Basic Research Program of China
(Grant No. 2012CB822100), and the State Key Laboratory of Natural
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tet.2012.06.035.