Expanded potential of seleno-carbohydrates as a molecular tool for X-ray structural determination of a carbohydrate-protein complex with single/multi-wavelength anomalous dispersion phasing

Article abstract of DOI:10.1016/j.bmc.2014.02.023

Seleno-lactoses have been successfully synthesized as candidates for mimicking carbohydrate ligands for human galectin-9 N-terminal carbohydrate recognition domain (NCRD). Selenium was introduced into the mono- or di-saccharides using p-methylselenobenzoi

Full text of DOI:10.1016/j.bmc.2014.02.023

Bioorganic & Medicinal Chemistry 22 (2014) 2090–2101
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Expanded potential of seleno-carbohydrates as a molecular tool for
X-ray structural determination of a carbohydrate–protein complex
with single/multi-wavelength anomalous dispersion phasing
Tatsuya Suzuki a,b, , Hisayoshi Makyio c, , Hiromune Ando a,b, , Naoko Komura a,b, Masanori Menjo a,
⇑
Yusuke Yamada c, Akihiro Imamura a, Hideharu Ishida a, Soichi Wakatsuki c,d,e, Ryuichi Kato c,
,
⇑
Makoto Kiso a,b
a Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan
b Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan
c Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho,
Tsukuba, Ibaraki 305-0801, Japan
d Photon Science, SLAC Natl. Accelerator Laboratory Structure Science, 2575 Sand Hill Road, MS 69, Menlo Park, CA 94025-7015, USA
e Department of Structural Biology, Stanford University, Beckman Center B105, 279 Campus Drive, Stanford, CA 94305-5126, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Seleno-lactoses have been successfully synthesized as candidates for mimicking carbohydrate ligands for
human galectin-9 N-terminal carbohydrate recognition domain (NCRD). Selenium was introduced into
the mono- or di-saccharides using p-methylselenobenzoic anhydride (Tol2Se) as a novel selenating
reagent. The TolSe-substituted monosaccharides were converted into selenoglycosyl donors or acceptors,
which were reacted with coupling partners to afford seleno-lactoses. The seleno-lactoses were converted
to the target compounds. The structure of human galectin-9 NCRD co-crystallized with 6-MeSe-lactose
was determined with single/multi-wavelength anomalous dispersion (SAD/MAD) phasing and was
similar to that of the co-crystal with natural lactose.
Received 4 February 2014
Accepted 18 February 2014
Available online 25 February 2014
Keywords:
Seleno-carbohydrate
Carbohydrate–protein complex
X-ray crystallography
SAD/MAD phasing
Lectin
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
selenium-containing molecules are likely to have great potential
for structural analysis of biomolecules by NMR5 and X-ray spec-
Since Wrede first reported the synthesis of selenium-containing
carbohydrates (seleno-carbohydrates),1 various seleno-carbohy-
drates have been synthesized by a number of research groups.2
With the exception of seleno-glycoside synthetic intermediates2a-
troscopy. In particular, the anomalous dispersion characteristics
have been successfully used in single-wavelength anomalous dis-
persion (SAD) and multi-wavelength anomalous dispersion
(MAD) phasing methods for X-ray crystallography. In protein X-
ray crystallography, methionine is replaced with selenomethionine
(SeMet) by a recombinant technique, which allows the structural
analysis of the SeMet-labeled proteins by the SAD/MAD phasing
method. This technique only requires crystals of SeMet-labeled
protein for structural determination whilst the conventional meth-
od such as multiple isomorphous replacement (MIR) requires more
crystals, which are native and heavy atom (Hg etc.)-soaked crys-
tals. Therefore, SAD/MAD methods are widely used and have al-
lowed a dramatic increase in the number of protein structures
solved.6 However, the production of the sufficient quantities of Se-
Met-labeled proteins for X-ray crystallography remains a chal-
lenge, mainly due to the low expression level of the labeled
proteins and the high production costs in insect or mammalian cell
expression systems. Structural changes or destabilization of
j
and selenylsulfide-linked glycoproteins found as metabolites in
the liver or urine and recently synthesized by Davis and co-work-
ers,3 the seleno-carbohydrates synthesized to date are unnatural.
Seleno-carbohydrates are expected to function as useful mimics
of biologically significant carbohydrates because of the inherent
chemical and physical properties of selenium. However, their use
in biological studies has been limited since the pioneering work
on the inhibition of glycosidase published by Pinto’s research
group.4
Because selenium has a spin of 1/2 (77Se) and exhibits anoma-
lous dispersion characteristics in response to X-ray irradiation,
⇑
Corresponding authors. Tel.: +81 58 239 3452; fax: +81 293 3452 (H.A.).
E-mail address: hando@gifu-u.ac.jp (H. Ando).
Both authors contributed equally to this study.
http://dx.doi.org/10.1016/j.bmc.2014.02.023
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

T. Suzuki et al. / Bioorg. Med. Chem. 22 (2014) 2090–2101
2091
contaminant
O
proteins caused by SeMet labeling also limits the application of
SAD/MAD.
However, if seleno-carbohydrates serve as a mimic of a ligand
O
SeSeK
SeK
5
6
for the protein in carbohydrate–protein complexes, the three
dimensional structure of the carbohydrate–protein complex could
be determined by the SAD/MAD phasing method without a SeMet-
labeled protein. Recently, it was demonstrated SAD/MAD phasing
using methyl seleno-glycosides of monosaccharides as ligand
mimetics.7 However, further investigation using more than
seleno-monosaccharide has not been carried out, probably
because of the synthetic difficulties in the seleno-derivatization
of carbohydrates. Determining high resolution three-dimensional
structures of carbohydrate–protein complexes is invaluable for
understanding biological processes that are mediated through
carbohydrate-protein interactions on the cell membrane, including
cell adhesion, cell proliferation, cell differentiation, and pathogen-
host cell interactions. It is also useful for developing drugs based on
the biological functions of carbohydrates. Therefore, in this study,
to complete the proof-of-concept demonstration of SAD/MAD
phasing with seleno-carbohydrates, we synthesized various
seleno-lactose derivatives as lactose mimics through a facile and
mild selenation method developed by our research group,
co-crystallized the seleno-lactose derivatives with a carbohydrate-
binding protein and determined the protein structure by using
the SAD/MAD phasing method.
(TolSeK)
O
RX
O
O
SeTol
SeR
Br
O
O
O
nucleophile
Se
Se
in situ generation
of
acyl selenolate anion
7
(Tol2Se)
Scheme 1. Reported method for the introduction of selenium into a carbohydrate
with TolSeK 5 and the strategy for using Tol2Se 7 as a TolSeꢀ equivalent. Tol =
p-methylbenzoyl.
highly reactive acyl selenolate anion in situ, which would subse-
quently react with the sugar electrophile to produce a seleno-
carbohydrate. According to the modified protocol reported by
Ishihara et al.,10 Tol2Se (7) could be obtained on a large scale as
pure crystals (Scheme 2).
Screening identified piperidine (1.0 equiv) and N,N-diisopropyl-
ethylamine (DIEA; 1.0 equiv) as the conditions that activated Tol2-
Se (7) (1.0 equiv) most effectively to afford the toluylselenylated
sugar. For this system, morpholine could also be used as a nucleo-
phile. However, when DIEA was replaced with an inorganic base
such as Cs2CO3, the reaction yield decreased considerably. Presum-
ably, this is because of the instability of the cesium acyl selenolate
generated from 7. This agrees well with the recently reported re-
sult that trialkylamines such as DIEA can form a more stable salt
with acylselenolate anions than metals can.11 This method was
2. Results and discussion
2.1. Synthesis of seleno-lactoses
Of the possible seleno-lactose analogues, seleno-glycoside ana-
logues 1 and 2, and methylseleno (MeSe)-substituted analogues 3
and 4 were selected as lactose mimics (Fig. 1). Based on our X-
ray crystallography results for a synthesized glycan–protein com-
plex,8 the 2-(trimethylsilyl)ethyl group was chosen as the aglycon
of seleno-lactoses 2–4. X-ray analysis of these seleno-lactoses co-
crystallized with a lactose-binding protein will provide informa-
tion about whether the binding site allows differences in the van
der Waals radius (O 1.52 Å, Se 1.90 Å) and the angle of the glyco-
sidic bond (CAOAC 112°, CASeAC 96°) between native lactose
and seleno-lactoses.
first used in the synthesis of 1-methylseleno-lactoside
1
(Scheme 3). Therefore, the anomeric center of 1-bromo-lactose
derivative 912 underwent substitution with a toluylselenyl (TolSe)
group by reaction with Tol2Se (7) in the presence of piperidine and
DIEA in DMF, giving 1-seleno-lactoside 10 in 76% yield. Next, the
TolSe group in compound 10 was converted into a methylselenyl
(MeSe) group via the in situ reaction of the glycosyl selenolate an-
ion with MeI in the presence of Cs2CO3, following a previously re-
ported method,9 to afford compound 11 in high yield. Full
deprotection of 11 under Zemplén conditions delivered 1-methyl-
seleno-lactoside 1.
We have previously reported a facile, mild method for the syn-
thesis of seleno-glycosides using potassium p-methylselenobenzo-
ate 5 as the key selenation reagent (Scheme 1).9 Afterward, we
noticed that this reagent was frequently contaminated with
byproducts, such as diselenide 6, which were produced during its
preparation. Because of its reactivity, complete purification of 5
is not possible at large scales. Impurities in 5 produced several
inseparable byproducts during selenation. To solve this problem,
we envisioned the use of p-methylselenobenzoic anhydride (Tol2-
Se) 710 as the synthetic equivalent of the acyl selenolate anion, in
light of the known stability of 7. It was anticipated that reactive
anhydride 7 would readily react with a nucleophile to generate a
Scheme 4 summarizes the synthesis of seleno-lactose 2. To con-
struct the intra-residual seleno-glycoside in target compound 2,
toluylseleno-galactoside 13 was prepared by the reaction of 1-bro-
mo-galactose tetraacetate 12 with 7. Then, seleno-glycoside 15
was synthesized by the previously reported reaction of compounds
13 and 14.9 Compound 15 was then de-O-acetylated to give 2.
Taking advantage of the facile, mild toluylselenation with Tol2-
Se (7), 6-methylseleno-glucosyl acceptor 23, which is the key inter-
mediate for the synthesis of target compound 3, could also be
derived from known 4,6-benzylidene-glucoside 16 (Scheme 5).13
To boost the reactivity of the C-4 hydroxyl group for glycosylation,
the 2,3-diol in 16 was converted into p-methoxybenzyl ethers, giv-
ing compound 17. Following acid hydrolysis of the benzylidene
acetal of 17, the C-6 hydroxyl group was selectively substituted
with bromine by treatment with CBr4 and Ph3P to afford com-
pound 19. Next, the substitution reaction of 6-bromo-glucose 19
HO
HO
HO
OH
O
OH
O
OH
O
OH
O
HO
O
HO
Se
a
OSE
OSE
SeMe
OSE
b
HO
OH
OH
OH
OH
1
2
HO
HO
HO
HO
1. NaBH4/ EtOH, 0 °C
2. TolCl/ THF, 0°C
3. I2, KI/ EtOH, 0°C
OH
O
SeMe
O
OH
OH
O
O
O
HO
O
HO
O
P(OEt)3
O
SeMe
Se Se
Se
7
OH
OH
OH
Toluene
50 °C
90%
3
4
8
Figure 1. Structures of seleno-lactoses synthesized in this study. SE = 2-
(trimethylsilyl)ethyl.
Scheme 2. Preparation of Tol2Se 7.

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T. Suzuki et al. / Bioorg. Med. Chem. 22 (2014) 2090–2101
Br
BzO
HO
R
RO
BzO
BzO
BzO
SeTol
O
OBz
O
OBz
O
BzO
d
b
a
O
BzO
O
BzO
O
a
SeTol
b
BzO
OSE
BzO
OSE
BzO
96%
78%
O
OBz
O
OBz
76%
OBz
OBz
OBz
OBz
9
BzO
27
28
AcO
c
29 R = H
R = OH
10
c
a
30
96%
AcO
R = Ac
R = Br
87%
OBz
O
BzO
b
BzO
SeMe
O
SeMe
O
SeMe
1
g
BzO
O
f
O
OBz
99%
88%
OBz
BzO
OR
BzO
quant.
71%
OBz
R = SE
BzO
33
O
NH
CCl3
11
31
e
32 R = H (α and β)
81%
(α:β = 20/1)
Scheme 3. Synthesis of seleno-lactose 1. Reagents and conditions: (a) 7, piperidine,
DIEA/DMF, rt, 76%; (b) MeI, MeNHNH2, Cs2CO3/DMF, rt, 99%; (c) NaOMe, MeOH/THF
(2:1), rt, 88%. Bz = benzoyl, DIEA = N,N-diisopropylethylamine.
AcO
SeMe
OR
O
RO
OAc
O
a
OSE
b
BzO
O
O
OAc
HO
OBz
OSE
OMBn
34
MBnO
h
83%
35
36
R = MBn
R = H
i
4
AcO
quant.
AcO
TfO
OSE
14
O
OAc
AcO
AcO
AcO
AcO
OAc
O
OAc
O
Scheme 6. Synthesis of seleno-lactose 4. Reagents and conditions: (a) CBr4, PPh3/
Pyr, 65 °C, 87%; (b) 7, piperidine, DIEA/DMF, 100 °C, 96%; (c) Ac2O/Pyr, rt, 96%; (d)
MeI, MeNHNH2, Cs2CO3/DMF, rt, 78%; (e) TFA/CH2Cl2, 0 °C, 81%; (f) CCl3CN, DBU/
CH2Cl2, 0 °C, quant.; (g) 34, TMSOTf, 4 Å MS/CH2Cl2, ꢀ40 °C, 71%; (h) TFA/CH2Cl2,
ꢀ20 °C, 83%; (i) NaOMe, MeOH/THF (2:1), sonication, quant.
a
SeTol
85%
piperazine
Cs2CO3
DMF
AcO
AcO
Br
12
13
quant.
AcO
OAc
OAc
O
b
O
AcO
a
OSE
2
b
Se
AcO
quant.
OAc
OAc
hemiacetal 32, which was functionalized as a glycosyl imidate do-
nor to give 33. Based on the synthesis of 3, the 2,3-di-MBn deriva-
tive of glucoside 34 was prepared from compound 17 as a coupling
partner for 33.17 Examination of the glycosylation of 34 with 33
promoted by TMSOTf revealed that ꢀ40 °C was the optimal reac-
tion temperature, although it took a longer time to reach comple-
tion (51 h). This reaction produced disaccharide 35 in 71% yield.
We also examined the glycosidation of fluoride donor 37 by
using a common promoter system, Cp2ZrCl2 (2.5 equiv) and AgOTf
(5.0 equiv)18 (Scheme 7). Even with an acid-resistant, highly nucle-
ophilic glycosyl acceptor 38, the glycosidation reaction only began
to proceed at room temperature and took a long time (24 h), pro-
viding glycosylated product 39 in 53% yield. Because these reaction
conditions probably affect the MBn groups of glycosyl acceptor 33,
fluoride 37 was not used for the synthesis of 35. Using the proce-
dure for the full deprotection of 25 on seleno-lactose derivative
35 produced target compound 4 in high yield.
The glycosidations of glycosyl donors 33 and 37 indicated that
the MeSe group at the C-6 position may affect the reactivity of
the compounds. To confirm this, glycosylation reactions using lac-
tosyl imidate donors 40,19 41, and 42 were performed.20 All gly-
cosylations were carried out in CH2Cl2 at 0 °C in the presence of
a catalytic amount of TMSOTf. The results clearly indicate that
the reactivity of 6-MeSe-lactosyl donor 41 was substantially lower
than that of 40 and 42 (Table 1). Thus, lactosyl donor 41 required
four-fold the equivalents of TMSOTf and around eleven-fold longer
than 60-MeSe-lactosyl donor 42 to complete the glycosylation.
Comparing entries 1 and 3 indicates that the MeSe group reduced
the reaction rate, probably because of the coordination of the
highly nucleophilic selenium and the Lewis acid.
15
Scheme 4. Synthesis of seleno-lactose 2. Reagents and conditions: (a) 7, piperidine,
DIEA/DMF, rt, 85%; (b) NaOMe, MeOH/THF (2:1), rt, quant.
R
O
Ph
O
d
b
O
HO
O
OSE
OMBn
18 R = OH
19
OSE
MBnO
RO
96%
64%
(2 steps)
OR
16 R = H
c
a
R = Br
17 R = MBn
93%
SeMe
O
SeTol
O
f
h
RO
RO
OSE
OSE
OMBn
MBnO
MBnO
89%
79%
OMBn
g
87%
22 R = Ac
23 R = H
e
20
R = H
21 R = Ac
98%
BzO
OBz
OR
BzO
BzO
OBz
O
O
RO
a
OSE
b
BzO
O
O
OBz
SeMe
j
BzO
O
NH
CCl3
25 R = MBn
26 R = H
i
3
24
quant.
quant.
Scheme 5. Synthesis of seleno-lactose 3. Reagents and conditions: (a) p-methoxy-
benzyl chloride, NaH/DMF, rt; (b) TFA/CH2Cl2, ꢀ20 °C, 64% (2 steps); (c) CBr4, PPh3/
Pyr, 65 °C, 93%; (d) 7, piperidine, DIEA/DMF, 60 °C, 96%; (e) Ac2O/Pyr, rt, 98%; (f)
MeI, MeNHNH2, Cs2CO3/DMF, rt, 89%; (g) NaOMe/MeOH, rt, 87%; (h) 24, TMSOTf,
4 Å MS/CH2Cl2, 0 °C, 79%; (i) TFA/CH2Cl2, ꢀ20 °C, quant.; (j) NaOMe, MeOH/THF
(2:1), sonication, quant. MBn = p-methoxybenzyl, TMSOTf = trimethylsilyl trifluo-
romethanesulfonate, MS = molecular sieves, TFA = trifluoroacetic acid.
with Tol2Se (7) in the presence of piperidine and DIEA at 70 °C in
DMF afforded toluylseleno-glucose 20 in 96% yield. Acetylation of
the C-4 hydroxyl group in 20, followed by conversion of the TolSe
group into a MeSe group gave 22 in 89% yield. The acetyl group was
deprotected, producing 23 in high yield (75% from 20). The 6-
MeSe-glucosyl acceptor was subjected to glycosylation with
galactosyl imidate donor 24.14 Equimolar amounts of 23 and 24
were reacted in CH2Cl2 in the presence of TMSOTf as a catalyst.15
When this reaction was performed at 0 °C, the best yield of disac-
charide 25 was 79%. The MBn protecting groups were quantita-
tively removed from the hydroxyl groups with trifluoroacetic
acid in CH2Cl2 at ꢀ20 °C without affecting the MeSe group. Com-
pound 3 was obtained by subsequent de-O-benzoylation.
In the case of 6-MeSe-lactosyl donor 41, the coordination may
induce a Seꢁ ꢁ ꢁO interaction,21 which renders the glycosyl donor
much less reactive. A similar deactivation may have occurred dur-
ing the glycosidation of donors 33 and 37 (Scheme 8). Although the
OH
O
AcO
BnO
BnO
SeMe
O
b
BnO
AcO
BzO
O
a
BzO
SeMe
O
a
OMe
38
The synthesis of 60-methylseleno-lactose 4 was achieved by the
glycosylation of the C-4 hydroxyl group of a glycosyl acceptor with
the 6-MeSe-galactosyl donor (Scheme 6). The 4,6-diol derivative of
galactoside 2716 was converted into 6-MeSe derivative 31 in rela-
tively high yields via a similar route to that for compound 20
(63% over 4 steps). Acidic treatment of compound 31 afforded
32
BzO
BnO
BnO
O
Cp2ZrCl2, AgOTf
MS4Å/ CH2Cl2
0 °C to RT
97%
BzO
37
F
BnO
OMe
39
53%
(α:β = 1/1.7)
Scheme 7. Glycosidation of 6-MeSe-galactosyl fluoride donor 37. Reagents and
conditions: (a) DAST/CH2Cl2, 0 °C, 97%. DAST = diethylaminosulfur trifluoride.

T. Suzuki et al. / Bioorg. Med. Chem. 22 (2014) 2090–2101
2093
Table 1
Comparison of the glycosylations using lactosyl 40, 6-MeSe-lactosyl 41, and 60-MeSe-lactsoyl 42 imidates
TMSOTf
O
β-glycoside
40 - 42
+
HO
NHTFAc
4Å MS (AW-300)
CH2Cl2
0 °C
Entry
1
Donor
BzO
Equiv of TMSOTf
0.1
Time
Product (% yield)
OBz
O
BzO
O
O
O
NH
BzO
O
BzO
O
OBz
40 (α:β= 20/1)
30 min
43 (97)
Cl
C
3
OBz
BzO
BzO
OBz
O
BzO
BzO
NH
O
O
2
3
0.4
0.1
8 h
44 (66)
45 (90)
CCl3
OBz
SeMe
41 (α:β= 20/1)
AcO
BzO
SeMe
O
BzO
BzO
NH
CCl3
O
O
45 min
OBz
OAc
(α:β= 20/1)
42
TFAc = trifluoroacetyl.
X-ray crystallography.23 First, we attempted to co-crystallize hu-
man galectin-9 NCRD with seleno-lactoses 1 to 4. Crystals of hu-
man galectin-9 NCRD with seleno-lactoses 1–3 were obtained
under different conditions; however, seleno-lactose 4 crystals were
not obtained. Although anomalous selenium signals were observed
from the crystals of human galectin-9 NCRD with seleno-lactoses 1
to 3 in the X-ray absorption fine structure (XAFS) experiments, we
could not obtain crystals with seleno-lactoses 1 and 2 with suffi-
cient quality for phase determination. In the seleno-lactose 1 crys-
tal, not all the selenium positions could be correctly identified in
the asymmetric unit. The affinity of seleno-lactose 1 for human
galectin-9 NCRD may be slightly weaker than that of the other se-
leno-lactoses. It was difficult for the phase determination and
model building of human galectin-9 NCRD with seleno-lactose 2
because of the slight low resolution (2.7–2.8 Å). However, the
structure of human galectin-9 co-crystallized with 6-MeSe-lactose
3 was successfully determined at a higher resolution (1.4 Å).
Good anomalous signals were obtained from all the crystals by
XAFS before the diffraction data were collected. The structure of
human galectin-9 NCRD with 6-MeSe-lactose 3 was solved by
the SAD method and also by the MAD method (data not shown).
In this case, the SAD method was sufficient to determine the struc-
ture of galectin-9 with 6-MeSe-lactose 3. Therefore, the structural
details described hereafter are from the SAD data.
E+
E
(TMS+, Ag+ or Cp(Cl)Zr+)
SeMe
O
SeMe
O
BzO
BzO
BzO
BzO
deactivated form
Lg
Lg
E+
SeMe
O
SeMe
O
R-OH
E
BzO
OR
BzO
BzO
Lg
BzO
Scheme 8. Proposed mechanism for the diminished reactivity of the 6-MeSe-
glycosyl donor based on the results in this study. Lg = leaving group.
presence of the MeSe group affected the reactivity of the glycosyl
donor, seleno-lactoses 1 to 4 were obtained.
2.2. Structure of human galectin-9 N-terminal carbohydrate
recognition domain with seleno-lactose
We attempted to use seleno-lactoses 1 to 4 to solve the struc-
ture of human galectin-9 N-terminal carbohydrate recognition do-
main (NCRD).22 Some crystal structures of human galectin-9 NCRD
with various carbohydrates, including lactose, have been solved by
Figure 2. Comparison of overall structure of human galectin-9 NCRD with
lactose (PDB_id: 2EAK), shown in pink. -Lactose is represented as a stick structure. (b) Cartoon representation of human galectin-9 NCRD with 6-MeSe-lactose 3, shown in
orange. Carbon atoms in 6-MeSe-lactose 3 are shown in green in the stick structure (PDB id: 3WLU).
a-lactose and seleno-lactose 3. (a) Cartoon representation of human galectin-9 NCRD with a-
a

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T. Suzuki et al. / Bioorg. Med. Chem. 22 (2014) 2090–2101
Figure 3. Comparison of the binding site of human galectin-9 NCRD with 6-MeSe-lactose 3 and
galectin-9 NCRD, shown in pink. -Lactose and residues that interacted with -lactose are shown as stick structures. (b) Cartoon representation of 6-MeSe-lactose binding
site of human galectin-9 NCRD. 6-MeSe-lactose and residues that interact with 6-MeSe-lactose are shown as stick structures. The colors of galectin-9 and 6-MeSe-lactose are
the same as in Figure 2. (c) Schematic diagram of -lactose created in Ligplot. Green dotted lines indicate hydrogen bonds between -lactose and the galectin-9 NCRD
residues. Lengths of the hydrogen bonds are shown in green (Å). (d) Schematic diagram of 6-MeSe-lactose created in Ligplot.
a-lactose. (a) Cartoon representation of the a-lactose binding site of human
a
a
a
a
Four molecules of galectin-9 with 6-MeSe-lactose 3 are present
in an asymmetric unit. There are no significant differences between
the four galectin-9 structures with 6-MeSe-lactose 3 in the asym-
metric unit. The RMSD values obtained by secondary structure
matching were between 0.303 and 0.548 Å. The positions of the
anomalous signals at high occupancies were consistent with the
positions of the selenium atoms on 6-MeSe-lactose 3 bound to hu-
man galectin-9 NCRD. The selenium atom in seleno-lactose 3 has
multiple conformations in the binding forms. The details of the
structure determination are described in the experimental section
and the refinement statistics are presented in the Supplementary
Material.
the residues on the ligand binding site of human galectin-9 NCRD
between 6-MeSe-lactose 3 and the -lactose binding forms (Fig. 3).
The same residues form the hydrogen bonds to the sugar chain
backbone in both structures. The relative positions of the protein
to the ligand are very similar in both structures.
a
3. Conclusions
We have shown that Tol2Se (7) functions as a synthetic equiva-
lent of the toluylselenolate anion, which enabled the efficient
incorporation of selenium at the electrophilic site of carbohydrate
derivatives. The considerable advantage of selenation with 7 over
the widely used method with RSeSeR and NaBH4 is the compatibil-
ity with ester groups. Although the 6-MeSe glycosyl donors
showed moderate reactivity in the coupling reaction, the compat-
ibility of the seleno-carbohydrate units with conventional chemis-
try for carbohydrate synthesis allowed four types of seleno-lactose
(1–4) to be synthesized.
The overall structure of human galectin-9 NCRD with 6-MeSe-
lactose 3 (PDB_ID: 3WLU) is almost same as the structure of
human galectin-9 NCRD with
a-lactose (PDB_ID: 2EAK) (Fig. 2).
6-MeSe-lactose 3 is located on the same
a
-lactose binding site in
the structure of human galectin-9 NCRD. The difference in RMSD
values between the structures obtained by secondary structure
matching is less than 0.617 Å. There are no major distortions of

T. Suzuki et al. / Bioorg. Med. Chem. 22 (2014) 2090–2101
2095
Our case study of the structural determination of a carbohy-
drate–protein complex has extended the potential of seleno-
carbohydrate as molecular tool for SAD/MAD phasing. Because
the proteins do not need to be modified with SeMet, this phasing
method does not interfere with protein production and does not al-
ter the structural and chemical properties of the protein. Further-
more, the correct ligand configuration can be confirmed by
detecting the anomalous signal from the selenium atoms incorpo-
rated in the seleno-carbohydrates, even if the collected data is dif-
fracted at low resolution. This might be useful for drug design
based on carbohydrate-protein recognition. However, since the
proper positioning of selenium atom in the carbohydrate residue
cannot be predicted in every case of co-crystallization with protein,
the creation of a library of seleno-carbohydrates is necessary for
analyzing the structures of unknown carbohydrate-binding pro-
teins. Therefore, we are currently assembling a diverse collection
of seleno-carbohydrates by using the facile selenation method
developed in this study.
data (1H, 13C, 77Se NMR) of compound 7 were identical to those
reported;10 HRMS: m/z C16H14O2SeNa+: 341.0051 [M+Na]+; found:
341.0051.
4.1.2. p-Methylbenzoyl (2,3,4,6-tetra-O-benzoyl-b-D-
galactopyranosyl)-(1?4)-2,3,6-tri-O-benzoyl-1-seleno-b-D-
glucopyranoside (10)
Compound 9 (298 mg, 264
added dropwise to a solution of 7 (101 mg, 318
propylethylamine (48 L, 279 mol) and piperidine (30
302 mol) in degassed DMF (2.7 mL) as bubbled with argon gas.
l
mol) in degassed DMF (2.7 mL) was
mol), N,N-diiso-
L,
l
l
l
l
l
The mixture was stirred for 40 min at rt (completion of the reaction
was confirmed by TLC analysis; EtOAc/hexane, 2:3), then the reac-
tion mixture was diluted with EtOAc and the solution was washed
with 2M aqueous HCl, water, saturated aqueous NaHCO3 and brine,
dried (Na2SO4), and concentrated. The residue was purified by col-
umn chromatography on silica gel (EtOAc/toluene, 1:30) to give 10
(250 mg, 76%). [a]
D = +46.5° (c 1.0, CHCl3); 1H NMR (500 MHz,
CDCl3):d 8.02–7.13 (m, 39H, Ar), 5.92 (t, 1H, J1,2 = J2.3 = 8.9 Hz, H-
2a), 5.79–5.71 (m, 4H, H-1a, H-3a, H-2b, H-4b), 5.35 (dd, 1H,
J2,3 = 10.3 Hz, J3,4 = 3.4 Hz, H-3b), 4.86 (d, 1H, J1,2 = 7.9 Hz, H-1b),
4.57 (dd, 1H, Jgem = 12.4 Hz, J5,6a = 1.6 Hz, H-6aa), 4.50 (dd, 1H,
J5,6b = 4.2 Hz, H-6ba), 4.24 (t, 1H, J3,4 = J4,5 = 9.6 Hz, H-4a), 4.02 (m,
1H, H-5a), 3.88 (t, 1H, J5,6a = J5,6b = 6.9 Hz, H-5b), 3.75–3.68 (m,
2H, H-6ab, H-6bb), 2.34 (s, 3H, ArCH3); 13C NMR (125 MHz, CDCl3):
d 190.7, 165.8, 165.6, 165.4, 165.4, 165.3, 165.2, 164.8, 145.3,
135.5, 133.5, 133.4, 133.3, 133.3, 133.2, 133.1, 130.0, 129.9,
129.8, 129.8, 129.7, 129.6, 129.5, 129.5, 128.9, 128.9, 128.7,
128.6, 128.6, 128.5, 128.5, 128.3, 128.2, 127.6, 101.0, 79.9, 78.5,
75.7, 74.2, 71.8, 71.3, 70.7, 69.8, 67.5, 62.6, 61.1, 21.7; 77Se NMR
(94 MHz, CDCl3): d 626.2; HRMS: m/z calcd for C69H56O18SeNa+:
1275.2524 [M+Na]+; found: 1275.2524.
4. Experimental section
4.1. General procedures
All reactions were performed in round-bottom flask fitted with
balloon filled with argon, otherwise specified. Transfer of air and
moisture sensitive liquids were performed via cannula under a po-
sitive pressure of argon. When necessary, reaction mixtures were
sonicated with AS ONE US CLEANER USD-4R by following the re-
ported procedure.24 TLC analysis was performed on Merck TLC (sil-
ica gel 60F254 on glass plate). Compounds were visualized by
exposure to UV light (254 nm) or by spraying either with H2SO4
solution in EtOH (10%) or with Ninhydrine Spray which was pur-
chased from Wako Pure Chemical Industries Ltd, followed by heat-
ing. Flash column chromatography on silica gel (Fuji Silysia, 80
mesh and 300 mesh) or size exclusion chromatography on Sepha-
dex (Pharmacia LH-20) was performed with the solvent systems
(v/v) specified. Quantity of silica gel was usually estimated as
100 to 150-fold weight of sample to be charged. Dichloromethane,
N,N-dimethylformamide (DMF), methanol, tetrahydrofuran (THF)
and toluene as reaction media were purchased from Wako Pure
Chemical Industries Ltd, dried over 3 Å or 4 Å molecular sieves
and used without purification. When necessary, solvents were de-
gassed prior to use by sonication under reduced presseure for
20 min, followed by bubbling argon through the solvents for
30 min. Molecular sieves were purchased from Wako Chemical
Inc. and dried at 300 °C for 2 h in muffle prior to use. 1H, 13C and
77Se NMR spectra were recorded with JEOL JNM-ECA400, JNM-
ECA500, JNM-ECA600 and Bruker Avance III 500 spectrometers.
1H NMR chemical shifts are expressed in ppm (d) relative to the
signal of Me4Si as an internal standard. 13C NMR chemical shifts
are expressed in ppm (d) relative to the signal of the solvent as a
standard. 77Se NMR chemical shifts are expressed in ppm (d) rela-
tive to the external standard. High-resolution mass spectrometry
(HRMS) was performed with a Bruker Daltonics micrOTOF (ESI-
TOF) mass spectrometer. Specific rotations were measured with a
Horiba SEPA-300 high-sensitivity polarimeter.
4.1.3. Methyl (2,3,4,6-tetra-O-benzoyl-b-
(1?4)-2,3,6-tri-O-benzoyl-1-seleno-b- -glucopyranoside (11)
Methyl hydrazine (2.5 L, 64 mol) was added to a solution of
10 (40 mg, 32 mol), cesium carbonate (21 mg, 48 mol) and
methyl iodide (4.0 L, 64 mol) in degassed DMF (1.8 mL) in ice
D-galactopyranosyl)-
D
l
l
l
l
l
l
bath as bubbled with argon gas. The mixture was stirred for
40 min at rt (completion of the reaction was confirmed by TLC
analysis; EtOAc/toluene, 1:10), then the reaction mixture was di-
luted with EtOAc and the solution was washed with 2M aqueous
HCl, water, saturated aqueous NaHCO3 and brine, dried (Na2SO4),
and concentrated. The residue was purified by column chromatog-
raphy on silica gel (EtOAc/toluene, 1:25) to give 11 (37 mg, 99%).
[a]
D = +47.3° (c 0.9, CHCl3); 1H NMR (500 MHz, CDCl3): d 8.02–
7.13 (m, 35H, Ph), 5.80 (t, 1H, J2,3 = J3,4 = 9.3 Hz, H-3a), 5.74–5.70
(m, 2H, H-2b, H-4b), 5.53 (t, 1H, J1,2 = 9.8 Hz, H-2a), 5.38 (dd, 1H,
J2,3 = 10.3 Hz, J3,4 = 3.4 Hz, H-3b), 4.89–4.85 (m, 2H, H-1a, H-1b),
4.60 (dd, 1H, Jgem = 12.3 Hz, J5,6a = 1.5 Hz, H-6aa), 4.49 (dd, 1H,
J5,6b = 4.3 Hz, H-6ba), 4.25 (t, 1H, J4,5 = 9.6 Hz, H-4a), 3.91 (t, 1H,
J5,6a = J5,6b = 6.8 Hz, H-5b), 3.86 (m, 1H, H-5a), 3.79–3.70 (m, 2H,
H-6ab, H-6bb), 2.34 (s, 3H, SeMe); 13C NMR (125 MHz, CDCl3): d
166.8, 166.5, 166.4, 165.4, 165.3, 165.2, 164.8, 133.5, 133.3,
133.3, 133.3, 133.2, 133.1, 129.9, 129.8, 129.7, 129.6, 129.6,
129.5, 129.4, 129.1, 128.8, 128.7, 128.6, 128.6, 128.5, 128.5,
128.4, 128.2, 100.9, 78.1, 77.2, 75.8, 73.8, 71.8, 71.3, 70.9, 69.9,
67.5, 62.5, 61.0, 29.6, 2.4; 77Se NMR (94 MHz, CDCl3): d 211.2;
HRMS: m/z calcd for C62H52O17SeNa+: 1171.2262 [M+Na]+; found:
1171.2262.
4.1.1. p-Methylselenobenzoic anhydride (7)
Triethylphosphite (8.7 mL, 50 mmol) was added dropwise to a
solution of 8 (19.9 g, 50 mmol) in toluene (250 mL) at rt. The mix-
ture was stirred for 2 h at rt (completion of the reaction was con-
firmed by TLC analysis; CH2Cl2/hexane, 1:2), then the reaction
mixture was evaporated. The residue was dissolved in Et2O and fil-
tered through Celite. Combined filtrate and washings were directly
crystallized from Et2O/hexane to give 7 (14.3 g, 90%). Spectroscopic
4.1.4. Methyl (b-D-galactopyranosyl)-(1?4)-1-seleno-b-D-
glucopyranoside (1)
Sodium methoxide (28% in MeOH, 8 mg, 39
l
mol) was added to
L) and MeOH
L). The reaction mixture was stirred for 4 days (completion
a solution of 11 (45 mg, 39
(870
lmol) in THF (430 l
l

2096
T. Suzuki et al. / Bioorg. Med. Chem. 22 (2014) 2090–2101
of the reaction was confirmed by TLC analysis; CHCl3/MeOH/H2O,
5:4:1). Then the reaction mixture was concentrated. The residue
was purified by column chromatography on silica gel (CHCl3/
MeOH/H2O, 8:5:1) and Sephadex LH-20 (MeOH/H2O, 8:1) to give
for 6 h at 50 °C (completion of the reaction was confirmed by TLC
analysis; EtOAc/hexane, 1:2), then coevaporation with toluene.
The residue was dissolved in EtOAc and the solution was washed
with water, saturated aqueous Na2CO3, and brine, dried (Na2SO4),
and concentrated. The residue was purified by column chromatog-
raphy on silica gel (EtOAc/hexane, 1:2) to give 18 (608 mg, 64% in 2
1 (15 mg, 88%). [
a
]
D = ꢀ1.0° (c 1.0, MeOH); 1H NMR (500 MHz,
D2O): d 4.75 (d, 1H, J1,2 = 10.0 Hz, H-1a), 4.48 (d, 1H, J1,2 = 7.8 Hz,
H-1b), 4.00–3.70 (m, 10H, H-3a, H-4a, H-5a, H-6aa, H-6ba, H-3b, H-
4b, H-5b, H-6ab, H-6bb), 3.57 (dd, 1H, J2,3 = 10.0 Hz, H-2b), 3.50
(dd, 1H, J2,3 = 9.9 Hz, H-2a), 2.15 (s, 3H, SeMe); 13C NMR
(125 MHz, D2O): d 104.3, 81.4, 79.6, 77.1, 76.8, 74.0, 73.6, 72.4,
70.0, 62.5, 61.7 3.2; 77Se NMR (94 MHz, D2O): d 183.4; HRMS: m/
z calcd for C13H24O10SeNa+: 443.0427 [M+Na]+; found: 443.0429.
steps). [
a]
D = ꢀ27.5° (c 0.7, CHCl3); 1H NMR (600 MHz, CDCl3): d
7.31–6.86 (m, 8H, Ar), 4.90–4.87 (2 d, 2H, Jgem = 11.6 Hz, ArCH2),
4.67 (d, 1H, ArCH2), 4.59 (d, 1H, ArCH2), 4.44 (d, 1H, J1,2 = 7.5 Hz,
H-1), 4.00 (m, 1H, TMSCH2CH2), 3.86 (m, 1H, H-6a), 3.80 (s, 3H,
ArOCH3), 3.79 (s, 3H, ArOCH3), 3.74 (m, 1H, H-6b), 3.63 (m, 1H,
TMSCH2CH2), 3.51–3.48 (near t, 1H, J3,4 = J4,5 = 8.9 Hz, J4,OH = 2.1 Hz,
H-4), 3.40 (t, 1H, J2,3 = 8.9 Hz, H-3), 3.36 (t, 1H, H-2), 3.32–3.29 (m,
1H, H-5), 2.40 (d, 1H, OH), 2.15 (t, 1H, J6,OH = 6.2 Hz, OH), 1.07–1.04
(m, 2H, TMSCH2), 0.04 (s, 9H, TMS); 13C NMR (150 MHz, CDCl3): d
159.3, 159.2, 130.6, 130.5, 129.8, 129.6, 114.0, 113.8, 103.4, 83.4,
81.6, 74.8, 74.7, 74.3, 70.4, 67.8, 62.7, 55.2, 18.6, ꢀ1.5; HRMS:
m/z calcd for C27H40O8SiNa+: 543.2390 [M+Na]+; found: 543.2390.
4.1.5. p-Methylbenzoyl 2,3,4,6-tetra-O-acetyl-1-seleno-b-D-
galactopyranoside (13)
12 (90 mg, 220
dropwise to a solution of 7 (84 mg, 264
ylamine (45 L, 264 mol) and piperidine (25
l
mol) in degassed DMF (2.2 mL) was added
mol), N,N-diisopropyleth-
L, 253 mol) in de-
l
l
l
l
l
gassed DMF (2.2 mL) as bubbled with argon gas. The mixture was
stirred for 1.5 h at rt (completion of the reaction was confirmed by
TLC analysis; EtOAc/hexane, 2:3), then the reaction mixture was
diluted with EtOAc and the solution was washed with 2M aqueous
HCl, water, saturated aqueous NaHCO3 and brine, dried (Na2SO4),
and concentrated. The residue was purified by column chromatog-
raphy on silica gel (acetone/hexane, 1:5) to give 13 (99 mg, 85%).
Spectroscopic data (1H, 13C, 77Se NMR) of compound 13 were iden-
tical to those reported9; HRMS: m/z calcd for C22H26O10SeNa+:
553.0583 [M+Na]+; found: 553.0583.
4.1.8. 2-(Trimethylsilyl)ethyl 6-bromo-6-deoxy-2,3-di-O-(p-
methoxybenzyl)-b-
Carbontetrabromide (114 mg, 345
phine (89 mg, 340 mol) were added to a solution of 18 (117 mg,
225 mol) in pyridine (2.3 mL) in ice bath, and stirred for 10 min.
D-glucopyranoside (19)
lmol) and triphenylphos-
l
l
Then reaction mixture was warmed to 65 °C. Stirring was contin-
ued for 1 h (completion of the reaction was confirmed by TLC anal-
ysis; EtOAc/hexane, 1:1). Then the reaction mixture was quenched
by addition of MeOH and the residual solvent was removed by
coevaporation with toluene. The residue was purified by column
chromatography on silica gel (EtOAc/toluene, 1:20) to give 19
4.1.6. 2-(Trimethylsilyl)ethyl Se-(b-
4-dexoy-4-seleno-b- -glucopyranoside (2)
Sodium methoxide (28% in MeOH, 8 mg, 44
a solution of 15 (35 mg, 44 mol) in THF (490
(980 L). The reaction mixture was stirred for 20 h (completion
D-galactopyranosyl)-(1?4)-
D
(122 mg, 93%). [
a
]
D = ꢀ25.4° (c 1.0, CHCl3); 1H NMR (400 MHz,
lmol) was added to
CDCl3): d 7.31–6.86 (m, 8 H Ar), 4.89 (2 d, 2H, ArCH2), 4.66 (d,
1H, ArCH2), 4.56 (d, 1H, ArCH2), 4.43 (d, 1H, J1,2 = 6.8 Hz, H-1),
4.03 (m, 1H, TMSCH2CH2), 3.79 (s, 6H, ArOCH3), 3.71–3.64 (m,
2H, H-6a, TMSCH2CH2), 3.45 (m, 1H, J5,6b = 5.9 Hz, H-6b), 3.42–
3.36 (m, 4H, H-2, H-3, H-4, H-5), 2.24 (s, 1H, OH), 1.10–1.04 (m,
2H, TMSCH2), 0.04 (s, 9H, TMS); 13C NMR (100 MHz, CDCl3): d
159.4, 159.2, 130.5, 130.4, 129.8, 129.6, 114.0, 113.8, 103.1, 83.1,
81.5, 74.7, 74.2, 71.8, 67.5, 55.2, 32.6, 18.4, ꢀ1.5; HRMS: m/z calcd
for C27H39BrO7SiNa+: 605.1546 [M+Na]+; found: 605.1546.
l
lL) and MeOH
l
of the reaction was confirmed by TLC analysis; CHCl3/MeOH,
2:1). Then the reaction mixture was neutralized with Dowex-50
(H+) and filtered through cotton, and removed resin was washed
with MeOH. The combined filtrate and washings were concen-
trated. The residue was purified by column chromatography on
Sephadex LH-20 (MeOH) to give 2 (22 mg, quant.). [
a
]
D = ꢀ41.7°
(c 0.5, MeOH); 1H NMR (500 MHz, CD3OD):
d
4.73 (d, 1H,
J1,2 = 10.8 Hz, H-1b), 4.25 (d, 1H, J1,2 = 8.0 Hz, H-1a), 4.08–3.43 (m,
12H, H-3a, H-4a, H-5a, H-6aa, H-6ba, H-3b, H-4b, H-5b, H-6ab, H-
6bb, TMSCH2CH2), 3.17 (t, 1H, J2,3 = 8.0 Hz, H-2a), 2.92 (t, 1H,
J2.3 = 10.8 Hz, H-2b), 1.05-0.91 (m, 2H, TMSCH2), 0.00 (s, 9H,
TMS); 13C NMR (125 MHz, CD3OD): d 104.4, 83.0, 82.8, 79.0, 77.2,
76.8, 76.7, 72.7, 71.6, 68.9, 64.9, 63.7, 45.1, 20.0, ꢀ0.5; 77Se NMR
(94 MHz, CD3OD): d 288.0; HRMS: m/z calcd for C17H34O10SeNa+:
529.0979 [M+Na]+; found: 529.0979.
4.1.9. 2-(Trimethylsilyl)ethyl 6-deoxy-2,3-di-O-(p-
methoxybenzyl)-6-(p-methylbenzoylseleno)-b-D-
glucopyranoside (20)
7
(702 mg, 2.21 mmol), N,N-diisopropylethylamine (377
lL,
2.22 mmol) and piperidine (220
lL, 2.22 mmol) were added to a
solution of 19 (1.03 g, 1.77 mmol) in degassed DMF (18 mL) at rt
as bubbled with argon gas. Then reaction mixture was warmed
to 60 °C. Stirring was continued for 45 min (completion of the reac-
tion was confirmed by TLC analysis; EtOAc/toluene, 1:5), then the
reaction mixture was diluted with EtOAc, and the solution was
washed with 2M aqueous HCl, water, saturated aqueous NaHCO3
and brine, dried (Na2SO4), and concentrated. The residue was puri-
fied by column chromatography on Sephadex LH-20 (MeOH/CHCl3,
1:1) and silica gel (EtOAc/toluene, 1:20) to give 20 (1.19 g, 96%).
4.1.7. 2-(Trimethylsilyl)ethyl 2,3-di-O-(p-methoxybenzyl)-b-D-
glucopyranoside (18)
NaH (60% in oil, 220 mg, 5.49 mmol) was added to a solution of
16 (674 mg, 1.83 mmol) in DMF at 0 °C. The mixture was stirred for
30 min, after which p-methoxybenzyl chloride (632 lL,
4.03 mmol) was added at 0 °C. Stirring was continued for 22 h at
rt (completion of the reaction was confirmed by TLC analysis;
EtOAc/hexane, 1:2), the reaction mixture was quenched by addi-
tion of saturated aqueous NH4Cl at 0 °C and the residual solvent
was removed by coevaporation with toluene. The residue was dis-
solved in EtOAc and the solution was washed with water and brine,
dried (Na2SO4), and concentrated. The residue was purified by col-
umn chromatography on silica gel (EtOAc/hexane, 1:10) to give the
crude product 17. The crude product 17 was pumped in high vac-
uum for 12 h. Then the crude product 17 was dissolved in AcOH
(16 mL) and H2O (4 mL) at rt. The reaction mixture was stirred
[a]
D = ꢀ8.9° (c 1.2, CHCl3); 1H NMR (600 MHz, CDCl3): d 7.82–
6.85 (m, 12H, Ar), 4.87–4.82 (m, 2H, ArCH2), 4.70–4.65 (m, 2H,
ArCH2), 4.41 (d, 1H, J1,2 = 8.3 Hz, H-1), 3.97 (m, 1H, TMSCH2CH2),
3.79 (2s, 6H, ArOCH3), 3.62 (m, 1H, TMSCH2CH2), 3.52 (dd, 1H,
Jgem = 13.1 Hz, J5,6a = 2.7 Hz, H-6a), 3.48 (m, 1H, H-5), 3.44–3.42
(m, 2H, H-3, H-4), 3.39 (dd, 1H, J5,6b = 5.5 Hz, H-6b), 3.35 (t, 1H,
J2,3 = 8.9 Hz, H-2), 2.97 (d, 1H, J4,OH = 2.8 Hz, OH), 2.40 (s, 3H,
ArCH3), 1.06–1.03 (m, 2H, TMSCH2), 0.03 (s, 9H, TMS); 13C NMR
(150 MHz, CDCl3): d 195.8, 159.2, 159.2, 144.8, 136.2, 130.7,
130.7, 129.8, 129.6, 129.4, 127.4, 113.9, 113.7, 103.0, 83.0, 81.6,
75.0, 74.3, 73.1, 67.5, 55.2, 27.4, 21.7, 18.5, ꢀ1.4; 77Se NMR

T. Suzuki et al. / Bioorg. Med. Chem. 22 (2014) 2090–2101
2097
(113 MHz, CDCl3): d 485.9; HRMS: m/z calcd for C36H46O9SeSiNa+:
725.2019 [M+Na]+; found: 725.2024.
(EtOAc/hexane, 1:6) to give 23 (36 mg, 87%). [
a
]
D = ꢀ29.7° (c 0.7,
CHCl3); 1H NMR (600 MHz, CDCl3): d 7.31–6.87 (m, 8H, Ar), 4.89
(2 d, 2H, ArCH2), 4.66 (d, 1H, ArCH2), 4.56 (d, 1H, ArCH2), 4.41 (d,
1H, J1,2 = 7.4 Hz, H-1), 4.00 (m, 1H, TMSCH2CH2), 3.80 (s, 6H,
ArOCH3), 3.65 (m, 1H, TMSCH2CH2), 3.43–3.35 (m, 4H, H-2, H-3,
H-4, H-5), 2.93 (dd, 1H, Jgem = 13.2 Hz, J5,6a = 2.9 Hz, H-6a), 2.69
(dd, 1H, J5,6b = 8.2 Hz, H-6b), 2.16 (s, 1H, OH), 2.07 (s, 3H, SeMe),
1.06 (t, 2H, TMSCH2), 0.04 (s, 9H, TMS); 13C NMR (150 MHz, CDCl3):
d 159.4, 159.2, 130.6, 129.8, 129.6, 114.0, 113.8, 103.1, 83.3, 81.8,
76.2, 74.7, 74.2, 73.3, 67.4, 55.2, 26.9, 18.5, 5.6, ꢀ1.5; 77Se NMR
(113 MHz, CDCl3): d 63.3; HRMS: m/z calcd for C30H44O8SeSiNa+:
621.1757 [M+Na]+; found: 621.1757.
4.1.10. 2-(Trimethylsilyl)ethyl 4-O-acetyl-6-deoxy-2,3-di-O-(p-
methoxybenzyl)-6-(p-methylbenzoylseleno)-b-D-
glucopyranoside (21)
Acetic anhydride (7.6 mL, 8.06 mmol) was added to a solution
of 20 (1.13 g, 1.61 mmol) in pyridine (16.1 mL) in ice bath. The
mixture was stirred for 17 h at rt (completion of the reaction was
confirmed by TLC analysis; EtOAc/toluene 1:5), then reaction mix-
ture was quenched by addition of MeOH and the residual solvent
was removed by coevaporation with toluene. The residue was dis-
solved in EtOAc, and the solution was washed with 2M aqueous
HCl, water, saturated aqueous NaHCO3 and brine, dried (Na2SO4),
and concentrated. The residue was purified by column chromatog-
raphy on silica gel (EtOAc/toluene, 1:30) to give 21 (1.17 g, 98%).
4.1.13. 2-(Trimethylsilyl)ethyl (2,3,4,6-tetra-O-benzoyl-b-
galactopyranosyl)-(1?4)-6-deoxy-2,3-di-O-(p-methoxybenzyl)-
6-methylseleno-b- -glucopyranoside (25)
Molecular sieves (MS4Å, 148 mg) were added to a solution of 24
(58 mg, 78 mol) and 23 (46 mg, 77 mol) in CH2Cl2 (1.5 mL). The
suspension was stirred for 2 h at rt. Then TMSOTf (1.5 L,
7.7 mol) was added to the solution at 0 °C. The mixture was stir-
D-
D
[
a]
D = ꢀ4.7° (c 0.2, CHCl3); 1H NMR (600 MHz, CDCl3): d 7.78–
6.83 (m, 12 H Ar), 4.91 (t, 1H, J3,4 = J4,5 = 9.8 Hz, H-4), 4.84 (d, 1H,
Jgem = 10.3 Hz, ArCH2), 4.72 (d, 1H, ArCH2), 4.63 (d, 1H, Jgem = 11.0 -
Hz, ArCH2), 4.54 (d, 1H, ArCH2), 4.37 (d, 1H, J1,2 = 8.3 Hz, H-1), 3.97
(m, 1H, TMSCH2CH2), 3.78 (s, 6H, ArOCH3), 3.66–3.62 (m, 1H,
TMSCH2CH2), 3.54 (t, 1H, J2,3 = 8.9 Hz, H-3), 3.48–3.43 (m, 2H, H-
2, H-5), 3.38 (dd, 1H, Jgem = 13.1 Hz, J5,6a = 2.8 Hz, H-6a), 3.05 (dd,
1H, J5,6b = 9.6 Hz, H-6b), 2.38 (s, 3H, ArCH3), 2.05 (s, 3H, Ac),
1.06–1.01 (m, 2H, TMSCH2), 0.26 (s, 9H, TMS); 13C NMR
(150 MHz, CDCl3): d 193.5, 170.0, 159.2, 159.1, 144.6, 136.2,
130.6, 129.8, 129.4, 127.2, 113.7, 102.9, 81.9, 81.1, 74.6, 74.5,
73.8, 73.6, 67.4, 55.2, 26.8, 21.7, 21.0, 18.4, ꢀ1.4; 77Se NMR
(113 MHz, CDCl3): d 499.1; HRMS: m/z calcd for C37H48O9SeSiNa+:
767.2131 [M+Na]+; found: 767.2130.
l
l
l
l
red for 14 h at 0 °C (completion of the reaction was confirmed by
TLC analysis; EtOAc/toluene, 1:8). The reaction mixture was
quenched by addition of saturated aqueous NaHCO3 (500 lL) then
filtered through Celite, and the removed molecular sieves were
washed with CHCl3. The combined filtrate and washings were ex-
tracted with CHCl3, and the organic layer was washed with satu-
rated aqueous NaHCO3, dried (Na2SO4), and concentrated. The
residue was purified by column chromatography on Sephadex
LH-20 (MeOH/CHCl3, 1:1) to give 25 (71 mg, 79%). [a]D = +36.0° (c
0.9, CHCl3); 1H NMR (500 MHz, CDCl3): d 8.02–6.75 (m, 28H, Ar),
5.91 (d, 1H, J3,4 = 3.4 Hz, H-4b), 5.78 (dd, 1H, J1,2 = 8.0 Hz,
J2,3 = 10.4 Hz, H-2b), 5.53 (dd, 1H, H-3b), 5.15 (d, 1H, H-1b), 4.93
(s, 2H, ArCH2), 4.82 (d, 1H, Jgem = 10.7 Hz, ArCH2), 4.61 (d, 1H,
ArCH2), 4.36–4.33 (m, 2H, H-1a, H-6ab), 4.26 (dd, 1H, Jgem = 11.2 Hz,
J5,6b = 7.8 Hz, H-6bb), 4.03 (t, 1H, H-5b), 3.92 (m, 1H, TMSCH2CH2),
3.78 (m, 3H, ArOCH3), 3.68–3.55 (m, 6H, H-3a, H-4a, ArOCH3,
TMSCH2CH2), 3.45–3.35 (m, 2H, H-2a, H-5a), 2.83 (dd, 1H,
Jgem = 12.7 Hz, J5,6a = 2.7 Hz, H-6aa), 2.47 (dd, 1H, J5,6b = 9.1 Hz, H-
6ba), 1.86 (s, 3H, SeMe), 1.04-0.98 (m, 2H, TMSCH2), 0.01 (s, 9H,
TMS); 13C NMR (125 MHz, CDCl3): d 165.7, 165.5, 165.4, 165.1,
159.2, 158.8, 133.5, 133.5, 133.3, 133.1, 131.1, 130.6, 129.9,
129.8, 129.7, 129.7, 129.7, 129.4, 129.0, 129.0, 128.7, 128.6,
128.4, 128.3, 113.7, 113.6, 102.9, 101.4, 82.3, 81.9, 81.8, 75.4,
74.5, 74.3, 71.8, 71.4, 70.6, 67.8, 67.3, 61.3, 55.2, 55.1, 26.5, 18.5,
5.3, 0.0, ꢀ1.5; 77Se NMR (94 MHz, CDCl3): d 66.8; HRMS: m/z calcd
for C62H68O16SeSiNa+: 1199.3334 [M+Na]+; found: 1199.3334.
4.1.11. 2-(Trimethylsilyl)ethyl 4-O-acetyl-6-deoxy-2,3-di-O-(p-
methoxybenzyl)-6-methylseleno-b-
Cesium carbonate (157 mg, 480
456 mol) and methyl hydrazine (24.0
to a solution of 21 (68 mg, 91 mol) in degassed DMF (1.8 mL) at
D
-glucopyranoside (22)
mol), methyl iodide (28.3
L, 456 mol) were added
l
lL,
l
l
l
l
rt as bubbled with argon gas. The mixture was stirred for 25 min
at rt (completion of the reaction was confirmed by TLC analysis;
EtOAc/toluene, 1:5). Then the reaction mixture was diluted with
EtOAc, and the solution was washed with 2M aqueous HCl, water,
saturated aqueous NaHCO3 and brine, dried (Na2SO4), and concen-
trated. The residue was purified by column chromatography on sil-
ica gel (EtOAc/hexane, 1:5) to give 22 (52 mg, 89%). [
a
]
D = ꢀ10.6° (c
0.9, CHCl3); 1H NMR (600 MHz, CDCl3): d 7.28–6.84 (m, 8 H Ar),
4.87–4.82 (m, 2H, H-4, ArCH2), 4.73 (d, 1H, ArCH2), 4.64 (d, 1H,
ArCH2), 4.54 (d, 1H, ArCH2), 4.41 (d, 1H, J1,2 = 7.6 Hz, H-1), 4.01
(m, 1H, TMSCH2CH2), 3.80 (s, 3H, ArOCH3), 3.79 (s, 3H, ArOCH3),
3.67 (m, 1H, TMSCH2CH2), 3.48 (m, 3H, H-2, H-3, H-5), 2.64 (dd,
1H, Jgem = 13.1 Hz, J5,6a = 8.9 Hz, H-6a), 2.58 (dd, 1H, J5,6b = 2.7 Hz,
H-6b), 2.06 (s, 3H, SeMe), 1.95 (s, 3H, Ac), 1.06 (t, 2H, TMSCH2),
0.04 (s, 9H, TMS); 13C NMR (150 MHz, CDCl3): d 169.8, 159.2,
159.1, 130.6, 130.0, 129.4, 113.7, 113.7, 103.0, 81.9, 81.1, 75.1,
74.6, 74.5, 73.9, 67.4, 55.2, 26.3, 21.0, 18.5, 5.7, ꢀ1.5; 77Se NMR
(113 MHz, CDCl3): d 76.9; HRMS: m/z calcd for C30H44O8SeSiNa+:
663.1863 [M+Na]+; found: 663.1868.
4.1.14. 2-(Trimethylsilyl)ethyl (2,3,4,6-tetra-O-benzoyl-b-D-
galactopyranosyl)-(1?4)-6-deoxy-6-methylseleno-b-D-
glucopyranoside (26)
Trifluoroacetic acid (290
lL) was added to a solution of 25
(52 mg, 44 mol) in CH2Cl2 (580
l
l
L) at ꢀ20 °C, and the mixture
was stirred for 75 min at ꢀ20 °C (completion of the reaction was
confirmed by TLC analysis; MeOH/CHCl3, 1:10). The reaction mix-
ture was quenched by addition of saturated aqueous NaHCO3
(3 mL) and extracted with CHCl3, and the organic layer was washed
with saturated aqueous NaHCO3, dried (Na2SO4), and concentrated.
The residue was purified by column chromatography on silica gel
4.1.12. 2-(Trimethylsilyl)ethyl 6-deoxy-2,3-di-O-(p-
methoxybenzyl)-6-methylseleno-b-
A catalytic amount of sodium methoxide (28% in MeOH) was
added to a solution of 22 (45 mg, 69 mol) in MeOH (1.4 mL).
D-glucopyranoside (23)
(EtOAc/hexane, 3:8) to give 26 (41 mg, quant.). [a]D = +78.4° (c
l
0.4, CHCl3); 1H NMR (500 MHz, CDCl3): d 8.10–7.23 (m, 20H, Ph),
6.00 (d, 1H, J3,4 = 3.1 Hz, H-4b), 5.87 (dd, 1H, J1,2 = 8.1 Hz,
J2,3 = 10.4 Hz, H-2b), 5.63 (dd, 1H, H-3b), 4.99 (d, 1H, H-1b), 4.64
(dd, 1H, Jgem = 11.5 Hz, J5,6a = 4.4 Hz, H-6ab), 4.53–4.45 (m, 2H, H-
5b, H-6bb), 4.36 (s, 1H, OH), 4.32 (d, 1H, J1,2 = 7.8 Hz, H-1a), 3.92
(m, 1H, TMSCH2CH2), 3.73 (t, 1H, J2,3 = J3,4 = 8.3 Hz, H-3a), 3.61
(m, 1H, TMSCH2CH2), 3.56–3.49 (m, 2H, H-4a, H-5a), 3.41 (t, 1H,
The mixture was stirred for 44 h at rt (completion of the reaction
was confirmed by TLC analysis; EtOAc/hexane, 1:5). Then the reac-
tion mixture was neutralized with Dowex-50 (H+), the mixture was
filtered through cotton, and removed resin was washed with
MeOH. The combined filtrate and washings were concentrated.
The residue was purified by column chromatography on silica gel

2098
T. Suzuki et al. / Bioorg. Med. Chem. 22 (2014) 2090–2101
H-2a), 2.58 (dd, 1H, Jgem = 12.7 Hz, J5,6a = 2.0 Hz, H-6aa), 2.49–2.45
(m, 2H, H-6ba, OH), 1.69 (s, 3H, SeMe), 1.06-0.94 (m, 2H, TMSCH2),
0.00 (s, 9H, TMS); 13C NMR (125 MHz, CDCl3): d 166.1, 165.4, 165.0,
133.8, 133.7, 133.4, 133.4, 130.0, 129.8, 129.7, 129.1, 128.7, 128.7,
128.7, 128.6, 128.5, 128.4, 128.3, 102.3, 101.6, 85.7, 74.7, 74.7,
73.7, 72.3, 71.5, 69.6, 68.0, 67.2, 62.4, 29.7, 25.9, 18.2, 5.0, 0.0,
ꢀ1.5; 77Se NMR (94 MHz, CDCl3): d 61.1; HRMS: m/z calcd for
raphy on silica gel (EtOAc/toluene, 1:15) to give 29 (6.52 g, 96%).
D = +36.8° (c 0.2, CHCl3); 1H NMR (400 MHz, CDCl3): d 7.99–
[a]
7.47 (m, 14H, Ar), 5.72 (dd, 1H, J1,2 = 8.1 Hz, J2,3 = 10.4 Hz, H-2),
5.30 (dd, 1H, J3,4 = 3.2 Hz, H-3), 4.70 (d, 1H, H-1), 4.36 (dd, 1H,
J4,OH = 6.0 Hz, H-4), 4.06 (m, 1H, TMSCH2CH2), 3.85 (t, 1H, J5,6a = -
J5,6b = 7.3 Hz, H-5), 3.63 (m, 1H, TMSCH2CH2), 3.50–3.39 (m, 2H,
H-6a, H-6b), 2.59 (d, 1H, OH), 2.41 (s, 3H, ArCH3), 1.00–0.84 (m,
2H, TMSCH2), ꢀ0.07 (s, 9H, TMS); 13C NMR (100 MHz, CDCl3): d
194.3, 165.9, 165.3, 145.1, 136.0, 133.3, 133.0, 129.9, 129.7,
129.5, 129.1, 129.0, 128.4, 128.2, 127.4, 100.7, 74.5, 69.7, 68.4,
67.4, 24.5, 21.7, 17.9, ꢀ1.5; 77Se NMR (75 MHz, CDCl3): d 530.7;
HRMS: m/z calcd for C33H38O8SeSiNa+: 693.1393 [M+Na]+; found:
693.1390.
C
46H52O14SeSiNa+: 959.2184 [M+Na]+; found: 959.2184.
4.1.15. 2-(Trimethylsilyl)ethyl (b-
deoxy-6-methylseleno-b- -glucopyranoside (3)
Sodium methoxide (28% in MeOH, 7 mg, 38 mol) was added to
a solution of 26 (36 mg, 39 mol) in THF (420 L) and MeOH
(840 L). The reaction mixture was sonicated for 5.5 h (completion
D-galactopyranosyl)-(1?4)-6-
D
l
l
l
l
of the reaction was confirmed by TLC analysis; CHCl3/MeOH/H2O,
5:4:1). Then the reaction mixture was neutralized with Dowex-
50 (H+) and filtered through cotton, and the removed resin was
washed with MeOH. The combined filtrate and washings were con-
centrated. The residue was purified by column chromatography on
silica gel (MeOH/CHCl3, 1:7) and Sephadex LH-20 (MeOH) to give 3
4.1.18. 2-(Trimethylsilyl)ethyl 4-O-acetyl-2,3-di-O-benzoyl-6-
deoxy-6-(p-methylbenzoylseleno)-b-D-galactopyranoside (30)
Acetic anhydride (3.9 mL, 41.3 mmol) was added to a solution
of 29 (5.48 g, 8.18 mmol) in pyridine (40.9 mL) in ice bath. The
reaction mixture was stirred for 25 h at rt (completion of the reac-
tion was confirmed by TLC analysis, EtOAc/hexane, 1:3), which was
then quenched by addition of MeOH, and the residual solvent was
removed by coevaporation with toluene. The residue was dissolved
in EtOAc and the solution was washed with 2M aqueous HCl, water,
saturated aqueous NaHCO3 and brine, dried (Na2SO4), and concen-
trated. The residue was purified by column chromatography on sil-
(20 mg, quant.). [a]
D = +3.5° (c 0.4, MeOH); 1H NMR (500 MHz, CD3
OD): d 4.34 (d, 1H, J1,2 = 7.6 Hz, H-1b), 4.29 (d, 1H, J1,2 = 7.9 Hz,
H-1a), 3.95 (m, 1H, TMSCH2CH2), 3.82 (d, 1H, J3,4 = 2.9 Hz, H-4b),
3.78 (dd, 1H, Jgem = 11.5 Hz, J5,6a = 7.6 Hz, H-6ab), 3.71–3.65 (m,
2H, H-6bb, TMSCH2CH2), 3.60–3.51 (m, 3H, H-5a, H-2b, H-5b),
3.50–3.43 (m, 3H, H-3a, H-4a, H-3b), 3.23 (t, 1H, H-2a), 3.18 (dd,
1H, Jgem = 13.0 Hz, J5,6a = 2.5 Hz, H-6aa), 2.79 (dd, 1H, J5,6b = 8.1 Hz,
H-6ba), 2.06 (s, 1H, SeMe), 1.02 (m, 2H, TMSCH2), 0.0 (s, 9H,
TMS); 13C NMR (125 MHz, CD3OD): d 105.5, 103.5, 84.6, 77.1,
77.0, 76.3, 74.9, 74.8, 72.5, 70.2, 68.0, 62.5, 27.5, 19.1, 5.2, ꢀ1.4;
77Se NMR (94 MHz, CDCl3): d 56.5; HRMS: m/z calcd for C18H36O10
SeSiNa+: 543.1135 [M+Na]+; found: 543.1135.
ica gel (EtOAc/hexane, 1:8) to give 30 (5.60 g, 96%). [a]D = +30.8° (c
1.3, CHCl3); 1H NMR (400 MHz, CDCl3): d 7.97–7.25 (m, 14H, Ar),
5.76 (d, 1H, J3,4 = 3.3 Hz, H-4), 5.67 (dd, 1H, J1,2 = 7.8 Hz,
J2,3 = 10.5 Hz, H-2), 5.40 (dd, 1H, H-3), 4.73 (d, 1H, H-1), 4.07 (m,
1H, TMSCH2CH2), 3.97 (t, 1H, J5,6a = J5,6b = 7.3 Hz, H-5), 3.64 (m,
1H, TMSCH2CH2), 3.36–3.25 (m, 2H, H-6a, H-6b), 2.41 (s, 3H,
ArCH3), 2.16 (s, 3H, Ac), 1.02–0.85 (m, 2H, TMSCH2), ꢀ0.07 (s, 9H,
TMS); 13C NMR (100 MHz, CDCl3): d 193.0, 170.1, 165.5, 165.2,
145.0, 135.9, 133.2, 133.0, 129.7, 129.6, 129.5, 129.0, 128.3,
128.2, 127.3, 100.7, 73.2, 72.1, 69.6, 69.1, 67.7, 60.3, 24.4, 21.7,
20.6, 17.9, 14.1, ꢀ1.5; 77Se NMR (75 MHz, CDCl3): d 495.6; HRMS:
4.1.16. 2-(Trimethylsilyl)ethyl 2,3-di-O-benzoyl-6-bromo-6-
deoxy-b-
Carbontetrabromide (262 mg, 790
phine (310 mg, 1.18 mmol) were added to a solution of 27
(384 mg, 787 mol) in pyridine (5.3 mL) in ice bath, and stirred
D-galactopyranoside (28)
l
mol) and triphenylphos-
m/z calcd for
C
35H40O9SeSiNa+: 735.1504 [M+Na]+; found:
l
735.1504.
for 10 min. Then the reaction mixture was warmed to 65 °C. Stir-
ring was continued for 1 h (completion of the reaction was con-
firmed by TLC analysis; EtOAc/toluene, 1:5), then the reaction
mixture was quenched by addition of MeOH, and the residual sol-
vent was removed by coevaporation with toluene. The residue was
purified by column chromatography on silica gel (EtOAc/toluene,
4.1.19. 2-(Trimethylsilyl)ethyl 4-O-acetyl-2,3-di-O-benzoyl-6-
deoxy-6-methylseleno-b- -galactopyranoside (31)
Cesium carbonate (289 mg, 885 mol), methyl iodide (53.9
865 mol) and methyl hydrazine (34.2 L, 650 mol) were added
to a solution of 30 (308 mg, 432 mol) in degassed DMF (8.7 mL)
D
l
lL,
l
l
l
l
1:20) to give 28 (379 mg, 87%). [
a]
D = +63.8° (c 1.1, CHCl3); 1H
at rt as bubbled with argon gas. The mixture was stirred for 2 h
NMR (400 MHz, CDCl3): d 7.99–7.36 (m, 10H, Ph), 5.69 (dd, 1H,
J1,2 = 7.8 Hz, J2,3 = 10.5 Hz, H-2), 5.32 (dd, 1H, J3,4 = 3.2 Hz, H-3),
4.72 (d, 1H, H-1), 4.47 (dd, 1H, J4,OH = 6.0 Hz, H-4), 4.07 (m, 1H,
TMSCH2CH2), 3.93 (t, 1H, J5,6a = J5,6b = 6.9 Hz, H-5), 3.69–3.57 (m,
3H, H-6a, H-6b, TMSCH2CH2), 2.25 (d, 1H, OH), 1.00-0.81 (m, 2H,
TMSCH2), ꢀ0.07 (s, 9H, TMS); 13C NMR (100 MHz, CDCl3): d
165.8, 165.3, 133.5, 133.1, 129.9, 129.7 128.9, 128.5, 128.3, 100.8,
74.6, 74.3, 69.5, 67.6, 67.3, 28.9, 17.9, ꢀ1.5; HRMS: m/z calcd for
at rt, while monitoring the reaction by TLC analysis (EtOAc/toluene,
1:10). Then another amount of methyl iodide (53.9
lL, 865 lmol)
and methyl hydrazine (34.2 L, 650 mol) were added to the reac-
l
l
tion mixture and then bubbling with argon gas was stopped. Stir-
ring was continued for another 15h, then the reaction mixture was
diluted with EtOAc, and the solution was washed with 2M aqueous
HCl, water, saturated aqueous NaHCO3 and brine, dried (Na2SO4),
and concentrated. The residue was purified by column chromatog-
raphy on silica gel (EtOAc/hexane, 1:3) to give 31 (206 mg, 78%).
C
25H31BrO7SiNa+: 573.0915 [M+Na]+; found:573.0912.
[a]
D = +31.2° (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3): d 7.98–
4.1.17. 2-(Trimethylsilyl)ethyl 2,3-di-O-benzoyl-6-deoxy-6-(p-
methylbenzoylseleno)-b- -galactopyranoside (29)
(4.12 g, 13.0 mmol), N,N-diisopropylethylamine (2.2 mL,
7.33 (m, 10H, Ph), 5.69–5.64 (m, 2H, H-4, H-2), 5.41 (dd, 1H,
J2,3 = 10.5 Hz, J3,4 = 2.8 Hz, H-3), 4.74 (d, 1H, J1,2 = 8.2 Hz, H-1),
4.06 (m, 1H, TMSCH2CH2), 3.98 (m, 1H, J5,6a = 7.8 Hz, J5,6b = 6.0 Hz,
H-5), 3.64 (m, 1H, TMSCH2CH2), 2.84 (dd, 1H, Jgem = 12.8 Hz, H-
6a), 2.60 (dd, 1H, H-6b), 2.14 (s, 3H, Ac), 2.09 (s, 3H, SeMe),
1.02–0.85 (m, 2H, TMSCH2), ꢀ0.07 (s, 9H, TMS); 13C NMR
(100 MHz, CDCl3) d 170.2, 165.6, 165.2, 133.3, 133.1, 132.3,
129.7, 129.6, 129.0, 128.4, 128.4, 128.3, 100.8, 74.6, 72.1, 69.6,
69.3, 67.7, 24.6, 20.6, 17.9, 5.6, ꢀ1.5; 77Se NMR (75 MHz, CDCl3):
d 73.2; HRMS: m/z calcd for C28H36O8SeSiNa+: 631.1237 [M+Na]+;
found: 631.1237.
D
7
13.0 mmol) and piperidine (1.3 mL, 13.0 mmol) were added to a
solution of 28 (5.61 g, 10.2 mmol) in degassed DMF (67 mL) at rt
as bubbled with argon gas. Then mixture was warmed to 100 °C
and stirred for 45 min (completion of the reaction was confirmed
by TLC analysis; EtOAc/hexane, 1:2). The reaction mixture was di-
luted with EtOAc, and the solution was washed with 2M aqueous
HCl, water, saturated aqueous NaHCO3 and brine, dried (Na2SO4),
and concentrated. The residue was purified by column chromatog-

T. Suzuki et al. / Bioorg. Med. Chem. 22 (2014) 2090–2101
2099
4.1.20. 4-O-Acetyl-2,3-di-O-benzoyl-6-deoxy-6-methylseleno-
galactopyranose (32)
D
-
128.8, 128.5, 128.4, 113.7, 113.5, 102.9, 100.6, 82.2, 81.5, 77.3,
74.6, 74.4, 72.3, 72.1, 70.2, 68.6, 67.5, 62.4, 55.3, 55.2, 29.7, 23.9,
20.7, 20.6, 18.4, 5.5, 1.0, 0.0, ꢀ1.5; 77Se NMR (113 MHz, CDCl3): d
64.8; HRMS: m/z calcd for C52H64O16SeSiNa+: 1075.3021 [M+Na]+;
found: 1075.3021.
Trifluoroacetic acid (2.1 mL) was added to a solution of 31
(190 mg, 312 mol) in CH2Cl2 (4.1 mL) at 0 °C. The mixture was
l
stirred for 2 h at rt (completion of the reaction was confirmed by
TLC analysis; MeOH/CHCl3, 1:50), then the reaction mixture was
coevaporation with toluene. The residue was purified by column
chromatography on silica gel (EtOAc/hexane, 1:20) to give 32
4.1.23. 2-(Trimethylsilyl)ethyl (4-O-acetyl-2,3-di-O-benzoyl-6-
deoxy-6-methylseleno-b-
acetyl-b- -glucopyranoside (36)
Trifluoroacetic acid (350 L) was added to a solution of 35
(55 mg, 52 mol) in CH2Cl2 (700
L) at ꢀ20 °C, and the mixture
D-galactopyranosyl)-(1?4)-6-O-
(128 mg, 81%):
a
-isomer; 1H NMR (600 MHz, CDCl3) d 8.00–7.35
D
(m, 10H, Ph), 5.88 (dd, 1H, J2,3 = 11.0 Hz, J3,4 = 2.8 Hz, H-3), 5.75–
5.71 (m, 2H, H-1, H-4), 5.58 (dd, 1H, J1,2 = 3.4 Hz, H-2), 4.54 (t,
1H, J5,6a = J5,6b = 6.9 Hz, H-5), 3.36 (br s, 1H, OH), 2.74 (dd, 1H,
Jgem = 13.1 Hz, H-6a), 2.58 (dd, 1H, H-6b), 2.16 (s, 3H, Ac), 2.05 (s,
3H, SeMe); 77Se NMR (113 MHz, CDCl3):d 64.4; HRMS: m/z calcd
for C23H24O8SeNa+: 531.0534 [M+Na]+; found: 531.0534.
l
l
l
was stirred for 2.5 h at ꢀ20 °C (completion of the reaction was con-
firmed by TLC analysis; MeOH/CHCl3, 1:15). The reaction mixture
was quenched by addition of saturated aqueous NaHCO3 (3 mL)
and extracted with CHCl3, and the organic layer was washed with
saturated aqueous NaHCO3, dried (Na2SO4), and concentrated. The
residue was purified by column chromatography on silica gel
4.1.21. 4-O-Acetyl-2,3-di-O-benzoyl-6-deoxy-6-methylseleno-D-
galactopyranosyl trichloroacetimidate (33)
(EtOAc/hexane, 3:8) to give 36 (35 mg, 83%). [a]D = +43.8° (c 0.7,
Trichloroacetonitrile (70.8
lL, 706
lmol) and DBU (3.2
l
L,
CHCl3); 1H NMR (500 MHz, CDCl3): d 7.95–7.34 (m, 10H, Ph),
5.72–7.68 (m, 2H, H-2b, H-4b), 5.43 (dd, 1H, J2,3 = 10.3 Hz,
J3,4 = 3.4 Hz, H-3b), 4.91 (d, 1H, J1,2 = 8.0 Hz, H-1b), 4.32 (d, 1H,
OH), 4.28 (d, 1H, J1,2 = 7.5 Hz, H-1a), 4.17 (dd, 1H, Jgem = 12.0 Hz,
J5,6a = 1.9 Hz, H-6aa), 4.02 (m, 1H, H-5b), 3.95–3.88 (m, 2H, H-6ba,
TMSCH2CH2), 3.75 (m, 1H, H-2a), 3.61–3.54 (m, 2H, H-3a, TMSCH2-
CH2), 3.49 (m, 1H, H-5a), 3.41 (t, 1H, J3,4 = J4,5 = 8.6 Hz, H-4a), 2.79
(dd, 1H, Jgem = 13.1 Hz, J5,6a = 8.3 Hz, H-6ab), 2.66 (dd, 1H,
J5,6b = 5.5 Hz, H-6bb), 2.57 (br s, 1H, OH), 2.17 (s, 3H, Ac), 1.69 (s,
3H, SeMe), 1.77 (s, 3H, Ac), 1.04-0.89 (m, 2H, TMSCH2CH2), ꢀ0.01
(s, 9H, TMS); 13C NMR (125 MHz, CDCl3): d 170.0 169.9, 165.4,
165.1, 133.5, 133.4, 129.7, 129.6, 129.0, 128.7, 128.6, 128.5,
128.4, 128.2, 101.7, 101.6, 80.9, 75.1, 73.5, 73.3, 71.9, 71.6, 69.4,
68.9, 67.4, 62.2, 24.7, 20.5, 20.4, 18.1, 5.6, ꢀ1.5; 77Se NMR
(94 MHz, CDCl3): d 61.1; HRMS: m/z calcd for C46H52O14SeSiNa+:
835.1876 [M+Na]+; found: 835.1876.
210 mol) were added to a solution of 32 (36 mg, 71
l
l
mol) in CH2-
Cl2 (1.4 mL) at 0 °C. The mixture was stirred for 70 min at 0 °C
(completion of the reaction was confirmed by TLC analysis;
MeOH/CHCl3, 1:15). The reaction mixture was concentrated and
the residue was purified by column chromatography on silica
gel (EtOAc/hexane, 1:2) to give 33 (46 mg, quant.,
a/b = 20/1).
a
-isomer; [
a]
D = +75.0° (c 0.2, CHCl3); 1H NMR (400 MHz, CDCl3):
d 8.62 (s, 1H, NH), 8.00–7.26 (m, 10H, Ph), 6.76 (d, 1H, J1,2 = 2.3 Hz,
H-1), 5.92–5.82 (m, H-2, H-3, H-4), 4.52 (t, 1H, J5,6a = J5,6b = 6.9 Hz,
H-5), 2.76 (dd, 1H, Jgem = 12.8 Hz, H-6a) 2.58 (dd, 1H, H-6b), 2.19 (s,
3H, Ac), 2.03 (s, 3H, SeMe); 13C NMR (100 MHz, CDCl3): d 170.0,
165.7, 165.5, 160.6, 133.5, 133.4, 129.8, 129.6, 129.0, 128.8,
128.5, 128.4, 93.8, 90.9, 72.5, 69.5, 68.7, 67.6, 24.0, 20.6, 5.3, 0.0;
77Se NMR (75 MHz, CDCl3): d 71.3; HRMS: m/z calcd for C25H24Cl3
O8Se Na+: 673.9631 [M+Na]+; found: 673.9630.
4.1.22. 2-(Trimethylsilyl)ethyl (4-O-acetyl-2,3-di-O-benzoyl-6-
4.1.24. 2-(Trimethylsilyl)ethyl (6-deoxy-6-methylseleno-b-
galactopyranosyl)-(1?4)-b- -glucopyranoside (4)
Sodium methoxide (28% in MeOH, 9 mg, 48 mol) was added to
a solution of 36 (39 mg, 48 mol) in THF (530 L) and MeOH
(1.1 mL). The reaction mixture was sonicated for 3.5 h (completion
of the reaction was confirmed by TLC analysis; CHCl3/MeOH/H2O,
5:4:1). Then the reaction mixture was neutralized with Dowex-
50 (H+) and filtered through cotton, and removed resin was washed
with MeOH. The combined filtrate and washings were concen-
trated. The residue was purified by column chromatography on
D-
deoxy-6-methylseleno-b-
acetyl-2,3-di-O-(4-methoxybenzyl)-b-
Molecular sieves (MS4Å, 206 mg) were added to a solution of 33
(48 mg, 74 mol) and 34 (61 mg, 109 mol) in CH2Cl2 (1.5 mL). The
suspension was stirred for 1 h at rt. Then TMSOTf (1.4
7.3
ture was stirred for 48 h at ꢀ40 °C, after which TMSOTf (1.4
7.3
D
-galactopyranosyl)-(1?4)-6-O-
D
D-glucopyranoside (35)
l
l
l
l
l
l
L,
l
mol) was added to the solution at ꢀ40 °C. The reaction mix-
l
L,
l
mol) was added to the solution at ꢀ40 °C. Stirring was contin-
ued for another 3 h at ꢀ40 °C (completion of the reaction was con-
firmed by TLC analysis; EtOAc/toluene, 1:4). The reaction mixture
Sephadex LH-20 (MeOH) to give 4 (24.8 mg, quant.). [
a
]
D = ꢀ6.7°
was quenched by addition of saturated aqueous NaHCO3 (500
l
L)
(c 0.5, MeOH); 1H NMR (500 MHz, CD3OD):
d
4.38 (d, 1H,
then filtered through Celite, and the removed molecular sieves
were washed with CHCl3. The combined filtrate and washings were
extracted with CHCl3, and the organic layer was washed with sat-
urated aqueous NaHCO3, dried (Na2SO4), and concentrated. The
residue was purified by column chromatography on Sephadex
LH-20 (MeOH/CHCl3, 1:1) and silica gel (EtOAc/toluene, 1:8) to give
J1,2 = 7.3 Hz, H-1b), 4.30 (d, 1H, J1,2 = 7.8 Hz, H-1a), 3.99 (m, 1H,
TMSCH2CH2), 3.95 (d, 1H, J3,4 = 2.6 Hz, H-4b), 3.91 (dd, 1H,
Jgem = 12.1 Hz, J5,6a = 5.6 Hz, H-6aa), 3.84 (dd, 1H, J5,6b = 4.2 Hz, H-
6ba), 3.70 (t, 1H, H-5b), 3.63 (m, 1H, TMSCH2CH2), 3.58–3.51 (m,
4H, H-3a, H-4a, H-2b, H-3b), 3.41 (m, 1H, H-5a), 3.23 (t, 1H, H-2a),
2.83–2.76 (m, 2H, H-6ab, H-6bb), 2.04 (s, 1H, SeMe), 1.09–0.94
(m, 2H, TMSCH2), 0.0 (s, 9H, TMS); 13C NMR (125 MHz, CD3OD): d
105.1, 103.7, 80.9, 76.5, 76.3, 76.1, 74.9, 72.2, 70.8, 68.2, 62.0,
25.5, 19.1, 4.5, ꢀ1.4; 77Se NMR (94 MHz, CDCl3): d 51.8; HRMS:
35 (55 mg, 71%). [a]
D = +40.3° (c 0.2, CHCl3); 1H NMR (600 MHz,
CDCl3): d 7.98–6.80 (m, 18H, Ar), 5.71 (d, 1H, J3,4 = 3.4 Hz, H-4b),
5.65 (d, 1H, J1,2 = 8.2 Hz, J2,3 = 10.3 Hz, H-2b), 5.35 (dd, 1H, H-3b),
4.94 (d, 1H, Jgem = 10.3 Hz, ArCH2), 4.91 (d, 1H, H-1b), 4.83 (d, 1H,
ArCH2), 4.81 (d, 1H, ArCH2), 4.62 (d, 1H, ArCH2), 4.32–4.30 (m,
2H, H-1a, H-6aa), 4.07 (dd, 1H, J5,6b = 4.8 Hz, H-6ba), 3.89 (m, 1H,
TMSCH2CH2), 3.84–3.74 (m, 8H, H-4a, H-5b, ArOCH3), 3.61–3.53
(m, 2H, H-3a, TMSCH2CH2), 3.39 (m, 1H, H-5a), 3.45 (t, 1H, H-2a),
2.58 (dd, 1H, Jgem = 13.1 Hz, J5,6a = 6.2 Hz, H-6ab), 2.49 (dd, 1H,
J5,6b = 7.6 Hz, H-6bb), 2.12 (s, 3H, Ac), 1.90 (s, 3H, Ac), 1.87 (s, 3H,
SeMe), 0.99 (t, 2H, TMSCH2), 0.00 (s, 9H, TMS); 13C NMR
(150 MHz, CDCl3): d 170.4, 169.9, 165.4, 165.1, 159.1, 158.9,
133.4, 133.3, 131.2, 130.5, 129.8, 129.7, 129.6, 128.9, 128.9,
m/z calcd for
C
18H36O10SeSiNa+: 543.1135 [M+Na]+; found:
543.1135.
4.1.25. 4-O-Acetyl-2,3-di-O-benzoyl-6-deoxy-6-methylseleno-D-
galactopyranosyl fluoride (37)
N,N-diethylaminosulfur trifluoride (39.6
lL, 300 lmol) was
added to a solution of 32 (101 mg, 199 mol) in CH2Cl2 (2.0 mL)
l
at 0 °C, and the mixture was stirred for 30 min at 0 °C (completion
of the reaction was confirmed by TLC analysis; EtOAc/hexane, 1:2),
then the reaction mixture was diluted with CHCl3, and the solution

2100
T. Suzuki et al. / Bioorg. Med. Chem. 22 (2014) 2090–2101
was washed with brine, dried (Na2SO4), and concentrated. The res-
idue was purified by column chromatography on silica gel (EtOAc/
4.3. Data collection and structure determination of co-cystal
with seleno-lactose (3)
toluene, 1:2) to give 37 (99 mg, 97%,
a/b = 1/1.7). a-isomer;
[a
]
D = +113.5° (c 0.8, CHCl3); 1H NMR (400 MHz, CDCl3): d 8.01–
The crystals were soaked in the cryo protectant buffer (10% w/v
PEG6000, 100 mM Tris–HCl pH 7.5 and 20% glycerol) in a minute
and flash-cooled in liquid nitrogen before data collection. The dif-
fraction data of crystals are collected at BL-17A beamline on Pho-
ton Factory of KEK (Tsukuba, Japan). The best data was collected
at 0.97889 Å for SAD phasing method. The data was processed by
XDS package.25 The scaling was performed with pointless and aim-
less in CCP4 suite.26 The maximum resolution is determined by a
correlation factor CC (1/2).27 The structure of human galectin-9
NCRD was solved using Autosol in phenix software.28 After Auto-
sol, 562 residues were built and R and Rfree was 0.20 and 0.21,
respectively. Further refinement was performed using Coot,29
manually. Finally, deposited structure was refined using Refmac30
with TLS option and no NCS restrains. Seleno-lactose 3 coordinate
and cif files were made using Jligand and phenix.elbow.31 The
geometry of refined structure was validated with MolProbity.32
The statistics of data collection and refinements were described
in the Supplementary Material.
7.36 (m, 10H, Ph), 5.98 (dd, 1H, J1,2 = 2.8 Hz, J1,F = 53.2 Hz, H-1),
5.86–5.81 (m, 2H, H-3, H-4), 5.63 (m, 1H, J2,F = 23.8 Hz, H-2), 4.47
(t, 1H, J5,6a = J5,6b = 7.7 Hz, H-5), 2.77 (dd, 1H, Jgem = 12.8 Hz, H-6a)
2.61 (dd, 1H, H-6b), 2.18 (s, 3H, Ac), 2.08 (s, 3H, SeMe); 13C NMR
(100 MHz, CDCl3): d 169.8, 165.9, 165.4, 133.4, 129.9, 129.6,
129.0, 128.7, 128.5, 128.4, 105.8, 103.5, 71.9, 69.2, 68.3, 68.1,
68.0, 23.8, 20.6, 5.4; 77Se NMR (75 MHz, CDCl3): d 64.8; HRMS:
m/z calcd for
C
23H23FO8SeNa+: 533.0485 [M+Na]+; found:
D = +74.4° (c 0.8, CHCl3); 1H NMR
533.0482; b-isomer;
[a]
(400 MHz, CDCl3): d 7.99–7.35 (m, 10 H Ph), 5.81–5.73 (m, 2H,
H-2, H-4), 5.60–5.45 (m, 2H, J1,F = 52.7 Hz, H-1, H-3), 4.10 (t, 1H,
J5,6a = J5,6b = 6.9 Hz, H-5), 2.87 (dd, 1H, Jgem = 13.3 Hz, H-6a), 2.66
(dd, 1H, H-6b), 2.17 (s, 3H, Ac), 2.09 (s, 3H, SeMe); 13C NMR
(100 MHz, CDCl3): d 169.9, 165.4, 165.1, 133.5, 129.8, 129.6,
128.8, 128.6, 128.5, 128.4, 108.2, 106.1, 74.9, 71.0, 70.9, 69.6,
69.3, 68.3, 23.9, 20.6, 5.7; 77Se NMR (75 MHz, CDCl3): d 71.3;
HRMS: m/z calcd for C23H23FO8SeNa+: 533.0485 [M+Na] +; found:
533.0485.
4.4. Structure analysis of human galectin-9 with seleno-lactose
(3)
4.1.26. Methyl (4-O-acetyl-2,3-di-O-benzoyl-6-deoxy-6-
methylseleno-b-
-glucopyranoside (39)
Molecular sieves (MS4Å, 106 mg) were added to a solution of 37
(52 mg, 102 mol) and 38 (47 mg, 102 mol) in CH2Cl2 (2 mL). The
suspension was stirred for 1 h at rt. Then Cp2ZrCl2 (74 mg,
254 mol) and AgOTf (131 mg, 510 mol) were added to the mix-
D-galactopyranosyl)-(1?6)-2,3,4-tri-O-benzyl-
RMSD value was calculated by SUPERPOSE in CCP4 suite.26
Hydrogen bonds between human galectin-9 NCRD and seleno-
lactose 3 were determined using Ligplot+33 and CONTACT programs.26
All figures of crystal structures were prepared using Pymol.34
a-D
l
l
l
l
Acknowledgements
ture at 0 °C. Stirring was continued for 24 h at rt (completion of the
reaction was confirmed by TLC analysis; EtOAc/hexane, 2:3). The
reaction mixture was filtered through Celite and the removed
molecular sieves were washed with CHCl3. The combined filtrate
and washings were washed with brine, dried (Na2SO4), and con-
centrated. The residue was purified by column chromatography
on Sephadex LH-20 (MeOH/CHCl3, 1:1) to give 39 (52 mg, 53%).
The iCeMS is supported by World Premier International Re-
search Center Initiative (WPI), MEXT, Japan. This work was finan-
cially supported in part by MEXT of Japan (a Grant-in-Aid for
Scientific Research (B) No. 22380067 to M.K., and a Grant-in-Aid
for Young Scientists (A) No. 23688014 and Grant-in-Aid on Innova-
tive Areas No. 24110505, Deciphering sugar chain-based signals
regulating integrative neuronal functions to H.A.). We thank Ms.
Kiyoko Ito (Gifu Univ.) for providing technical assistance.
[a]
D = +44.6° (c 0.9, CHCl3); 1H NMR (500 MHz, CDCl3): d 7.88–
7.06 (m, 25H, Ph), 5.72 (dd, 1H, J1,2 = 8.0 Hz, J2,3 = 10.3 Hz, H-2b),
5.66 (d, 1H, J3,4 = 2.9 Hz, H-4b), 5.41 (dd, 1H, H-3b), 4.88 (d, 2H,
Jgem = 10.9 Hz, PhCH2), 4.74–4.67 (m 3H, H-1b, PhCH2), 4.58 (d,
1H, PhCH2), 4.47 (d, 1H, J1,2 = 3.4 Hz, H-1a), 4.32 (d, 1H, PhCH2),
4.19 (m, 1H, H-6aa), 3.92–3.87 (m, 2H, H-3a, H-5b), 3.75–3.71 (m,
2H, H-5a, H-6ba), 3.43–3.36 (m, 2H, H-2a, H-4a), 3.20 (s, 3H,
OMe), 2.81 (dd, 1H, Jgem = 13.2 Hz, J5,6a = 8.0 Hz, H-6ab), 2.58 (dd,
1H, J5,6b = 5.7 Hz, H-6bb), 2.16 (s, 3H, Ac), 2.06 (s, 3H, SeMe); 13C
NMR (125 MHz, CDCl3): d 170.1, 165.6, 165.1, 138.8, 133.3, 133.1,
128.4, 128.3, 128.4, 128.1, 127.9, 127.6, 127.5, 101,6, 97.9, 81.9,
79.8, 77.4, 75.5, 74.7, 74.7, 73.3, 71.9, 69.4, 69.3, 68.5, 54.9, 24.5,
20.6, 5.7; 77Se NMR (113 MHz, CDCl3): d 72.6; HRMS: m/z calcd
for C51H54O13SeNa+: 977.2622, [M+Na]+; found: 977.2627.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.023.
References and notes
1. Wrede, F. Z. Physiol. Chem. 1921, 115, 284.
2. Recent reports on the use of seleno-carbohydrates as synthetic intermediates;
(a) Yamago, S.; Kokubo, K.; Murakami, H.; Mino, Y.; Hara, O.; Yoshida, J.-I.
Tetrahedron Lett. 1998, 39, 7905; (b) Jiaang, W.-T.; Chang, M.-Y.; Tseng, P.-H.;
Chen, S.-T. Tetrahedron Lett. 2000, 41, 3127; (c) Yamago, S.; Yamada, T.; Hara,
O.; Ito, H.; Mino, Y.; Yoshida, J.-I. Org. Lett. 2001, 3, 3867; (d) Ikeda, K.;
Sugiyama, Y.; Tanaka, K.; Sato, M. Bioorg. Med. Chem. Lett. 2002, 12, 2309; (e)
Gamblin, D. P.; Garnier, P.; van Kasteren, S.; Oldham, N. J.; Fairbanks, A. J.;
Davis, B. G. Angew. Chem., Int. Ed. 2004, 43, 828; (f) Mukherjee, C.; Tiwari, P.;
Misra, A. K. Tetrahedron Lett. 2006, 47, 441; (g) Tiwari, P.; Misra, A. K.
Tetrahedron Lett. 2006, 47, 2345; (h) Valerio, S.; Iadonisi, A.; Adinolfi, A.; Ravidà,
A. J. Org. Chem. 2007, 72, 6097; (i) Di Bussolo, V.; Fiasella, A.; Balzano, F.;
Barretta, G. U.; Crotti, P. J. Org. Chem. 2010, 75, 4284; (j) Kumar, A. A.; Illyes, T.
Z.; Kover, K. E.; Szilagyi, L. Carbohydr. Res. 2012, 360, 8; Selenocarbohydrates as
mimetics of carbohydrates; (k) Czernecki, S.; Randriamandimby, D. J.
Carbohydr. Chem. 1996, 15, 183; (l) Sivapriya, K.; Suguna, P.; Chandrasekaran,
S. Tetrahedron Lett. 2007, 48, 2091; (m) Abdo, M.; Knapp, S. J. Am. Chem. Soc.
2008, 130, 9234; (n) Kim, E. J.; Love, D. C.; Darout, E.; Abdo, M.; Rempel, B.;
Withers, S. G.; Rablen, P. R.; Hanover, J. A.; Knapp, S. Bioorg. Med. Chem. 2010,
18, 7058; (o) Fournière, V.; Cumpstey, I. Tetrahedron Lett. 2010, 51, 2127; (p)
Cumpstey, I.; Ramstadius, C.; Akhtar, T.; Goldstein, I. J.; Winter, H. C. Eur. J. Org.
Chem. 2010, 1951; (q) Braga, H. C.; Stefani, H. A.; Paixão, M. W.; Santos, F. W.;
Lüdtke, D. S. Tetrahedron 2010, 66, 3441; (r) Affeldt, R. F.; Braga, H. C.;
Baldassari, L. L.; Lüdtke, D. S. Tetrahedron 2012, 68, 10470.
4.2. Protein expression, purification and crystallization
The human galectin-9 NCRD was expressed as a glutathione S-
transferase (GST) fusion protein and purified as described.23 5 mg/
ml of human galectin-9 NCRD was mixed with seleno-lactose 1 to 4
(10 mM) before crystallization. The crystallization was manually
performed by using grid screening based on the crystallization
condition of human or mouse galectin-9 NCRD with lactose (pdb_i-
d:2EAK and 2D6M).23 Finally, the crystals of human galectin-9
NCRD with seleno-lactoses 1, 2 and 3 were obtained under the
optimized condition (1. 20% w/v PEG 3350 and 8% v/v Tacsimate
pH 8.0; 2. 20% w/v PEG 3350, 0.2 M Sodium Iodide and 100 mM
Tris-HCl pH 8.0; 3. 10% w/v PEG 6000 and 100 mM Tris–HCl pH
7.5, respectively) at 289 K for 2 or 3 days.

T. Suzuki et al. / Bioorg. Med. Chem. 22 (2014) 2090–2101
2101
3. Boutureira, O.; Bernardes, G. J. L.; Fernández-González, M.; Anthony, D. C.;
Davis, B. G. Angew. Chem., Int. Ed. 2012, 51, 1432.
16. Zhang, Z.; Magnusson, G. J. Org. Chem. 1996, 61, 2394.
17. See the Supplementary Material for the experimental details of the synthesis of
glucosyl acceptor 34.
18. Suzuki, K.; Maeta, H.; Suzuki, T.; Matsumoto, T. Tetrahedron Lett. 1989, 30,
6879.
19. Experimental details for the synthesis of compounds 41 and 42 and their
glycosidation reactions are reported in the Supplementary Material.
20. Boons, G. J. P. H.; van Delft, F. L.; van der Klein, P. A. M.; van der Marel, G. A.; van
Boom, J. H. Tetrahedron 1992, 48, 885.
21. (a) Kurose, N.; Takahashi, T.; Koizumi, T. J. Org. Chem. 1996, 61, 2932; (b)
Mukherjee, A. J.; Zade, S. S.; Singh, H. B.; Sunoj, R. B. Chem. Rev. 2010, 110, 4357.
22. Hirabayashi, J.; Kasai, K. Glycobiology 1993, 3, 297.
23. (a) Nagae, M.; Nishi, N.; Murata, T.; Usui, T.; Nakamura, T.; Wakatsuki, S.; Kato,
R. J. Biol. Chem. 2006, 281, 35884; (b) Nagae, M.; Nishi, N.; Nakamura-Tsuruta,
S.; Hirabayashi, J.; Wakatsuki, S.; Kato, R. J. Mol. Biol. 2008, 375, 119.
24. Deng, S.; Gangadharmath, U.; Chang, C.-W. T. J. Org. Chem. 2006, 71, 5179.
25. Kabsch, W. Acta. Cryst. 2010, D66, 125.
26. Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.; Emsley, P.; Evans, P. R.;
Keegan, R. M.; Krissinel, E. B.; Leslie, A. G.; McCoy, A.; McNicholas, S. J.;
Murshudov, G. N.; Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin,
A.; Wilson, K. S. Acta. Crystallogr. 2011, D67, 235.
4. (a) Mehta, S.; Andrews, J. S.; Johnson, B. D.; Svensson, B.; Pinto, B. M. J. Am.
Chem. Soc. 1995, 117, 9783; (b) Szczepina, M. G.; Johnston, B. D.; Yuan, Y.;
Svensson, B.; Pinto, B. M. J. Am. Chem. Soc. 2004, 126, 12458; (c) Liu, H.; Pinto, B.
M. J. Org. Chem. 2005, 70, 753; (d) Liu, H.; Nasi, R.; Jayakanthan, K.; Sim, L.;
Heipel, H.; Rose, D. R.; Pinto, B. M. J. Org. Chem. 2007, 72, 6562.
5. (a) Gettins, P.; Wardlaw, S. A. J. Biol. Chem. 1991, 266, 3422; (b) House, K. L.;
Dunlap, R. B.; Odom, J. D.; Wu, Z. P.; Hilvert, D. J. Am. Chem. Soc. 1992, 114,
8573; (c) Mobli, M.; Morgenstern, D.; King, G. F.; Alewood, P. F.; Muttenthaler,
M. Angew. Chem., Int. Ed. 2011, 50, 11952; (d) Schaefer, S. A.; Dong, M.;
Rubenstein, R. P.; Wilkie, W. A.; Bahnson, B. J.; Thorpe, C.; Rozovsky, S. J. Mol.
Biol. 2013, 425, 222.
6. (a) Taylor, G. Acta. Crystallogr. 2003, D59, 1881; (b) Hendrickson, W. A.; Teeter,
M. M. Nature 1981, 290, 107; (c) Hendrickson, W. A.; Pahler, A.; Smith, J. L.;
Satow, Y.; Merritt, E. A.; Phizackerley, R. P. Proc. Natl. Acad. Sci. U.S.A. 1989, 86,
2190; (d) Yang, W.; Hendrickson, W. A.; Crouch, R. J.; Satow, Y. Science 1990,
249, 1398; (e) Du, Q.; Carrasco, N.; Teplova, M.; Wilds, C. J.; Egli, M.; Huang, Z. J.
Am. Chem. Soc. 2002, 124, 24.
7. (a) Buts, L.; Loris, R.; De Genst, E.; Oscarson, S.; Lahmann, M.; Messens, J.;
Brosens, E.; Wyns, L.; De Greve, H.; Bouckaert, J. Acta. Crystallogr. 2003, D59,
1012; (b) Kostlánová, N.; Mitchell, E. P.; Lortat-Jacob, H.; Oscarson, S.;
Lahmann, M.; Gilboa-Garber, N.; Chambat, G.; Wimmerová, M.; Imberty, A. J.
Biol. Chem. 2005, 280, 27839.
8. (a) Fotinou, C.; Emsley, P.; Black, I.; Ando, H.; Ishida, H.; Kiso, M.; Sinha, K. A.;
Fairweather, N. F.; Isaacs, N. W. J. Biol. Chem. 2001, 276, 32274; (b) Attrill, H.;
Imamura, A.; Sharma, R. S.; Kiso, M.; Crocker, P. R.; van Aalten, D. M. F. J. Biol.
Chem. 2006, 281, 32774; (c) Stenmark, P.; Dupuy, J.; Imamura, A.; Kiso, M.;
Stevens, R. C. PLoS Pathog. 2008, 4, e1000129; (d) Neu, U.; Hengel, H.; Blaum, B.
S.; Schowalter, R. M.; Macejak, D.; Gilbert, M.; Wakarchuk, W. W.; Imamura, A.;
Ando, H.; Kiso, M.; Arnberg, N.; Garcea, R. L.; Peters, T.; Buck, C. B.; Stehle, T.
PLoS Pathog. 2012, 8, e1002738.
27. Karplus, P. A.; Diederichs, K. Science 2012, 336, 1030.
28. Adams, P. D.; Afonine, P. V.; Bunkóczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.;
Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.;
Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.;
Terwilliger, T. C.; Zwart, P. H. Acta. Crystallogr. 2010, D66, 213.
29. Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Acta. Crystallogr. 2010, D66,
486.
30. Murshudov, G. N.; Skubák, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.;
Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A. Acta. Crystallogr. 2011, D67,
355.
31. (a) Lebedev, A. A.; Young, P.; Isupov, M. N.; Moroz, O. V.; Vagin, A. A.;
9. (a) Kawai, Y.; Ando, H.; Ozeki, H.; Koketsu, M.; Ishihara, H. Org. Lett. 2005, 5,
4653; (b) Nanami, M.; Ando, H.; Kawai, Y.; Koketsu, M.; Ishihara, H. Tetrahedron
Lett. 2007, 48, 1113.
Murshudov, G. N. Acta Crystallogr. D Biol. Crystallogr. 2012, 68, 431; (b)
Moriarty, N. W.; Grosse-Kunstleve, R. W.; Adams, P. D. Acta. Crystalloge. 2009,
D65, 1074.
10. Ishihara, H.; Sato, S.; Hirabayashi, Y. Bull. Chem. Soc. Jpn. 1977, 50, 3007.
11. Surabhi, P.; Wu, X.; Hu, L. Tetrahedron Lett. 2006, 47, 4609.
12. Lichtenthaler, F. W.; Kaji, E.; Weprek, S. J. Org. Chem. 1985, 50, 3505.
13. Jansson, K.; Ahlfors, S.; Frejd, T.; Kihlberg, J.; Magnusson, G.; Dahmen, J.; Noori,
G.; Stenvall, K. J. Org. Chem. 1988, 53, 5629.
32. Chen, V. B.; Arendall, W. B., 3rd; Headd, J. J.; Keedy, D. A.; Immormino, R. M.;
Kapral, G. J.; Murray, L. W.; Richardson, J. S.; Richardson, D. C. Acta. Crystallogr.
2010, D66, 12.
33. Laskowski, R. A.; Swindells, M. B. J. Chem. Inf. Model. 2011, 51, 2778.
34. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.
14. Rio, S.; Beau, J. M.; Jacquinet, J. C. Carbohydr. Res. 1991, 219, 71.
15. (a) Schmidt, R. R.; Michel, J. Angew. Chem., Int. Ed. Engl. 1980, 19, 731; (b)
Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25, 212.