Synthesis of seleno-fucose compounds and their application to the X-ray structural determination of carbohydrate-lectin complexes using single/multi-wavelength anomalous dispersion phasing

Article abstract of DOI:10.1016/j.bmc.2016.12.021

Selenium-incorporated fucoses (seleno-fucoses) differing in the position of the seleno-substituent were synthesized and applied to the X-ray structural determination of a carbohydrate-lectin complex using single/multi-wavelength anomalous dispersion (SAD/

Full text of DOI:10.1016/j.bmc.2016.12.021

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of seleno-fucose compounds and their application to the X-ray
structural determination of carbohydrate-lectin complexes using
single/multi-wavelength anomalous dispersion phasing
a,c,1
d,1
a,c
d
d
Junpei Shimabukuro
, Hisayoshi Makyio , Tatsuya Suzuki , Yosuke Nishikawa , Masato Kawasaki ,
a
a,b
a,b,c,
⇑
d,
⇑
a,c
Akihiro Imamura , Hideharu Ishida , Hiromune Ando
, Ryuichi Kato , Makoto Kiso
a
Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan
Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki
05-0801, Japan
b
c
d
3
a r t i c l e i n f o
a b s t r a c t
Article history:
Selenium-incorporated fucoses (seleno-fucoses) differing in the position of the seleno-substituent were
synthesized and applied to the X-ray structural determination of a carbohydrate-lectin complex using
single/multi-wavelength anomalous dispersion (SAD/MAD) phasing. The hydroxyl groups at the C-1,
Received 10 November 2016
Revised 15 December 2016
Accepted 16 December 2016
Available online xxxx
-
2, -3 and -4 position of fucose were individually substituted with a methylseleno group via a transacetal-
À
ization reaction using MeSeCH
2
OBn or by an S
N
2 reaction with TolSe equivalents to afford the corre-
sponding MeSe-fucose. The three-dimensional structures of a fucose-binding lectin complexed with
several of these MeSe-fucoses have been determined by SAD/MAD phasing by utilizing the diffraction
of selenium in the bound MeSe-fucoses.
Keywords:
Seleno-carbohydrate
Carbohydrate-protein complex
X-ray crystallography
SAD/MAD phasing
Lectin
Ó 2016 Elsevier Ltd. All rights reserved.
1
. Introduction
Glycans are present mainly on the cell surface as complexes
of MAD since the 1990s has led to a considerable increase in the
number of solved protein structures. This approach typically
requires the modification of native methionines in proteins with
selenium-labeled methionines. The production of the seleno-
methionine substituted proteins is carried out in recombinant host
cells. However, the toxicity of selenium often impeded not only the
protein production but also cell growth, and sometimes it remains
a challenge to obtain sufficient quantity of selenium-labeled pro-
tein for X-ray crystallography.
covalently linked with proteins or lipids and are involved in biolog-
ical events such as antigen-antibody reactions, the targeting of
1
2
3
toxins to cells, and pathogen-host interactions. In the initial stage
of these cellular recognition events, most glycans serve as the
specific counterparts for membrane proteins to form glycan-pro-
tein complexes. Therefore, it is important to obtain structural
information regarding these complexes in order to understand
the molecular basis underlying the biological functions of glycans.
X-ray crystallography is the most powerful method for the
three-dimensional structural analysis of proteins but its applica-
tion is hampered by the need to determine the phase of each
reflection. The multi-wavelength anomalous dispersion (MAD)
These issues highlight the need to use non-labeled protein.
5
–8
9,10
Recently, several research groups,
including our group,
reported that selenium-containing carbohydrates (seleno-sugars)
serve as mimics of ligands in carbohydrate-protein complexes
and that the three-dimensional structures of native proteins com-
plexed with seleno-sugars were determined using single-wave-
length anomalous dispersion (SAD) and/or MAD phasing
methods. However, our previous study also revealed that the sub-
stitution of a hydroxyl group with a MeSe group in a sugar residue
4
phasing method uses the unique dispersion of specific atoms in
response to X-ray irradiation as phasing references. The application
significantly decreased the affinity to the sugar-binding protein,
⇑
Corresponding authors at: Department of Applied Bioorganic Chemistry, Gifu
University, 1-1 Yanagido, Gifu 501-1193, Japan (H. Ando).
depending on the position of the MeSe substitution.9 This
result suggests that the application of seleno-sugars to the X-ray
E-mail address: hando@gifu-u.ac.jp (H. Ando).
These authors contributed equally to this study.
1
http://dx.doi.org/10.1016/j.bmc.2016.12.021
0
968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
J. Shimabukuro et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
structure determination of unknown sugar-binding proteins will
be problematic.
SeMe
OH
O
O
SeMe
OH
We envisioned that a set of seleno-sugars consisting of possible
regioisomers with respect to the MeSe substitution could be used
to compensate for the unpredictable loss of affinity to proteins.
OH
HO
OH
HO
1
α
1β
OMe
OMe
OMe
OH
1
1
O
O
O
The first synthesis of seleno-trehalose was reported in 1921,
SeMe
OH
OH
HO
SeMe
HO
OH
MeSe
and since then many seleno-sugars have been synthesized, includ-
ing seleno-glycosides and C2- or C6-seleno-substituted deriva-
2
3
4
1
2,13
tives.
However, there are few reports regarding the
Fig. 1. Molecular structure of seleno-fucosides
substitution of a hydroxyl group within a pyranose such as glucose
or galactose with selenium, although substitutions with sulfur
have been reported by many research groups.14 In the present
study, we explored the synthesis of seleno-fucosides 1–4 in which
each hydroxyl group is substituted with a MeSe group (Fig. 1).
Moreover, to demonstrate the utility of the prepared compounds
in X-ray crystallography, we attempted to determine the three-
dimensional structure of a fucose-binding lectin, Aspergillus oryzae
SeMe
O
OR1
OR2
2
R O
1
R2
R
MeSe
O
NPh
CF3
a
7α PMB Ac
6
9
0%
8α
1α
H
H
Ac
H
O
TMSOTf
b
9%
1
5
lectin (AOL).
8
O
OPMB
TBME-CH2Cl2
(2:1)
MS AW-300
OAc
AcO
SeMe
5
O
1
OR
-
78 °C
7%
α:β = 66/21)
OR2
2
. Results and discussion
2
8
R O
R1
R2
(
a
7β PMB Ac
2.1. Synthesis of 1-MeSe-Fuc
8
6%
b
8
1
β
β
H
H
Ac
H
We chose our recently developed trans-acetalization reaction
quant.
between selenoacetal and glycosyl imidate16 to incorporate sele-
Scheme 1. Synthesis of
CH Cl
, À40 °C, 90%; for b-isomer: TFA, anisole, CH
MeOH, RT, 89% (1 ), quant. (1b). PMB = p-methoxybenzyl, TMSOTf = trimethylsilyl
a
and b-MeSe-Fuc 1
a
and 1b. (a) For
a
-isomer: TFA, anisole,
nium at the anomeric position of fucose simultaneously as
and b-selenoglycosides (Scheme 1). In this reaction, the selenium
atom attacks both the - and b-face of the anomeric carbon of
the oxocarbenium ion generated from a glycosyl imidate to afford
a-
2
2
2
Cl
2
, À20 °C, 86%; (b) NaOMe,
a
a
trifluoromethanesulfonate, TBME = tert-butylmethyl ether, MS = molecular sieves,
TFA = trifluoroacetic acid
1
7
a
- and b-selenoglycosides. Known fucosyl imidate 5 (Scheme 1)
16
was reacted with BOMSeMe 6, at À78 °C by the catalytic action
OMe
OMe
of TMSOTf in the presence of acid-washed molecular sieves (AW-
OMe
OH
ref. 19
O
3
00) in CH
methylseleno-fucosides 7
b = 66:21). In this reaction, the reported synergic solvent effect
of TBME and CH Cl was utilized to increase the production of
isomer. Next, 7
TFA) in the presence of anisole, followed by basic methanolysis
of the acetyl groups to give - and b-methylseleno-fucosides 1
2
Cl
2
-tert-butylmethyl ether (TBME) to afford 1-
O
d
O
O
a~c
81%
(3 steps)
a
and 7b in excellent yield (87%,
a
:
OO OH
O
OTf
11
OH
HO
1
8
9
10
2
2
a
-
O
O
a
and 7b were treated with trifluoroacetic acid
Se
OMe
(
OMe
1
2
O
SeR1
f
a
a
O
OH
SeMe
O
piperidine, DIEA
DMA
O
83%
and 1b, respectively, in good yield. The anomeric configurations
HO
1
of the target compounds were confirmed from their H NMR spec-
90 °C
2
13 R1 = Tol
R1 = Me
9
3% (2 steps)
e
tra. The J1,2 coupling constants of 1
indicating that 1 and 1b were - and b-glycosides, respectively.
The Se NMR spectra of 1 and 1b showed selenium signals at
5.4 ppm and 190.4 ppm, respectively.
a and 1b were 5.5 Hz and 9.5 Hz,
6
4%
1
4
a
a
7
7
a
Scheme 2. Synthesis of 2-MeSe-Fuc 2. (a) DMP, CSA, MeCN, RT; (b) Dess-Martin
periodinane, CH Cl , RT; (c) diisobutylaluminium hydride, CH Cl , À78 °C, 81% (3
7
2
2
2
2
steps); (d) Tf
2
2
O, py, CH Cl
2
, À20 °C ? RT; (e) MeI, MeNHNH
2
, Cs
2
3
CO , DMF, RT, 64%;
(
f) AcOH aq. 50 °C, 83%. DMP = 2,2-dimethoxypropane, CSA = (±)-camphorsulfonic
acid, DIEA = N,N-diisopropylethylamine, Tol = p-methylbenzoyl.
2.2. Synthesis of 2-MeSe-Fuc
The synthesis of 2-MeSe-Fuc 2 started with transforming Me-
a
-
2.3. Synthesis of 3-MeSe-Fuc
fucopyranoside 9 (Scheme 2). The taloside-type precursor 10 was
prepared by following reported methods.19 Next, triflation of the
C2-hydroxyl group of 10 was followed by a substitution reaction
Following the synthesis of 2-MeSe-Fuc 2, the synthesis of 3-
MeSe-Fuc 3 adopted the triflate of gulose 18 as an electrophile
for nucleophilic substitution with the TolSe group (Scheme 3). To
establish the configuration of the modified gulose, methyl fucoside
9 was transformed into C3-hydroxy derivative 15 through orthoe-
ster formation with the C3- and C4-hydroxyl groups, followed by
methoxymethylation and acidic treatment. Then, oxidation of the
C3-hydroxyl group of 15 with Dess-Martin periodinane and reduc-
9
,20
with Tol
2
Se
12 in the presence of piperidine and DIEA in
DMA, giving selenium-containing compound 13 in 93% yield over
two steps. Then, the TolSe group in 13 was converted into a
methylselenyl (MeSe) group via in situ reaction of the selenolate
2
1
2
anion with MeI, initiated by the action of piperidine and Cs CO3,
to afford compound 14 in 64% yield. Finally, acid hydrolysis of the
acetonide in 14 led to 2-MeSe-fucoside 2 in 83% yield. The equato-
rial orientation of the MeSe group was confirmed by the coupling
constant between H-2 and H-3 (J2,3 = 11.0 Hz) of compound 14.
4
tion with NaBH gave gulose derivative 17. Next, 17 was converted
into triflate 18, which was subsequently reacted with potassium p-
methylselenobenzoate 19. However, the reaction did not produce
the desired compound 20 and instead provided the olefinated
(2,3-ene) compound as the major byproduct.
7
7
The Se NMR spectrum of 14 showed a selenium signal at
02.8 ppm.
1
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J. Shimabukuro et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
3
OMe
OMe
The orientation of the TolSe group was determined from the cou-
a~c
2%
d
9
O
OMOM
O
pling constants J3,4 (4.5 Hz) and J4,5 (1.0 Hz), which indicated that
8
73%
OMOM
77
H-4 adopted an equatorial orientation. The Se NMR spectrum
OH
AcO
AcO
(
3 steps) AcO
O
of 37 showed a selenium signal at 465.9 ppm. Exchange of the pro-
tecting group with selenium was performed as described above,
yielding 38 as protected 4-MeSe-Fuc 4.
Finally, deprotection of 38 under Zemplén condition afforded 4
in 96% yield.
1
5
OMe
16
OMe
OH
O
OTf
O
e
f
OMOM
OMOM
8
2%
O
AcO
1
7
18
SeK
OMe
2.5. Co-crystallization of MeSe-Fuc derivatives with AOL and structural
analysis of the MeSe-Fuc-AOL complex by MAD/SAD phasing
1
9
O
OMOM
DMF
RT
SeTol
AcO
Each MeSe-fucose was co-crystallized with a fucose-specific
2
0
1
5
lectin, AOL. Very recently, we reported the structure of AOL co-
crystallized with 1 -MeSe-Fuc 1 or 1b-MeSe-Fuc 1b solved by
Scheme 3. Unsuccessful route to 3-MeSe-Fuc 3. (a) Triethyl orthoformate, CSA,
MeCN, RT; (b) MOMCl, DIEA, MeCN, RT; (c) then 2 M HCl aq. 82% (3 steps); (d) Dess-
a
a
MAD or SAD phasing using the selenium atoms in the carbohydrate
Martin periodinane, NaHCO
3
, CH
2
Cl
2
, RT, 73%; (e) NaBH
, À20 °C. MOM = methoxymethyl.
4
, MeOH, 0 °C, 82%; (f) Tf
2
O,
10
as anomalously scattering sites. Here, we used the SAD method
py, CH Cl
2
2
to determine the structure of AOL co-crystallized with 2-MeSe-
Fuc 2 (Fig. 2a). There are 12 AOL molecules in an asymmetric unit
and the root mean square deviation (RMSD) among the 12 mole-
cules is between 0.147 Å and 0.332 Å. The anomalous difference
Fourier map against the final refined model shows the anomalous
signal from 2-MeSe-Fuc 2 in all six fucose binding sites (Fig. 2b).
The interaction modes between 2-MeSe-Fuc 2 and AOL at the six
binding sites are almost the same. There is a hydrogen bond
between the ring oxygen of 2-MeSe-Fuc 2 and an arginine residue
of AOL. In addition, the C3- and C4-hydroxyls form hydrogen bonds
with the tryptophan and arginine/glutamine/glutamate residues of
AOL, respectively, and these interactions are the same as those in
We speculated that this result was attributable to dipolar repul-
sions (a) between the nucleophile and electron-withdrawing func-
tional group at C4, and (b) between the nucleophile and ring
2
2
oxygen at the transition state (Richardson-Hough rules). Based
on this speculation, we designed a new intermediate that was pro-
tected with an electron-donating group at the C4-hydroxyl group.
Furthermore, we assumed that ring formation at the C1 and C2
positions would suppress E2 elimination due to the ring strain of
the bicyclic structure. To embody this redesign, we chose MOM
as the C4-hydroxyl protecting group and acetonide as a ring moiety
fused at C1 and C2. The C3- and C4-hydroxyl groups were pro-
tected in advance of acetonide formation at C1 and C2 because
regioselective acetonide formation at C1 and C2 over C3 and C4
1
0
the 1-MeSe-Fuc complex.
We also obtained separate crystals of AOL in the presence of the
MeSe-fucoses 3-MeSe-Fuc 3 and 4-MeSe-Fuc 4 and subjected them
to X-ray diffraction experiments. In both cases, no anomalous sig-
nals from selenium were observed (data not shown), indicating the
absence of these MeSe-fucoses in the crystals due to their weak
binding affinity to AOL.
is difficult. First,
2
2
L
-fucopyranose 21 was converted to orthoester
2 over 3 steps (Scheme 4). Acetyl groups were removed to give
3, then 23 underwent a three-step protecting group manipula-
tion, including benzylation, hydrolysis of the orthoester, and
deacetylation, to yield 1,2-diol derivative 24. The resulting 24
was converted to 1,2-acetonide derivative 25.
2.6. Surface plasmon resonance analysis of the affinity of the MeSe-Fuc
Next, to introduce a MOM group at the C4-hydroxyl group, the
benzyl groups of 25 were removed and the retrieved C3-hydroxyl
group was selectively protected as TBDPS ether to afford 27. Then,
the MOM group was introduced at the free C4-hydroxyl group, fol-
lowed by removal of the TBDPS group to provide 29. As in the syn-
thesis of 2, compound 30 underwent oxidation and reduction at
the C3 position to give 6-deoxy gulose derivative 31. Next, the tri-
flation and toluoyl selenation reactions of 31 were carried out. To
our delight, the selenation reaction of 32 proceeded smoothly to
yield 3-TolSe-fucose derivative 33 in 75% yield (over two steps).
The Tol group of 33 was exchanged with a Me group, as in the
preparation of 2-MeSe-Fuc 2, thereby affording 34. The J2,3 value
of 34 (7.0 Hz) was larger than that of 31 (3.0 Hz). These values indi-
cated the equatorial orientation of the MeSe group of compound
derivatives to AOL
We succeeded in obtaining AOL co-crystalized with 1a-MeSe-
1
0
Fuc 1a, 1b-MeSe-Fuc 1b, and 2-MeSe-Fuc 2 (this work), but not
with 3-MeSe-Fuc 3 or 4-MeSe-Fuc 4. The affinity of the latter
MeSe-fucoses to AOL would be lower and hence we could not
obtain the ligand-bound crystals. To test this hypothesis, the inter-
action between one non-binding MeSe-fucose was measured using
surface plasmon resonance (SPR). SPR analysis showed consider-
able differences (ranging from a factor of 2–12) in the dissociation
d
constants (K ) between the co-crystallized (1a, 1b and 2) and non-
co-crystallized (3 and 4) MeSe-Fucs (Table 1 and Supporting Infor-
mation Figure S1). Co-crystals of AOL with MeSe-fucose are
obtained with high-affinity MeSe-fucoses but not with low-affinity
MeSe-fucoses. In addition, 2-MeSe-Fuc 2 was found to bind more
7
7
3
4. In addition, the Se NMR spectrum of 34 showed a selenium
signal at 138.7 ppm. Deprotection and Fischer glycosidation with
methanol afforded 3-MeSe-fucoside 3.
tightly than methyl-a-fucoside 9, which suggests that the MeSe
at the C2 position of 2 might enhance the interaction with AOL
through formation of an intramolecular hydrogen bond between
the compound 2 to AOL. There is no direct interaction between
selenium atom and AOL in the X-ray crystal structure. We hypoth-
esized that selenium atom increases the electronegativity of the
proton on the C3-hydroxyl group, and it increases the hydrogen
bond between the O3 and Trp94 (in the case of site 1). In contrast,
the incorporation of a MeSe group at the C3 or C4 positions would
interfere with hydrogen bonding with the amino acid residues of
AOL.
2
.4. Synthesis of 4-MeSe-Fuc
The synthesis of 4-MeSe-Fuc 4 commenced with protection of
the C3- and C4-hydroxyl groups of 9 as benzylidene acetals, fol-
lowed by conversion to 4-bromo derivative 3523 (Scheme 5) via
an oxidative ring opening reaction. After protection of the C2
hydroxyl group of 35 with an Ac group, installation of selenium
using potassium p-methylselenobenzoate21,24 gave 37 in 87% yield.
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4
J. Shimabukuro et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
OH
O
O
OH
a~c
0%
d
e~g
h
OMe
OMe
O
O
O
O
OBn
24
OH
O
O
OH
9
99%
83%
(3 steps)
86%
OH
HO
OAc
OH
HO
(3 steps) AcO
BnO
2
1
22
23
R2
R1
O
O
O
j
26
H
H
H
i
9%
m
n
O
O
OR1
86%
O
O
O
27 TBDPS
O
8
7%
48%
8
k
6%
OBn
2
MOMO
BnO
R O
9
8
28 TBDPS MOM
O
2
5
l
30
2
9
H
MOM
0%
O
R3 = OH, R4 = H
31
32 R3 = OTf, R4 = H
33 R3 = H, R4 = SeTol
O
OMe
OH
R3
o
q
r,s
O
O
SeMe
34
O
O
O
p
45%
R4
9%
(2 steps)
SeMe
MOMO
75%
MOMO
HO
(
2 steps)
3
Scheme 4. Synthesis of 3-MeSe-Fuc 3. (a) Ac
2
O, 4-DMAP, py-THF, RT; (b) HBr, Ac
2
O, CH
2
Cl
2
, RT; (c) MeOH, Et
3
N, nBu
, Pd-C, MeOH, 89%; (j) TBDPSCl, imidazole, DMF, À40 °C,
, RT, 87%; (n) NaBH , MeOH, 0 °C, 48%; (o) Tf O, py, CH Cl
, DMF, RT, 45%, (r) 80% TFA aq., RT; (s) MeOH/H SO , 9% (2 steps). DCE = 1,2-dichloroethane, 4-
DMAP = N,N-dimethyl-4-aminopyridine, TBDPSCl = tert-butyldiphenylchlorosilane
4
NBr, DCE, 50 °C, 90% (3 steps); (d) NaOMe, MeOH, RT,
9
8
9%; (e) BnBr, NaH, DMF, RT; (f) then 2 M HCl aq.; (g) NaOMe, MeOH, RT, 83% (3 steps); (h) DMP, CSA, RT, 86%; (i) H
6%; (k) MOMCl, DIEA, DCE, 45 °C, 96%; (l) Bu
2
n
4
NF, THF, RT, 80%; (m) Dess-Martin periodinane, NaHCO
, Cs CO
3 2
, CH Cl
2
4
2
2
2
,
À20 °C?RT; (p) TolSeK, DMF, RT, 75% (2 steps); (q) MeI, MeNHNH
2
2
3
2
4
OMe
OH
OMe
fucoses, and in determining the X-ray structure of the co-crystal
using a SAD/MAD phasing method. X-ray structural analysis of
AOL complexed with 2-MeSe-Fuc 2 showed that AOL recognizes
the ring oxygen, and 3- and 4-hydroxy groups of fucose, consistent
a,b
c
9
O
O
Br
Br
OAc
1
1%
95%
(
2 steps)
OBz
OBz
3
5
36
1
0
OMe
OMe
with our recently reported results and in accordance with the
d
f
6
,7
O
O
result reported by Imberty’s research group.
OAc
OH
8
7%
96%
OBz
MeSe OH
3- and 4-MeSe-Fuc 3 and 4 turned out not to be co-crystallized
with AOL. SPR analysis revealed that these compounds show dras-
tically reduced affinity to AOL compared to 2-MeSe-Fuc 2. These
results also emphasized the influence of selenium incorporation
on carbohydrate-protein interactions.
Our findings demonstrate that the widespread utility of the
seleno-sugar-based SAD/MAD phasing method for the structural
determination of unknown sugar-binding proteins requires estab-
lishment of a library that includes various positional isomers of
MeSe-substituted saccharides. In this study, we determined that
each secondary hydroxyl group in fucose can be replaced with a
MeSe group, although replacement of the C3- and C4-hydroxyls
proved difficult. We anticipate that the results obtained by this
synthetic study will serve to elaborate a method for synthesizing
RSe
e
37 R = Tol
R = Me
4
5
3%
3
8
Scheme 5. Synthesis of 4-MeSe-Fuc 4. (a) Benzaldehyde dimethyl acetal, CSA,
MeCN, 50 °C; (b) N-bromosuccinimide, CCl , reflux, 11% (2 steps); (c) Ac O, 4-DMAP,
py-THF, RT, 95%; (d) TolSeK, DMF, 60 °C, 87%; (e) MeI, MeNHNH , Cs CO , DMF, RT,
3%; (f) NaOMe, MeOH, RT, 96%. Bz = benzoyl.
4
2
2
2
3
5
3
. Conclusion
In conclusion, we succeeded in individually introducing a MeSe
group at each hydroxyl group of fucose, in co-crystallizing a
fucose-specific lectin, AOL, with one of the synthesized seleno-
(a)
(b)
IV
4
V
1
3
5
2
3
5
4
Arg25
III
Trp94
VI
6
2
N
Glu37
C
II
1
I
Fig. 2. X-ray crystal structure of AOL with 2-MeSe-Fuc 2. (a) Overall structure of the AOL monomer is represented by a ribbon model. Six 2-MeSe-Fuc molecules in the fucose
binding sites are represented by stick models colored light pink and labeled with Arabic numerals. AOL is depicted in gradated color from blue (N-terminal) to red (C-
terminal) and the six blades are labeled with Roman numerals (PDB id: 5H47). (b) Close-up view of one fucose binding site (site 1). 2mFo–DFc map of 2-MeSe-Fuc 2 is
contoured with a cyan mesh (1.5r) and the anomalous difference Fourier map is contoured in red (6r). Residues that contribute hydrogen bonds are represented by stick
models in green. Yellow dotted lines indicate hydrogen bonds.
Please cite this article in press as: Shimabukuro J., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.021

J. Shimabukuro et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
5
Table 1
confirmed by TLC analysis (EtOAc/Toluene = 1/8), and then the
reaction was quenched with 500
And the reaction mixture was filtered through a pad of Celite. Then,
the combined filtrate and washings were concentrated. The residue
was purified by column chromatography on silica gel (EtOAc/
Results of SPR analysis of the affinity of MeSe-Fucs to AOL.
lL of triethylamine at À78 °C.
K
d
SE
1
1
1
2
3
4
a
a
-Me-Fuc (9)
-MeSe-Fuc (1
71
85
121
52
501
624
±8.6
±18
±18
±2.5
±269
±108
a)
b-MeSe-Fuc (1b)
-MeSe-Fuc (2)
-MeSe-Fuc (3)
-MeSe-Fuc (4)
Toluene = 1/30) to give compound 7
a
(27.3 mg, 66%) and com-
-isomer (7 ); [
); H NMR (500 MHz, CDCl ) d 7.27–6.87 (2
d, 4 H, Ar), 5.87 (d, 1 H, J1,2 = 5.5 Hz, H-1), 5.29 (d, 1 H,
pound 7b (9.3 mg, 21%) as a yellow syrup.
a
a
a]
D
1
À165.2° (c 1.0, CHCl
3
3
SE = standard error.
J
J
(
(
6
1
3,4 = 3.0 Hz, H-4), 5.16 (dd, 1 H, J2,3 = 10.5 Hz, H-3), 4.64 (d, 1 H,
gem = 12.0 Hz, OCH Ar), 4.49 (d, 1 H, Jgem = 12.0 Hz, OCH Ar), 4.37
m, 1 H, H-5), 3.97 (dd, 1 H, H-2), 3.80 (s, 3 H, OCH ), 2.14–1.98
2 s, 6 H, 2 Ac), 1.90 (s, 3 H, SeCH ), 1.15 (d, 3 H, J5,6 = 6.5 Hz, H-
) d 170.4, 170.0, 159.4, 129.7, 129.3,
2
2
3
diverse seleno-sugars. We confirmed that the synthesized seleno-
fucoses were water-soluble, thereby allowing us to quantify the
binding affinities of the seleno-fucoses by SPR assay. We also antic-
ipate that SPR assay will be useful in the initial screening of seleno-
sugars as potential ligands to target sugar-binding proteins.
3
1
3
); C NMR (125 MHz, CDCl
13.8, 80.8, 72.9, 71.8, 71.4, 71.0, 66.2, 55.3, 20.8, 16.0, 1.63; Se
3
77
+
NMR (94 MHz, CDCl
69.0736, C19 Se calcd for [M + Na] 469.0736.
b-isomer (7b); [ + 3.6° (c 0.8, CHCl
); 1H NMR (500 MHz,
CDCl ) d 7.27–6.86 (2 d, 4 H, Ar), 5.27 (dd, 1 H, J3,4 = 3.0 Hz,
4,5 = 0.5 Hz, H-4), 4.98 (dd, 1 H, J2,3 = 9.5 Hz, H-3), 4.75 (d, 1 H,
gem = 10.5 Hz, OCH Ar), 4.66 (d, 1 H, J1,2 = 9.5 Hz, H-1), 4.59 (d, 1
Ar), 3.79–3.71 (m, 5 H, CH , H-2, H-5),
.17–1.99 (3 s, 9 H, 2 Ac, SeCH ), 1.38 (d, 3 H, J5,6 = 6.5 Hz, H-6);
): d 170.4, 170.1, 159.6, 130.1, 129.8,
13.9, 79.0, 76.4, 75.1, 74.7, 74.2, 71.2, 55.4, 29.8, 20.9, 20.9,
3
) d 91.3; HRMS (ESI) m/z: found [M + Na]
+
4
26 7
H O
a
]
D
3
4
. Experimental procedures
3
J
J
4.1. General procedures
2
H, Jgem = 10.0 Hz, OCH
2
3
All reactions were performed in round-bottom flask fitted with
2
3
1
3
balloon filled with argon unless otherwise stated. Transfer of air
and moisture sensitive liquids were performed via cannula under
a positive pressure of argon. When necessary, reaction mixtures
were sonicated with AS ONE US CLEANER USD-4R by following
the reported procedure.25 Reactions were monitored by thin layer
chromatography carried out on Merck silica gel plates (60F-254)
using ultraviolet light (254 nm) or by soak in a solution of 10%
C NMR (125 MHz, CDCl
3
1
1
7
7
6.5, 3.4; Se NMR (94 MHz, CDCl
3
) d 211.8; HRMS (ESI) m/z:
+
Se calcd for [M + Na]+
found [M + Na] 469.0736,
19 26 7
C H O
4
69.0736.
4.1.2. Methyl 3,4-di-O-acetyl-6-deoxy-1-seleno-a-L-galactopyranoside
2 4
H SO in ethanol followed by heating. Flash column chromatogra-
(8a)
phy on silica gel (Fuji Silysia, 80 mesh and 300 mesh) or size exclu-
sion chromatography on Sephadex (Pharmacia LH-20) was
performed with the solvent systems (v/v) specified. Quantity of sil-
ica gel was usually estimated as 25–200-fold weight of sample to
be charged. Dichloromethane, Dichloroethane (DCE), N,N-
dimethylformamide (DMF), N,N-dimethylacetamide (DMA),
methanol, tetrahydrofuran (THF), tert-butyl methyl ether (TBME)
and toluene as dry reaction media were purchased from Kanto
Chemical Co., Inc. (Tokyo, Japan) and used without further purifica-
tion. When necessary, solvents were degassed prior to use by son-
ication under reduced pressure for 30 min, followed by bubbling
argon through the solvents for 30 min. Molecular sieves were pur-
chased from Wako Chemicals Inc. (Miyazaki, Japan) and pre-dried
at 300 °C for 2 h in a muffle furnace, and dried in a flask at 300 °C
for 2 h in vacuo prior to use. Evaporation and concentration were
Trifluoroacetic acid (0.46 mL) was added to a mixture of com-
pound 7 (30 mg, 70 mol) and anisole (15 mg, 140 mol) in
CH Cl
(0.92 mL) at À40 °C, and then the mixture was stirred for
h. The completion of the reaction was confirmed by TLC analysis
EtOAc/n-Hexane = 1/1), and then the reaction mixture was co-
a
l
l
2
2
6
(
evaporated with toluene. The residue was purified by column chro-
matography on silica gel (EtOAc/n-Hexane = 1/2) to give com-
pound 8
a
(21 mg, 90%) as a yellow syrup. [
); H NMR (500 MHz, CDCl ) d 5.71 (d, 1 H, J1,2 = 5.5 Hz, H-
), 5.26 (dd, 1 H, J3,4 = 3.0 Hz, J4,5 = 1.0 Hz, H-4), 4.91 (dd, 1 H,
2,3 = 10.0 Hz, H-3), 4.34 (m, 1 H, H-5), 4.13 (m, 1 H, H-2), 2.16–
.05 (3 s, 9 H, 2 Ac, SeCH
), 1.18 (d, 3 H, J5,6 = 6.5 Hz, H-6); 13
NMR (125 MHz, CDCl ) d 171.0, 170.8, 86.9, 73.2, 71.1, 67.9, 67.4,
a
]
D
À273.2° (c 1.0,
1
CHCl
3
3
1
J
2
3
C
3
7
7
2
1.4, 21.0, 16.3, 4.3; Se NMR (94 MHz, CDCl
3
) d 75.2; HRMS
Se calcd for
+
(
[
ESI) m/z: found [M + Na] 349.0161,
M + Na] 349.0161.
C
11
H
18
O
6
1
13
77
+
carried out in vacuo. H, C and Se NMR spectra were recorded
1
on a Bruker Avance III 500 spectrometers. H NMR chemical shifts
are expressed in ppm (d) relative to the signal of Me
4
Si as an inter-
4
.1.3. Methyl 1-seleno-
Sodium methoxide (28% in MeOH, 2.00 mg, 10.0
added to a solution of compound 8 (16.0 mg, 49.0 mol) in MeOH
500 L). The reaction mixture was stirred for 20 min at ambient
a-L-galactopyranoside (1a)
1
3
nal standard. C NMR chemical shifts are expressed in ppm (d) rel-
l
mol) was
77
ative to the signal of the solvent as a standard. Se NMR chemical
shifts are expressed in ppm (d). The following abbreviations or
combinations thereof were used to explain the multiplicities:
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
br = broad. High-resolution mass spectrometry (HRMS) was per-
formed with a Bruker Daltonics micrOTOF (ESI-TOF) mass spec-
trometer. Specific rotations were measured with a Horiba SEPA-
a
l
(
l
temperature. The progress of the reaction was monitored by TLC
analysis (MeOH/Toluene = 1/5). The reaction mixture was neutral-
ized with Muromac (H ) and filtered through a pad of cotton, and
the removed resin was washed with MeOH. The combined filtrate
and washings were concentrated. The residue was purified by col-
+
3
00 high-sensitivity polarimeter.
umn chromatography on Sephadex LH-20 (H
give compound 1 (10.6 mg, 89%) as an amorphous white solid.
À327.6° (c 0.8, MeOH); H NMR (500 MHz, CD
(dd, 1 H, J1,2 = 9.5 Hz, H-1), 3.70–3.61 (m, 3 H, H-2, H-4, H-5),
3.47 (dd, 1 H, J3,4 = 3.5 Hz, H-3), 2.10 (s, 3 H, SeCH ), 1.28 (d, 3 H,
OD) d 85.3, 73.2, 73.2,
69.9, 69.4, 16.6, 1.4; Se NMR (94 MHz, CD OD) 75.4; HRMS
Se calcd for
2
O/MeOH = 1/4) to
a
1
4.1.1. Methyl 3,4-di-O-acetyl-6-deoxy-2-O-(4-methoxybenzyl)-1-
[a
]
D
3
OD) d 4.51
seleno-
A mixture of selenoacetal 6 (100 mg, 463
date 5 (50.0 mg, 90.0 mol) and AW-300 (90 mg) in CH
925
L) was stirred for 30 min, and cooled to À78 °C, to which
TMSOTf (10.2 L, 99.6 mol) was then added. The reaction mixture
was stirred at À78 °C for 21 h. The completion of the reaction was
a
-L
-galactopyranoside (7
a
) and -b-
L
-galactopyranoside (7b)
mol), glycosyl imi-
Cl -TBME
l
3
1
3
l
2
2
J5,6 = 6.5 Hz, H-6); C NMR (125 MHz, CD
3
7
7
(
l
3
+
l
l
(ESI) m/z: found [M + Na] 264.9948,
7
C H
14
O
4
+
[M + Na] 264.9950.
Please cite this article in press as: Shimabukuro J., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.021

6
J. Shimabukuro et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
4
.1.4. Methyl 3,4-di-O-acetyl-6-deoxy-1-seleno-b-
L
-galactopyranoside
H-2), 3.37 (s, 3 H, OCH
(CH C), 1.41 (d, 3 H, J5,6 = 6.5 Hz, H-6), 1.36 (s, 3 H, (CH
C NMR (125 MHz, CDCl ) d 192.5, 144.6, 136.2, 129.4, 127.5,
109.4, 109.1, 101.3, 75.9, 74.4, 63.1, 55.7, 44.9, 29.7, 28.1, 26.5,
3
), 2.39 (s, 3 H, Ar-CH
3
), 1.63 (s, 3 H,
(
8b)
Trifluoroacetic acid (0.6 mL) was added to
compound 7b (64 mg, 140 mol) and anisole (31
in CH Cl
completion of the reaction was confirmed by TLC analysis
MeOH/CHCl = 1/20), and then the reaction mixture was co-evap-
3
)
2
3 2
) C);
1
3
a
mixture of
L, 290 mol)
3
l
l
l
7
7
2
2
(1.9 mL) at À20 °C. The mixture was stirred for 5 h. The
21.7, 16.7; Se NMR (94 MHz, CDCl
3
) d 534.0; HRMS (ESI) m/z:
found [M + Na]+ 423.0681,
423.0681.
Se calcd for [M + Na]+
18 24 5
C H O
(
3
orated with toluene. The residue was purified by column chro-
matography on silica gel (EtOAc/n-Hexane = 1/2) to give
4.1.7. Methyl 2,6-dideoxy-3,4-O-isopropylidene-2-methylseleno-a-L-
compound 8b (40 mg, 86%) as a yellow syrup. [
a
]
D
À6.6° (c 0.8,
galactopyranoside (14)
1
CHCl
3
); H NMR (500 MHz, CDCl
3
) d 5.27 (d, 1 H, J3,4 = 3.0 Hz, H-
A solution of compound 13 (60 mg, 150
(2.0 mL) was added dropwise to a suspension of Cs
300 mol), N-methylhydrazine (12.0 mg, 230 mol) and methyl
iodide (19.0 L, 300 mol) in degassed DMF (1.0 mL) at ambient
l
mol) in degassed DMA
4
1
2
), 4.93 (dd, 1 H, J2,3 = 9.5 Hz, H-3), 4.60 (d, 1 H, J1,2 = 10.0 Hz, H-
), 3.90–3.83 (m, 2 H, H-2, H-5), 2.47 (d, 1 H, J2,OH = 2.0 Hz, OH),
2
CO (98 mg,
3
l
l
.16–2.06 (3 s, 9 H, 2 Ac, SeCH
3
), 1.21 (d, 3 H, J5,6 = 6.5 Hz, H-6);
l
l
1
3
C NMR (125 MHz, CDCl
5.6, 65.5, 18.7, 18.5, 14.3; Se NMR (94 MHz, CDCl
3
) d 168.4, 79.3, 78.9, 72.3, 71.8, 68.7,
temperature, and the mixture was stirred for 70 min. The progress
of the reaction was monitored by TLC analysis (EtOAc/n-Hex-
ane = 1/4). The reaction mixture was diluted with EtOAc, washed
77
6
3
) d 177.9;
Se calcd for
+
HRMS (ESI) m/z: found [M + Na] 349.0161, C11
H
18
O
6
+
[
M + Na] 349.0161.
3 2 4
with satd aq. NaHCO and brine, dried over Na SO and filtered
off. The filtrate was concentrated. The resulting residue was puri-
4.1.5. Methyl 1-seleno-b-
L-galactopyranoside (1b)
fied by column chromatography on silica gel (EtOAc/n-Hex-
Sodium methoxide (28% in MeOH, 10 mg, 40
l
mol) was added
ane = 1/6) to give compound 14 (29 mg, 64%) as a yellow syrup.
1
to a solution of compound 8b (40 mg, 120
l
mol) in THF (1.4 mL)
[
a
]
D
À141.7° (c 1.1, CHCl
3
); H NMR (500 MHz, CDCl
3
) d 4.81 (d,
and MeOH (2.7 mL). The reaction mixture was stirred for 30 min
at ambient temperature. The progress of the reaction was moni-
tored by TLC analysis (MeOH/Toluene = 1/5). The reaction mixture
1 H, J1,2 = 3.0 Hz, H-1), 4.43 (dd, 1 H, J2,3 = 9.5 Hz, J3,4 = 5.0 Hz, H-
3), 4.11 (m, 1 H, H-5), 3.96 (dd, 1 H, J4,5 = 2.5 Hz, H-4), 3.36 (s, 3
H, OCH
9 H, (CH
75.7, 62.8, 55.6, 43.1, 29.7, 28.6, 26.4, 16.7, 4.6;
(94 MHz, CDCl ) d 127.2; HRMS (ESI) m/z: found [M + Na]
319.0419, C11
3
), 2.82 (dd, 1 H, H-2), 2.14 (s, 3 H, SeCH
3 2 3
) C, H-6); C NMR (125 MHz, CDCl ) d 108.7, 101.6,
3
), 1.52–1.36 (m,
+
13
was neutralized with Muromac (H ) and filtered through a pad of
77
cotton, and the removed resin was washed with MeOH. The com-
bined filtrate and washings were concentrated. The resulting resi-
due was purified by column chromatography on Sephadex LH-20
Se NMR
+
3
+
20 4
H O Se calcd for [M + Na] 319.0419.
2
(H O/MeOH = 1/4) to give compound 1b (30 mg, quant.) as an
1
amorphous white solid. [
a
]
D
+21.9° (c 0.8, MeOH); H NMR
OD) d 4.51 (dd, 1 H, J1,2 = 9.5 Hz, H-1), 3.70–3.61
m, 3 H, H-2, H-4, H-5), 3.47 (dd, 1 H, J2,3 = 9.5 Hz, J3,4 = 3.5 Hz,
4.1.8. Methyl 2,6-dideoxy-2-methylseleno-
Acetic acid (270 L) was added to a suspension of compound 14
(10 mg, 34 mol) in H O (67 L) at 0 °C. The mixture was stirred
for 50 min at 50 °C. The completion of the reaction was confirmed
by TLC analysis (MeOH/CHCl = 1/10), and then the reaction mix-
a-L-galactopyranoside (2)
(
500 MHz, CD
3
l
(
l
2
l
1
3
H-3), 2.10 (s, 3 H, SeCH
3
), 1.28 (d, 3 H, J5,6 = 6.5 Hz, H-6);
OD) d 110.0, 105.3, 104.2, 101.3, 99.7, 45.0,
9.7; Se NMR (94 MHz, CD OD) d 190.4; HRMS (ESI) m/z: found
7 14 4
M + Na] 264.9948, C H O Se calcd for [M + Na] 264.9950.
C
NMR (125 MHz, CD
3
3
7
7
2
3
ture was co-evaporated with toluene. The resulting residue was
purified by column chromatography on silica gel (EtOAc/
+
+
[
Toluene = 2/1) to give compound 2 (7.2 mg, 83%) as an amorphous
1
4
.1.6. Methyl 2,6-dideoxy-3,4-O-isopropylidene-2-(4-methyl-
-galactopyranoside (13)
To a solution of compound 10 (96.0 mg, 440
10.0 mL) were added pyridine (148 L, 183 mol) and trifluo-
romethanesulfonic anhydride (154 L, 916
white solid. [
a
]
D
+21.9° (c 0.8, MeOH); H NMR (500 MHz, CD
3
OD)
benzoylseleno)-
a-L
d 4.77 (d, 1 H, J1,2 = 3.5 Hz, H-1), 3.91–3.84 (m, 2 H, H-3, H-5), 3.61
(d, 1 H, J3,4 = 2.5 Hz, H-4), 3.31 (s, 3 H, OCH ), 2.96 (dd, 1 H,
2,3 = 11.0 Hz, H-2), 2.06 (s, 3 H, SeCH ), 1.21 (d, 3 H, J5,6 = 6.5 Hz,
OD) d 103.3, 73.5, 72.5, 67.7, 55.8,
44.9, 16.9, 4.8; Se NMR (94 MHz, CD OD) d 102.8; HRMS (ESI)
Se calcd for [M + Na]+
l
mol) in CH
2
Cl
2
3
(
l
l
J
3
1
3
l
l
mol) at À20 °C, and
H-6); C NMR (125 MHz, CD
3
7
7
the mixture was stirred for 30 min at ambient temperature. The
progress of the reaction was monitored by TLC analysis (EtOAc/n-
3
+
8 16 4
m/z: found [M + Na] 279.0106, C H O
Hexane = 1/1). The reaction mixture was diluted with CH
washed with 2 M HCl aq., H O, satd aq. NaHCO and brine, dried
over Na SO , and filtered off. The filtrate was concentrated. The
2
Cl2,
279.0106.
2
3
2
4
4.1.9. Methyl 4-O-acetyl-6-deoxy-2-methoxymethyl-a-L-galacto-
resulting residue was exposed to high vacuum for 3 h to give crude
triflate derivative, which was used for next reaction without fur-
ther purification. The resulting triflate was dissolved in degassed
DMA (3.60 mL), which was then added dropwise to a solution of
pyranoside (15)
To a solution of compound 9 (2.44 g, 13.7 mmol) in acetonitrile
(137 mL) were added triethyl orthoformate (3.80 mL, 20.6 mmol)
and CSA (325 mg, 1.40 mmol) at 0 °C, and the mixture was stirred
for 7 h at ambient temperature. The progress of the reaction was
4
-methylselenobenzoic anhydride 12 (788 mg, 2.48 mmol), N,N-
diisopropylethylamine (444 L, 2.48 mmol) and piperidine
244 L, 2.48 mmol) in degassed DMF (6.40 mL) as bubbled with
l
3
monitored by TLC analysis (MeOH/CHCl = 10/1). The reaction mix-
(
l
ture was quenched by addition of triethylamine at 0 °C. Then, DIEA
(11.8 mL, 68.5 mmol) and MOMCl (2.10 mL, 27.4 mmol) were
added to the mixture, and stirred for 28 h at ambient temperature.
The progress of acetalization was checked by TLC analysis (EtOAc/
n-Hexane = 1/1). The reaction mixture was quenched by addition
of methanol at 0 °C. Then, 2 M HCl aq. was added to the mixture,
and stirred for 10 min. The progress of hydrolysis of the orthoester
was checked by TLC analysis (EtOAc/n-Hexane = 1/1). Then, the
argon gas. The mixture was stirred for 75 min at 90 °C. The pro-
gress of the reaction was monitored by TLC analysis (EtOAc/n-Hex-
ane = 1/3). Then, the reaction mixture was diluted with EtOAc. The
solution was washed with 2 M HCl aq., water, satd aq. NaHCO
3
and
brine, dried over Na SO and filtered off. The filtrate was concen-
2
4
trated. The resulting residue was purified by column chromatogra-
phy on silica gel (EtOAc/toluene = 1/5) to give compound 13
1
(
163 mg, 93%) as a yellow syrup. [
a
]
D
À66.0° (c 1.1, CHCl
3
);
H
solution was washed with water, satd aq. NaHCO
3
and brine, and
NMR (500 MHz, CDCl
J
3
) d 7.81–7.24 (2 d, 4 H, Ar), 4.76 (d, 1 H,
1,2 = 3.5 Hz, H-1), 4.38 (dd, 1 H, J2,3 = 11.0 Hz, J3,4 = 5.0 Hz, H-3),
dried over Na SO and filtered off. The filtrate was concentrated.
2
4
The resulting residue was purified by column chromatography on
4
.16 (m, 1 H, H-5), 4.04 (dd, 1 H, J4,5 = 2.0 Hz, H-4), 3.94 (dd, 1 H,
silica gel (EtOAc/Toluene = 1/5) to give compound 15 (2.97 g,
Please cite this article in press as: Shimabukuro J., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.021

J. Shimabukuro et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
7
8
(
4
OCH
2%) as a colorless syrup. [
500 MHz, CDCl ) d 5.27 (dd, 1 H, J3,4 = 3.5 Hz, J4,5 = 1.0 Hz, H-4),
.84 (d, 1 H, J1,2 = 3.5 Hz, H-1), 4.82 (d, 1 H, Jgem = 12.0 Hz, OCH
), 4.73 (d, 1 H, Jgem = 12.0 Hz, OCH OCH ), 4.12–4.04 (m, 2
H, H-3, H-5), 3.72 (dd, 1 H, J2,3 = 10.0 Hz, H-2), 3.43–3.41 (m, 6 H,
OCH ), 3.01 (s, 1 H, OH), 2.18 (s, 3 H, Ac), 1.16 (d, 3 H,
5,6 = 6.5 Hz, H-6); C NMR (125 MHz, CDCl
8.3, 73.0, 67.6, 64.5, 55.9, 55.3, 20.9, 16.2; HRMS (ESI) m/z: found
a
]
D
À28.9° (c 0.4, CHCl
3
); 1H NMR
24 h to give the crude penta-acetate derivative. The resulting resi-
due was dissolved in CH Cl (20.0 mL). To the solution were added
Ac O (2.88 mL, 30.5 mmol) and 25% HBr-HOAc solution (40.0 mL)
at ambient temperature. The progress of the reaction was moni-
tored by TLC analysis (EtOAc/n-Hexane = 1/5). The solution was
stirred for 16 h, and then the reaction mixture was diluted with
3
2
2
2
-
2
3
2
3
2
J
7
3
1
3
3
) d 177.2, 99.3, 98.1,
CH
satd aq. Na
2
Cl
2
and poured into ice water. The solution was washed with
CO and brine, dried over Na SO and filtered off. The
2
3
2
4
+
[
M + Na] 287.1103, C11
H
20
O
7
calcd for [M + Na]+ 287.1101.
filtrate was concentrated. The residue was exposed to high vacuum
for 12 h to give the crude fucosyl bromide. The resulting residue
4
.1.10. Methyl 4-O-acetyl-6-deoxy-2-O-methoxymethyl-
hexopyranosid-3-ulose (16)
To a suspension of compound 15 (620 mg, 2.35 mmol) and
NaHCO (983 mg, 11.7 mmol) in CH Cl (23.5 mL) was added
a
-
L
-xylo-
3
was dissolved in DCE (95.3 mL). To the solution were added Et N
(8.50 mL, 61.0 mmol), MeOH (1.40 mL, 33.6 mmol) and Bu NBr
4
(4.93 g, 15.3 mmol) at ambient temperature. The solution was stir-
red for 5 h at 50 °C. The completion of the reaction was confirmed
by TLC analysis (EtOAc/n-Hexane = 1/3), and then the reaction mix-
ture was filtered through a pad of Celite. The filtrate was washed
n
3
2
2
Dess-Martin periodinane (1.29 g, 3.05 mmol) at 0 °C. The reaction
mixture was warmed to ambient temperature and stirred for
1
(
8 h. The progress of the reaction was monitored by TLC analysis
EtOAc/n-Hexane = 2/1). After the mixture was diluted with Et O,
was added and the aqueous layer was extracted
with 2 M HCl aq., water, satd aq. Na
2 3 2
CO and brine, dried over Na -
2
SO and filtered off. The filtrate was concentrated. The residue was
4
satd aq. Na
2
2
S O
3
purified by column chromatography on silica gel (EtOAc/n-Hex-
with Et
dried over Na
2
O. The combined organic layers were washed with brine,
SO and filtered off. The filtrate was concentrated.
ane = 1/5) to give compound 22 (8.65 g, 90%) as a colorless syrup.
H NMR (500 MHz, CDCl ) d 5.80 (d, 1 H, J1,2 = 5.0 Hz, H-1), 5.27–
3
1
2
4
The resulting residue was purified by column chromatography on
silica gel (Acetone/Toluene = 1/5) to give compound 16 (449 mg,
5.25 (m, 1 H, H-4), 5.04 (dd, 1 H, J2,3 = 7.0 Hz, J3,4 = 3.0 Hz, H-3),
4.29–4.23 (m, 2 H, H-2, H-5), 3.30 (s, 3 H, OCH ), 2.14 (s, 3 H,
Ac), 2.06 (s, 3 H, Ac), 1.20 (d, 3 H, J5,6 = 6.5 Hz, H-6); HRMS (ESI)
3
1
7
(
J
4
1
1
3%) as a colorless syrup. [
500 MHz, CDCl
4,5 = 1.5 Hz, H-4), 4.74–4.72 (m, 3 H, H-2, 2 OCH
a
]
D
+3.0° (c 0.7, CHCl
3
); H NMR
+
20 8
m/z: found [M + Na] 327.1050, C13H O
calcd for [M + Na]+
3
) d 5.11 (d, 1 H, J1,2 = 4.0 Hz, H-1), 5.08 (d, 1 H,
OCH ), 4.28–
), 2.19 (s, 3 H, Ac),
.27 (d, 3 H, J5,6 = 6.5 Hz, H-6); 13C NMR (125 MHz, CDCl
) d
97.8, 169.6, 101.8, 96.5, 78.9, 77.4, 67.7, 56.0, 55.7, 20.7, 15.6;
2
3
327.1045.
.24 (m, 1 H, H-5), 3.43–3.42 (m, 6 H, 2 OCH
3
3
4.1.13. 6-Deoxy-1,2-O-(1-methoxymethylidene)-a-L-galactopyranose
(23)
+
HRMS (ESI) m/z: found [M + Na] 285.0945, C11
H
18
O
7
calcd for
Sodium methoxide (28% in MeOH, 21.0 mg, 400
added to a solution of compound 22 (1.00 g, 3.28 mol) in MeOH
6.56 mL). The reaction mixture was stirred for 30 min at ambient
lmol) was
+
[
M + Na] 285.0945.
(
4
.1.11. Methyl 4-O-acetyl-6-deoxy-2-O-methoxymethyl-
a-L-
temperature. The progress of the reaction was monitored by TLC
gulopyranoside (17)
A solution of compound 16 (50 mg, 0.19 mmol) in MeOH
1.9 mL) was cooled to 0 °C, and NaBH (7.9 mg, 0.21 mmol) was
added. The reaction mixture was stirred for 5 min at 0 °C. The pro-
gress of the reaction was monitored by TLC analysis (EtOAc/n-Hex-
analysis (EtOAc/n-Hexane = 1/1). The reaction mixture was neu-
tralized with Muromac (H ) and filtered through a pad of cotton,
and the removed resin was washed with MeOH. The combined fil-
trate and washings were concentrated. The residue was purified by
+
(
4
column chromatography on silica gel (MeOH/CHCl
compound 23 (720 mg, 99%) as a colorless syrup. H NMR
(500 MHz, CDCl ) d 5.74 (d, 1 H, J1,2 = 5.0 Hz, H-1), 4.25 (t, 1 H,
2,3 = 5.0 Hz, H-2), 4.04–4.00 (m, 1 H, H-5), 3.88–3.85 (m, 1 H, H-
3), 3.78–3.75 (m, 1 H, H-4), 3.38 (m, 1 H, OH), 3.29 (s, 3 H,
OCH ), 2.84 (s, 1 H, OH), 1.32 (d, 3 H, J5,6 = 6.5 Hz, H-6); HRMS
3
= 1/20) to give
1
ane = 2/1). The reaction mixture was quenched with satd aq. NH
and diluted with EtOAc. The solution was washed with water and
brine, dried over Na SO and filtered off. The filtrate was concen-
4
Cl
3
2
4
J
trated. The resulting residue was purified by column chromatogra-
phy on silica gel (EtOAc/n-Hexane = 3/1) to give compound 17
3
+
9 16 6
(ESI) m/z: found [M + Na] 243.0839, C H O
calcd for [M + Na]+
(
41 mg, 82%) as an amorphous white solid. [
a
]
D
À43.7° (c 0.4,
), d 4.98 (dd, 1 H, J3,4 = 3.5 Hz,
4,5 = 1.0 Hz, H-4), 4.87 (d, 1 H, J1,2 = 3.5 Hz, H-1), 4.81 (d, 1 H,
gem = 12.0 Hz, OCH OCH ), 4.74 (d, 1 H, Jgem = 12.0 Hz, OCH OCH ),
.35–4.30 (m, 2 H, H-3, H-5), 4.00 (m, 1 H, H-3), 3.84 (t, 1 H,
2,3 = 3.5 Hz, H-3), 3.68 (d, 1 H, J3, OH = 8.0 Hz, OH), 3.47–3.43 (m,
1
CHCl
J
J
4
J
3
); H NMR (500 MHz, CDCl
3
243.0842.
2
3
2
3
4.1.14. 3,4-Di-O-benzyl-6-deoxy-
L
-galactopyranose (24)
BnBr (834 L, 6.99 mmol) was added to a mixture of compound
l
23 (700 mg, 3.17 mmol) and NaH (1.20 g, 6.99 mmol) in DMF
(16.0 mL) at 0 °C. The mixture was stirred for 24 h at ambient tem-
perature. The progress of the reaction was monitored by TLC anal-
ysis (EtOAc/Toluene = 1/1). The reaction mixture was quenched by
addition of MeOH at 0 °C and diluted with EtOAc. Then, 2 M HCl aq.
was added to the mixture, and stirred for 10 min. The progress of
hydrolysis of the orthoester was checked by TLC analysis (EtOAc/
Toluene = 1/1). Then, the solution was washed with water, satd
1
3
6
H, 2 OCH
3
), 2.14 (s, 3 H, Ac), 1.16 (d, 3 H, J5,6 = 6.5 Hz, H-6);
C
NMR (125 MHz, CDCl
3
) d 170.0, 100.1, 95.9, 73.6, 71.0, 68.3, 60.5,
+
5
5.9, 55.7, 20.9, 15.7; HRMS (ESI) m/z: found [M + Na] 287.1103,
+
C
11
20 7
H O calcd for [M + Na] 287.1101.
4
.1.12. 1,2-O-Diacetyl-6-deoxy-1,2-O-(1-methoxymethylidene)-
a-L-
galactopyranose (22)
Compound 21 (5.00 g, 30.5 mmol) was dissolved in pyridine
3 2 4
aq. NaHCO and brine, and dried over Na SO and filtered off.
(
(
30.0 mL) and THF (30.0 mL). To the solution were added 4-DMAP
37.0 mg, 300 lmol) and Ac O (23.0 mL, 244 mmol) at ambient
2
The filtrate was concentrated. The residue was exposed to high
vacuum for 3 h to give the crude mono-acetate derivative. The
resulting residue was dissolved in MeOH (6.30 mL), and sodium
temperature. The solution was stirred for 13 h. The completion of
the reaction was confirmed by TLC analysis (MeOH/CHCl = 1/10),
3
methoxide (28% in MeOH, 61.2 mg, 317 lmol) was added at ambi-
and then the reaction was quenched with methanol at 0 °C. The
ent temperature. The mixture was stirred at ambient temperature
reaction mixture was co-evaporated with toluene. The residue
for 20 min. Then, the reaction mixture was neutralized with Muro-
mac (H ) and filtered through a pad of cotton, and the removed
resin was washed with MeOH. The combined filtrate and washing
were concentrated. The resulting residue was purified by column
+
was diluted with EtOAc, washed with 2 M HCl aq., H
NaHCO and brine, dried over Na SO and filtered off. The filtrate
was concentrated. The residue was exposed to high vacuum for
2
O, satd aq.
3
2
4
Please cite this article in press as: Shimabukuro J., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.021

8
J. Shimabukuro et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
+
chromatography on silica gel (MeOH/CHCl
3
= 1/25) to give com-
isomer;
) d 7.37–7.23 (m, 10 H, Ar), 5.31 (d, 1 H,
1,2 = 3.5 Hz, H-1), 4.93–4.63 (m, 4 H, 2 ArCH ), 4.15 (dd, 1 H,
27.1, 19.4, 16.5; HRMS (ESI) m/z: found [M + Na] 465.2068,
C H O Si calcd for [M + Na] 465.2068.
25 34 5
+
pound 24 (911 mg, 83%) as an amorphous white solid.
a
1
H NMR (500 MHz, CDCl
3
J
J
J
6
[
2
4
.1.18. 3-O-tert-Butyldiphenylsilyl-6-deoxy-1,2-O-isopropylidene-4-
2,3 = 9.5 Hz, H-2), 3.74 (dd, 1 H, J3,4 = 2.5 Hz, H-3), 3.68 (d, 1 H,
O-methoxymethyl- -galactopyranose (28)
a-L
4,5 = 1.5 Hz, H-4), 3.53 (m, 1 H, H-5), 1.18 (d, 3 H, J5,6 = 6.5 Hz, H-
Compound 27 (1.90 g, 4.29 mmol) was dissolved in DCE
21.0 mL), and MOMCl (3.30 mL, 42.9 mmol) and DIEA (7.40 mL,
2.9 mmol) were added at ambient temperature. The mixture
+
); HRMS (ESI) m/z: found [M + Na] 367.1516, C20
H
24
O
5
calcd for
(
4
+
M + Na] 367.1516.
was stirred at 45 °C for 3 h. The progress of the reaction was mon-
itored by TLC analysis (EtOAc/n-Hexane = 1/8). The reaction mix-
ture was quenched with MeOH at 0 °C, and then co-evaporated
with toluene. The residue was purified by column chromatography
4
.1.15. 3,4-Di-O-benzyl-6-deoxy-1,2-O-isopropylidene-
galactopyranose (25)
To a solution of compound 24 (1.83 g, 5.31 mmol) in DMP
26.6 mL) was added CSA (12.0 mg, 500 mol). The reaction mix-
a-L-
(
l
on silica gel (EtOAc/n-Hexane = 1/15) to give compound 28 (2.01 g,
ture was stirred for 4 h at ambient temperature. The progress of
the reaction was monitored by TLC analysis (EtOAc/Toluene = 1/2).
Then, triethylamine was added to the reaction mixture, which was
concentrated. The resulting residue was purified by column chro-
matography on silica gel (EtOAc/Toluene = 1/5) to give compound
1
9
6%) as an amorphous white solid. [
a
]
D
À20.3° (c 0.6, CHCl
3
);
H
NMR (500 MHz, CDCl
J
J
3
) d 7.74–7.35 (m, 10 H, Ar), 5.51 (d, 1 H,
1,2 = 4.5 Hz, H-1), 4.95–4.66 (2 d, 2 H, OCH ), 4.15 (dd, 1 H,
2,3 = 6.5 Hz, H-2), 3.93 (dd, 1 H, J3,4 = 2.5 Hz, H-3), 3.86 (m, 1 H,
), 1.24–
, H-6); C NMR (125 MHz, CDCl
d 136.4, 136.1, 133.7, 133.3, 130.0, 129.8, 127.8, 127.5, 108.5, 97.6,
2
H-5), 3.49 (t, 1 H, J4,5 = 2.5 Hz, H-4), 3.37 (s, 3 H, OCH
3
2
5 (1.76 g, 86%) as an amorphous solid. [
a]
D
À59.2° (c 1.0, CHCl
) d 7.41–7.26 (m, 10 H, Ar), 5.58 (d, 1 H,
1,2 = 4.5 Hz, H-1), 4.99–4.65 (m, 4 H, 2 ArCH ), 4.30 (dd, 1 H,
2,3 = 6.5 Hz, H-2), 4.03 (m, 1 H, H-5), 3.68–3.66 (m, 2 H, H-3, H-
), 1.45 (1 s, 3 H, (CH C), 1.39 (1 s, 3 H, (CH C), 1.25 (d, 3 H,
5,6 = 6.5 Hz, H-6); 1 C NMR (125 MHz, CDCl
) d 138.6, 138.5,
28.5, 128.4, 128.3, 128.2, 127.7, 127.6, 108.7, 97.5, 81.1, 77.7,
7.6, 75.6, 74.4, 71.7, 70.2, 28.4, 27.3, 16.7; HRMS (ESI) m/z: found
3
);
t
13
1
.10 (m, 18 H, Bu, (CH
3
)C, OCH
3
3
)
1
H NMR (500 MHz, CDCl
3
J
J
4
J
1
7
2
9
7.0, 78.2, 76.0, 75.3, 69.8, 56.2, 27.9, 27.1, 27.0, 19.4, 16.7; HRMS
+
Si calcd for [M + Na]+
(
ESI) m/z: found [M + Na] 509.2330, C27H O
38 6
3
)
2
3 2
)
5
09.2330.
3
3
4.1.19. 6-Deoxy-1,2-O-isopropylidene-4-O-methoxymethyl-a-L-
+
+
galactopyranose (29)
To a solution of compound 28 (2.0 g, 4.1 mmol) in THF (21 mL)
[
M + Na] 407.1829, C23
H
28
O
5
calcd for [M + Na] 407.1829.
n
4
was added Bu NF (1.0 M in THF, 8.2 mL, 8.2 mmol). The reaction
4
.1.16. 6-Deoxy-1,2-O-isopropylidene- -galactopyranose (26)
a-L
was stirred for 8 h at ambient temperature. The progress of the
reaction was monitored by TLC analysis (EtOAc/Toluene = 1/1).
The reaction mixture was diluted with EtOAc. The solution was
washed with water and brine, dried over Na SO and filtered off.
2 4
The filtrate was concentrated. The resulting residue was purified
by column chromatography on silica gel (EtOAc/Toluene = 1/1) to
A flask containing a suspension of compound 25 (3.30 g,
.59 mmol) and 10% Pd on activated carbon (3.30 g) in MeOH
8
(
430 mL) was evacuated under vacuum and flushed with H
2
. The
mixture was stirred for 24 h under an atmospheric H
2
pressure.
The progress of the reaction was monitored by TLC analysis
EtOAc/Toluene = 1/1). The reaction mixture was filtered through
a pad of Celite, and the residue was washed with CHCl . The com-
(
give compound 29 (1.0 g, 80%) as a colorless syrup. [
a
]
D
À57.1°
) d 5.56 (d, 1 H,
), 4.18 (m, 1 H, H-5),
.04 (dd, 1 H, J2,3 = 6.5 Hz, H-2), 3.88–3.85 (m, 1 H, H-3) 3.66 (t, 1
H, J3,4 = J4,5 = 2.5 Hz, H-4), 3.58 (d, 1 H, J3,OH = 6.5 Hz, OH), 3.47 (s,
H, OCH ), 1.54 (1 s, 3 H, (CH C), 1.39 (1 s, 3 H, (CH C), 1.30
d, 3 H, J5,6 = 6.5 Hz, H-6); C NMR (125 MHz, CDCl ) d 108.5,
8.4, 97.4, 79.3, 71.0, 68.6, 56.3, 28.1, 27.0, 16.4; HRMS (ESI) m/z:
3
1
(
3 3
c 0.5, CHCl ); H NMR (500 MHz, CDCl
bined filtrate and washings were concentrated. The resulting resi-
due was purified by column chromatography on silica gel (MeOH/
J
1,2 = 4.5 Hz, H-1), 4.84–4.63 (2 d, 2 H, OCH
2
4
CHCl
white solid. [
.57 (d, 1 H, J1,2 = 5.5 Hz, H-1), 4.14–4.07 (m, 2 H, H-2, H-5), 3.88
m, 1 H, H-3), 3.77 (s, 1 H, H-4), 2.68–2.23 (2 s, 2 H, OH), 1.52
1 s, 3 H, (CH C), 1.38 (1 s, 3 H, (CH C), 1.33 (d, 3 H,
) d 108.2, 97.5, 75.7,
3
= 1/25) to give compound 26 (1.55 g, 89%) as an amorphous
1
a
]
D
À52.5° (c 0.6, CHCl
3 3
); H NMR (500 MHz, CDCl ) d
3
(
3
3
)
2
3 2
)
5
(
(
1
3
3
9
3
)
2
3 2
)
+
+
1
3
20 6
found [M + Na] 271.1152, C11H O calcd for [M + Na] 271.1152.
J5,6 = 6.5 Hz, H-6); C NMR (125 MHz, CDCl
3
+
7
2
1.6, 69.4, 67.6, 27.8, 26.8, 16.4; HRMS (ESI) m/z: found [M + Na]
+
27.0890, C H
9 16
O
5
calcd for [M + Na] 227.0890.
4.1.20. 6-Deoxy-1,2-O-isopropylidene-4-O-methoxymethyl-
hexopyrano-3-ulose (30)
a
-
L
-xylo-
Cl
4
.1.17. 3-O-tert-Butyldiphenylsilyl-6-deoxy-1,2-O-isopropylidene-
a-l-
To a solution of compound 29 (840 mg, 3.39 mmol) in CH
(42.0 mL) were added NaHCO (1.63 g, 20.3 mmol) and Dess-Mar-
2
2
galactopyranose (27)
3
Compound 26 (2.80 g, 13.7 mmol) was dissolved in DMF
68.0 mL), and imidazole (2.30 g, 34.3 mmol) and TBDPSCl
3.60 mL, 13.7 mmol) were added at À40 °C. The mixture was stir-
tin periodinane (2.20 mg, 5.08 mmol) at 0 °C. The reaction mixture
was warmed to ambient temperature and stirred for 12 h. The pro-
gress of the reaction was monitored by TLC analysis (EtOAc/
(
(
red at À40 °C for 15 h. The progress of the reaction was monitored
by TLC analysis (EtOAc/n-Hexane = 1/5). The reaction mixture was
quenched by addition of MeOH at À40 °C and diluted with EtOAc.
Toluene = 1/8). After the mixture was diluted with Et
Na was added and the aqueous layer was extracted with
Et O. The combined organic layers were washed with brine, dried
over Na SO and filtered off. The filtrate was concentrated. The
2
O, satd aq.
2 2 3
S O
2
The solution was washed with satd aq. NaHCO
3
and brine, dried
2
4
over Na
resulting residue was purified by column chromatography on silica
gel (Et O/Toluene = 1/15) to give compound 27 (5.20 g, 86%) as an
amorphous white solid. [
500 MHz, CDCl ) d 7.74–7.37 (m, 10 H, Ar), 5.53 (d, 1 H,
2
SO
4
and filtered off. The filtrate was concentrated. The
resulting residue was purified by column chromatography on silica
gel (EtOAc/Toluene = 1/20) to give compound 30 (728 mg, 87%) as
1
2
a colorless syrup. [
CDCl
OCH
4,5 = 6.5 Hz, H-4), 3.43 (s, 3 H, OCH
1.40 (1 s, 3 H, (CH
C), 1.34 (d, 3 H, J5,6 = 6.5 Hz, H-6); 13C NMR
(125 MHz, CDCl ) d 202.7, 111.3, 99.9, 96.8, 79.3, 77.4, 70.5, 56.3,
26.7, 26.4, 15.4; HRMS (ESI) m/z: found [M + Na] 269.0995,
a]
D
3
+38.7° (c 1.0, CHCl ); H NMR (500 MHz,
1
a
]
D
À41.5° (c 0.4, CHCl
3
); H NMR
3
) d 5.75 (d, 1 H, J1,2 = 4.5 Hz, H-1), 4.79–4.69 (2 d, 2 H,
), 4.60 (m, 1 H, H-5), 4.47 (d, 1 H, H-2), 4.24 (d, 1 H,
), 1.58 (1 s, 3 H, (CH C),
(
3
2
J1,2 = 5.0 Hz, H-1), 4.04 (t, 1 H, J2,3 = 5.0 Hz, H-2), 3.89 (dd, 1 H,
J
3
3 2
)
J
3,4 = 4.0 Hz, H-3), 3.83 (m, 1 H, H-5), 3.39 (d, 1 H, H-4), 2.48 (d, 1
3 2
)
t
13
H, J4,OH = 2.5 Hz, OH), 1.33–1.12 (m, 18 H, (CH
3
)
2
C, H-6, Bu);
C
3
+
3
NMR (125 MHz, CDCl ) d 136.2, 136.0, 135.8, 133.5, 132.8, 130.2,
+
1
30.1, 128.0, 127.8, 107.4, 97.6, 75.6, 74.0, 70.0, 67.4, 27.7, 27.3,
11 18 6
C H O calcd for [M + Na] 269.0996.
Please cite this article in press as: Shimabukuro J., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.021

J. Shimabukuro et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
9
4
.1.21. 6-Deoxy-1,2-O-isopropylidene-4-O-methoxymethyl-
gulopyranose (31)
A solution of compound 30 (1.00 g, 4.06 mmol) in MeOH
40.6 mL) was cooled to 0 °C, and NaBH (169 mg, 4.47 mmol)
was added. The reaction mixture was stirred for 5 min. The pro-
gress of the reaction was monitored by TLC analysis (Et O/
CHCl = 1/20). The reaction mixture was quenched with satd aq.
NH Cl and diluted with EtOAc (80.0 mL). The solution was washed
with water and brine, dried over Na SO and filtered off. The fil-
trate was concentrated. The resulting residue was purified by col-
umn chromatography on silica gel (Et O/CHCl = 1/20) to give
compound 31 (485 mg, 48%) as an amorphous white solid. [
a
-
L
-
NMR (500 MHz, CDCl
4.67 (2 d, 2 H, OCH ), 4.36 (dd, 1 H, J2,3 = 7.0 Hz, H-2), 4.18 (m, 1
H, H-5), 3.88 (t, 1 H, J3,4 = 3.5 Hz, H-4), 3.44 (s, 3 H, OCH ), 3.12
(dd, 1 H, H-3), 2.15 (s, 3 H, SeCH ), 1.53 (1 s, 3 H, (CH C), 1.38
(1 s, 3 H, (CH C), 1.28 (d, 3 H, J5,6 = 6.5 Hz, H-6); C NMR
(125 MHz, CDCl ) d 108.2, 97.9, 92.3, 76.5, 76.0, 69.2, 56.6, 43.8,
28.0, 26.8, 16.9, 5.4; Se NMR (94 MHz, CDCl
3
) d 5.53 (d, 1 H, J1,2 = 4.5 Hz, H-1), 4.86–
2
3
(
4
3
3 2
)
1
3
3 2
)
2
3
7
7
3
3
) d 138.7; HRMS
Se calcd for [M
+
4
(ESI) m/z: found [M + Na] 349.0525, C12
+ Na] 349.0525.
H
22
O
5
+
2
4
2
3
4.1.24. Methyl 3,6-dideoxy-3-methylseleno-
Trifluoroacetic acid (11 mL) was added to a suspension of com-
pound 34 (30 mg, 90 mol) in H O (2.8 mL) at ambient tempera-
a-L-galactopyranoside (3)
a]
D
1
À20.3° (c 1.0, CHCl
3
); H NMR (500 MHz, CDCl
1,2 = 5.0 Hz, H-1), 4.80–4.65 (2 d, 2 H, OCH ), 4.46 (dd, 1 H,
2,3 = 3.0 Hz, H-2), 4.31 (m, 1 H, H-5), 4.13 (bs, 1 H, OH) 3.79 (dd,
H, J3,4 = 6.5 Hz, H-3), 3.56 (t, 1 H, J4,5 = 6.0 Hz, H-4), 3.47 (s, 3 H,
OCH ), 1.57 (1 s, 6 H, (CH C), 1.38 (1 s, 6 H, (CH C), 1.22 (d, 3
H, J5,6 = 6.5 Hz, H-6); 13C NMR (125 MHz, CDCl
) d 110.0, 98.4,
7.7, 83.1, 74.6, 71.2, 66.6, 56.0, 26.1, 25.3, 15.1; HRMS (ESI) m/z:
3
) d 5.50 (d, 1 H,
l
2
J
J
1
2
ture, and then the mixture was stirred for 19.5 h. The completion
of the reaction was confirmed by TLC analysis (MeOH/
CHCl = 1/10), and then the reaction was quenched with triethy-
3
lamine. The reaction mixture was co-evaporated with toluene.
The residue was exposed to high vacuum for 24 h, and dissolved
3
3
)
2
3
)
2
3
9
in MeOH (550
acid (16 L). The mixture was stirred for 5 h at ambient tempera-
ture. The completion of the reaction was confirmed by TLC analysis
(MeOH/CHCl = 1/100), and then the reaction mixture was neutral-
lL). To the stirred solution was added 95% sulfonic
+
+
found [M + Na] 271.1153, C11
H
20
O
6
calcd for [M + Na] 271.1152.
l
4
.1.22. 3,6-Dideoxy-1,2-O-isopropylidene-4-O-methoxymethyl-3-(4-
methylbenzoylseleno)- -galactopyranose (33)
To a solution of compound 31 (75.0 mg, 302
1.50 mL) were added pyridine (260 L, 1.03 mmol) and trifluo-
romethanesulfonic anhydride (92.0
3
a-L
ized with 1 M NaOH aq. and co-evaporated with toluene. The resi-
due was purified by column chromatography on silica gel (MeOH/
2 2
lmol) in CH Cl
(
l
CHCl
white solid. [
d 4.77 (d, 1 H, J1,2 = 4.0 Hz, H-1), 3.97 (m, 1 H, H-5), 3.85 (m, 1 H,
H-2), 3.76 (s, 1 H, H-4), 3.46 (s, 3 H, OCH ), 3.24 (dd, 1 H,
2,3 = 11.5 Hz, J3,4 = 2.5 Hz, H-3), 2.42–2.14 (2 d, 2 H, OH), 2.07 (s,
3
= 1/300) to give compound 3 (3.2 mg, 9%) as an amorphous
1
lL, 1.03 mmol) at À20 °C, and
a
]
D
À32.5° (c 0.1, CHCl
3 3
); H NMR (500 MHz, CDCl )
the mixture was stirred for 150 min at ambient temperature. The
progress of the reaction was monitored by TLC analysis (EtOAc/n-
Hexane = 1/4). The reaction mixture was diluted with CH
washed with 2 M HCl aq., H O, satd aq. NaHCO and brine, dried
over Na SO and filtered off. The filtrate was concentrated. The
3
2
Cl
2
, and
J
1
3
2
3
3
3 H, SeCH ), 1.31 (d, 3 H, J5,6 = 6.5 Hz, H-6); C NMR (500 MHz,
77
2
4
CDCl
(94 MHz, CDCl
Se calcd for [M + Na] 279.0106.
3
) d 99.1, 71.0, 67.7, 66.2, 55.3, 48.6, 16.7, 3.3; Se NMR
+
residue was exposed to high vacuum for 3 h.
3
) d HRMS (ESI) m/z: found [M + Na] 279.0106,
+
The dried residue was dissolved in degassed DMF (500
l
L),
8 16 4
C H O
which was then added dropwise to a solution of potassium 4-
methylselenobenzoate 19 (215 mg, 906 mol) in degassed DMF
1.00 mL) as bubbled with argon gas. The mixture was stirred for
l
4.1.25. Methyl 2-O-acetyl-3-O-benzoyl-4-bromo-4,6-dideoxy-
galactopyranoside (36)
a-L-
(
3
h at ambient temperature. The completion of the reaction was
Compound 35 (490 mg, 1.40 mmol) was dissolved in pyridine
confirmed by TLC analysis (EtOAc/Toluene = 1/10), and then the
reaction mixture was diluted with EtOAc. The solution was washed
(2.40 mL) and THF (2.40 mL). To the solution were added 4-DMAP
(1.20 mg, 10.0 lmol) and Ac O (286 lL, 2.84 mmol) at ambient
2
with water and brine, dried over Na
trate was concentrated. The resulting residue was purified by col-
umn chromatography on silica gel (EtOAc/toluene = 1/50) to give
2
SO
4
and filtered off. The fil-
temperature. The solution was stirred for 3 h. The completion of
the reaction was confirmed by TLC analysis (EtOAc/n-Hex-
ane = 1/5), and then the reaction was quenched with methanol at
0 °C. The reaction mixture was co-evaporated with toluene. The
compound 33 (97.0 mg, 75%) as a yellow syrup. [
a]
D
À10.0° (c
) d 7.82–7.24 (2 d, 4 H, Ar),
.56 (d, 1 H, J1,2 = 4.5 Hz, H-1), 4.75–4.61 (2 d, 2 H, OCH ), 4.42–
.33 (m, 3 H, H-2, H-4, H-5), 4.08 (t, 1 H, J2,3 = J3,4 = 4.0 Hz, H-3),
.40 (s, 3 H, OCH ), 2.40 (s, 3 H, ArCH ), 1.56 (1 s, 3 H, (CH C),
.35 (1 s, 3 H, (CH C), 1.29 (d, 3 H, J5,6 = 6.5 Hz, H-6); C NMR
) d 193.7, 145.0, 136.2, 129.6, 127.5, 108.8, 97.8,
1
0
5
4
3
1
.5, CHCl
3
); H NMR (500 MHz, CDCl
3
residue was diluted with EtOAc, washed with 2 M HCl aq., H
2
O,
2
satd aq. NaHCO and brine, dried over Na SO and filtered off.
3
2
4
The filtrate was concentrated. The resulting residue was purified
by column chromatography on silica gel (EtOAc/Toluene = 1/5) to
3
3
3 2
)
1
3
3
)
2
give compound 36 (523 mg, 95%) as a yellow syrup. [
(c 2.2, CHCl ); H NMR (500 MHz, CHCl
3 3
a
]
D
À53.4°
) d 7.82–7.24 (m, 5 H,
Ar), 5.83 (dd, 1 H, J2,3 = 10.0 Hz, J3,4 = 10.5 Hz, H-3), 5.00 (dd, 1 H,
1,2 = 3.5 Hz, H-2), 4.95 (d, 1 H, H-1), 4.13 (m, 1 H, H-5), 3.76 (t, 1
H, J4,5 = 10.5 Hz, H-4), 3.45 (s, 3 H, OCH ), 1.96 (s, 3 H, Ac), 1.46
d, 3 H, J5,6 = 6.5 Hz, H-6); C NMR (125 MHz, CDCl ) d 170.3,
165.2, 133.3, 129.8, 129.4, 128.4, 97.0, 72.0, 72.0, 55.4, 52.7, 20.7,
1
(
125 MHz, CDCl
3
7
7
9
7.3, 74.5, 74.4, 67.7, 56.5, 44.1, 27.2, 26.5, 21.9, 16.6; Se NMR
+
(
94 MHz, CDCl
3
) d 539.5; HRMS (ESI) m/z: found [M + Na]
J
+
4
53.0787, C19
H
26
O
6
Se calcd for [M + Na] 453.0787.
3
1
3
(
3
4
.1.23. 3,6-Dideoxy-1,2-O-isopropylidene-4-O-methoxymethyl-3-
-galactopyranose (34)
To a solution of compound 33 (200 mg, 470
DMF (3.2 mL) were added Cs CO (300 mg, 930
hydrazine (32 mg, 700 mol) and methyl iodide (58
7
7
methyseleno-
a-
L
19.1; Se NMR (94 MHz, CDCl
3
) d 464.1; HRMS (ESI) m/z: found
calcd for [M + Na] 367.0152.
+
+
l
l
mol) in degassed
mol), N-methyl-
L, 930 mol)
[M + Na] 367.0152, C14
H17BrO
5
2
3
l
l
l
4.1.26. Methyl 2-O-acetyl-3-O-benzoyl-4,6-dideoxy-4-(4-methyl-
benzoylseleno)- -galactopyranoside (37)
at ambient temperature, and the mixture was stirred for 2 h. The
progress of the reaction was monitored by TLC analysis (EtOAc/n-
Hexane = 1/4). The reaction mixture was diluted with EtOAc, and
a-L
Compound 36 (60 mg, 0.16 mmol) was dissolved in degassed
DMF (1.6 mL) at ambient temperature. To the stirred solution
was added potassium p-methylselenobenzoate 19 (110 mg,
the resulting solution was washed with satd aq. NaHCO
3
and brine,
dried over Na SO and filtered off. The filtrate was concentrated.
2
4
470 lmol). The reaction mixture was warmed to 60 °C and stirred
The resulting residue was purified with column chromatography
for 7 h. The progress of the reaction was monitored by TLC analysis
(EtOAc/n-Hexane = 1/10). The reaction mixture was co-evaporated
with toluene, and then the residue was diluted with EtOAc. The
on silica gel (EtOAc/n-Hexane = 1/6) to give compound 34
(
67 mg, 45%) as a yellow syrup. [
a]
D
À10.3° (c 1.0, CHCl
); 1
H
3
Please cite this article in press as: Shimabukuro J., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.021

1
0
J. Shimabukuro et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
solution was washed with water and brine, dried over Na
filtered off. The resulting residue was purified by column chro-
matography on silica gel (EtOAc/n-Hexane = 1/10) to give com-
2
SO
4
and
were indexed, then integrated using X-ray diffraction and spec-
2
7,28
troscopy.
less programs in the CCP4 suite.
Scaling was performed using the Pointless and Aim-
2
9,30
Structural calculation was
3
1
pound 37 (58 mg, 87%) as a yellow syrup. [
a
]
D
À92.3° (c 2.2,
) d 7.95–7.21 (m, 9 H, Ar), 5.79
3,4 = 4.5 Hz, H-3), 5.11 (dd, H,
1,2 = 4.0 Hz, H-2) 4.94 (d, 1 H, H-1), 4.53 (dd, 1 H, J4,5 = 1.0 Hz, H-
mainly performed using the Phenix software suite. The selenium
sites and initial phase angles were determined by Phenix
(Autosol). Eighty-six selenium sites were found and had a figure
of merit (FOM) of 0.286. After subjecting the data to Phenix (Auto-
sol), a clear electron density map and an initial model were con-
1
CHCl
dd,
3
); H NMR (500 MHz, CHCl
H, 2,3 = 11.0 Hz,
3
3
2
(
1
J
J
1
J
4
2
), 4.49 (m, 1 H, H-5), 3.43 (s, 3 H, OCH
.01 (s, 3 H, Ac), 1.33 (d, 3 H, J5,6 = 6.5 Hz, H-6);
) d 170.5, 165.3, 144.7, 136.1, 132.8, 129.7,
3
), 2.38 (s, 3 H, ArCH
3
),
1
3
33
C NMR
structed using Phenix (Autobuild) and the model had an FOM
(
125 MHz, CDCl
3
of 0.779. Further manual refinement was performed using Coot34
after rigid body and simulated annealing refinement with Phenix
1
2
29.6, 129.3, 128.3, 128.2, 127.3, 97.5, 71.0, 69.4, 65.0, 55.4, 52.3,
1.7, 20.8, 19.2; 7 Se NMR (94 MHz, CDCl
7
(Refine). The final restrained refinement was performed using
35
3
) d 465.9; HRMS (ESI)
+
+
36
m/z: found [M + Na] 529.0737, C24
H
26
O
7
Se calcd for [M + Na]
Refmac with automatically optimized weight. The data collection
5
29.0736.
and refinement statistics are summarized in Supporting Informa-
tion Table S1. The coordinates of AOL co-crystallized with 2-
MeSe-Fuc 2 have been deposited under the accession code 5H47.
4.1.27. Methyl 3-O-benzoyl-4,6-dideoxy-4-methylseleno-a-L-
galactopyranoside (38)
To a solution of compound 37 (80.0 mg, 186
l
mol) in degassed
mol), N-methyl-
mol) and methyliodide (23.3 L,
mol) at ambient temperature, and the mixture was stirred
for 1 h. The progress of the reaction was monitored by TLC analysis
EtOAc/Toluene = 1/1). The reaction mixture was extracted with
EtOAc twice, and the combined organic layer was washed with
satd aq. NaHCO and brine, dried over Na SO and filtered off.
4.3. Surface plasmon resonance (SPR) analysis of AOL-Se-Fuc binding
DMF (3.70 mL) were added Cs
hydrazine (14.6 L, 279
72
2 3
CO (121 mg, 372 l
l
l
l
All SPR experiments were performed with a Biacore T200 (GE
Healthcare) at 25 °C. A research grade CM5 sensor chip was acti-
vated with an EDC/NHS solution (1-ethyl-3-(3-dimethylamino-
propyl) carbodiimide hydrochloride, N-hydroxysuccinimide) for
3
l
(
10 min, and 2.4
(10 mM HEPES, 150 mM NaCl, pH 6.5) was injected at a flow rate
of 10 L/min to immobilize the AOL on the sensor chip. The unre-
lL of 5 mg/mL AOL in 10 mM HBS-N buffer
3
2
4
The filtrate was concentrated. The resulting residue was purified
by column chromatography on silica gel (EtOAc/n-Hexane = 1/16)
l
acted species on the sensor surface were blocked using 1 M etha-
nolamine. The blank channel was treated identically except that
AOL was not injected.
to give compound 38 (39.6 mg, 53%) as a yellow syrup. [
À56.7° (c 3.1, CHCl ); 8.07–7.45 (m, 5 H, Ar), 5.54–5.46 (m, 2 H,
H-2, H-3), 4.91 (d, 1 H, J1,2 = 3.5 Hz, H-1), 4.27 (m, 1 H, H-5), 3.45
a]
D
3
A serial dilution series of carbohydrate solutions (150
centrations of 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500 and
1000 M) in running buffer (10 mM HEPES, 150 mM NaCl, pH
7.4) were injected for 2 min at 30 L/min, then dissociation was
lL, con-
(
dd, 1 H, J3,4 = 4.0 Hz, J4,5 = 2.0 Hz, H-4), 3.40 (s, 3 H, OCH
s, 3 H, Ac), 1.89 (s, 3 H, SeCH ) 1.26 (d, 3 H, J5,6 = 6.5 Hz, H-6);
) d 170.4, 165.8, 133.4, 129.7, 129.5,
3
), 2.03
(
3
l
1
3
C NMR (125 MHz, CDCl
28.6, 97.5, 72.0, 69.9, 65.3, 55.3, 51.5, 20.9, 19.6, 6.0; Se NMR
3
l
77
1
monitored for the following 20 min. The equilibrium response
(after subtracting the response of the reference surface) in each
experiment was used to calculate the dissociation constant. The
results were analyzed using the Biacore T-200 evaluation software
(GE Healthcare). Sensorgram curves were curve-fitted to a 1:1
steady-state equilibrium binding model.
+
(
3
94 MHz, CDCl ) d 37.6; HRMS (ESI) m/z: found [M + Na]
+
4
22 6
25.0474, C17H O Se calcd for [M + Na] 425.0474.
4
.1.28. Methyl 4,6-dideoxy-4-methylseleno-
Sodium methoxide (28% in MeOH, 2.00 mg, 10.0
added to a solution of compound 38 (17.6 mg, 40.0 mol) in MeOH
200 L). The reaction mixture was stirred for 10 min at ambient
a
-L
-galactopyranoside (4)
l
mol) was
l
(
l
Acknowledgement
temperature. The progress of the reaction was monitored by TLC
analysis (EtOAc/n-Hexane = 1/1). Then, the reaction mixture was
concentrated. The residue was purified by column chromatography
on silica gel (MeOH/Toluene = 1/8) and Sephadex LH-20 (MeOH/
The iCeMS is supported by World Premier International
Research Center Initiative (WPI), Ministry of Education, Culture,
Sports, Science, and Technology (MEXT), Japan. This work was
financially supported in part by MEXT, Japan (Grants-in-Aid for Sci-
entific Research (B) No. 15H04495 to H.A., and Grant-in-Aid on
Innovative Areas No. 26110704, Deciphering sugar chain-based
signals regulating integrative neuronal functions to H.A.). We also
thank Kiyoko Ito for technical assistance.
2
H O = 4/1) to give compound 4 (10.2 mg, 96%) as an amorphous
1
white solid. [
a]
D
À47.2° (c 1.1, CHCl
3 3
); H NMR (500 MHz, CDCl )
d 4.72 (d, 1 H, J1,2 = 4.0 Hz, H-1), 4.10 (m, 1 H, H-5), 3.81 (dd, 1 H,
J2,3 = 10.0 Hz, H-2), 3.48 (dd 1 H, J3,4 = 3.0 Hz, H-3), 3.11 (dd, 1 H,
J
4,5 = 1.5 Hz, H-4), 2.07 (s, 3 H, SeCH ), 1.26 (d, 3 H, J5,6 = 6.5 Hz,
) d 99.4, 72.1, 71.1, 65.9, 57.8,
5.4, 19.6, 7.1; Se NMR (94 MHz, CDCl ) d 3.7; HRMS (ESI) m/z:
3
1
3
H-6); C NMR (125 MHz, CDCl
5
3
7
7
3
Supplementary material
+
+
found [M + Na] 279.0106, C
.2. Co-crystallization and X-ray crystallography
Purified AOL was purchased from Tokyo Chemical Industry.
8 16 4
H O Se calcd for [M + Na] 279.0106.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.12.021.
4
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Please cite this article in press as: Shimabukuro J., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.12.021