Synthesis and evaluation of a radioiodinated trisaccharide derivative as a synthetic substrate for a sensitive N-acetylglucosaminyltransferase v radioassay

Article abstract of DOI:10.1016/j.bmc.2011.05.047

N-acetylglucosaminyltransferase V (GnT-V) is one of the most relevant glycosyltransferases to tumor invasion and metastasis. Based on previous findings of molecular recognition between GnT-V and synthetic substrates, we designed and synthesized a p-iodophenyl-derivatized trisaccharide, 2-(4-iodophenyl)ethyl 6-O-[2-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)- α-d-mannopyranosyl]-β-d-glucopyranoside (IPGMG, 1) and its radiolabeled form, [¹²⁵I]IPGMG ([¹²⁵I]1), for use in assays of GnT-V activity in vitro. The tributyltin derivative, 2-[4-(n-tributylstannyl)phenyl]ethyl 6-O-[2-O-(3,4,6-tri-O-acetyl-2-acetamido-2- deoxy-β-d-glucopyranosyl)-3,4,6-tri-O-acetyl-α-d-mannopyranosyl]-2,3, 4-tri-O-acetyl-β-d-glucopyranoside (21), was synthesized as a precursor for the preparation of [¹²⁵I]1. The iododestannylation of 21 using hydrogen peroxide as an oxidant followed by deacetylation yielded [ ¹²⁵I]1. When [¹²⁵I]1 was incubated in GnT-V-expressing cells with a UDP-GlcNAc donor, the production of β1-6GlcNAc-bearing IPGMG (IPGGMG, 2) was confirmed by radio-HPLC. In kinetic analysis, 1 was found to be a good substrate with a K_m of 23.7 μM and a V_max of 159 pmol/h. μg protein. [¹²⁵I]1 would therefore be a useful synthetic substrate for the quantitative determination of GnT-V activity.

Full text of DOI:10.1016/j.bmc.2011.05.047

Bioorganic & Medicinal Chemistry 19 (2011) 4312–4321
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of a radioiodinated trisaccharide derivative
as a synthetic substrate for a sensitive N-acetylglucosaminyltransferase
V radioassay
Takahiro Mukai a, Masayori Hagimori b, Kenji Arimitsu c, Takahiro Katoh c, Misa Ukon b, Tetsuya Kajimoto c,
Hiroyuki Kimura b, Yasuhiro Magata d, Eiji Miyoshi e, Naoyuki Taniguchi f, Manabu Node c, Hideo Saji b,
⇑
a Department of Biophysical Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan
b Department of Patho-functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
c Department of Pharmaceutical Manufacturing Chemistry, Kyoto Pharmaceutical University, Misasagi Nakauchi-cho, Yamashina-ku, Kyoto 607-8414, Japan
d Laboratory of Genome Bio-Photonics, Photon Medical Research Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan
e Department of Molecular Biochemistry & Clinical Investigation, Osaka University Graduate School of Medicine, 1-7 Yamadaoka, Suita 565-0871, Japan
f System Glycobiology Research Group Advanced Science Institute, RIKEN 2-1 Hirosawa, Wako 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
N-acetylglucosaminyltransferase V (GnT-V) is one of the most relevant glycosyltransferases to tumor inva-
sion and metastasis. Based on previous findings of molecular recognition between GnT-V and synthetic sub-
strates, we designed and synthesized a p-iodophenyl-derivatized trisaccharide, 2-(4-iodophenyl)ethyl 6-O-
Received 26 March 2011
Revised 21 May 2011
Accepted 23 May 2011
Available online 27 May 2011
[2-O-(2-acetamido-2-deoxy-b-
and its radiolabeled form, [125I]IPGMG ([125I]1), for use in assays of GnT-V activity in vitro. The tributyltin
derivative, 2-[4-(n-tributylstannyl)phenyl]ethyl 6-O-[2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b-
D-glucopyranosyl)-a-D-mannopyranosyl]-b-D-glucopyranoside (IPGMG, 1)
D-
Keywords:
glucopyranosyl)-3,4,6-tri-O-acetyl-
a
-
D
-mannopyranosyl]-2,3,4-tri-O-acetyl-b-
D
-glucopyranoside
(21),
N-acetylglucosaminyltransferase V
Radiolabeled substrate
Radioassay
was synthesized as a precursor for the preparation of [125I]1. The iododestannylation of 21 using hydrogen
peroxide as an oxidant followed by deacetylation yielded [125I]1. When [125I]1 was incubated in GnT-V-
expressing cells with a UDP-GlcNAc donor, the production of b1–6GlcNAc-bearing IPGMG (IPGGMG, 2)
was confirmed by radio-HPLC. In kinetic analysis, 1 was found to be a good substrate with a Km of
23.7
the quantitative determination of GnT-V activity.
lM and a Vmax of 159 pmol/h. l
g protein. [125I]1 would therefore be a useful synthetic substrate for
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
a1–6 side chain of the trimannosyl core of N-glycans, to form a
GlcNAcb1–6Man linkage. This activity has been assessed predomi
nantly using radiolabeled donors or fluorescent-labeled acceptors.
In the former case, the production of sugar chains, the non-reducing
ends of which are modified with radiolabeled GlcNAc by the enzy-
matic reaction, is measured through the use of a UDP-[3H]GlcNAc
or UDP-[14C]GlcNAc donor.2,11,12 However, there is a problem with
specificity since UDP-GlcNAc functions as a substrate for many gly-
cosyltransferases. In the latter case, a GnT-specific acceptor, Glc-
The sugar chains of glycoproteins on cell surfaces, which are con-
structed by glycosyltransferases and glycosidases, change during
particular biological events including development, carcinogenesis,
and malignant transformation. Numerous studies conducted in the
last two decades have demonstrated that N-acetylglucosaminyl-
transferase V (GnT-V) is relevant to tumor invasion and metastasis.
Since Cummings first reported its expression in mouse lymphoma
cells in 1982,1 GnT-V has been shown to be highly expressed in
various cancer cell lines and tissues from the early stages of tumor-
igenesis.2–8 In addition, studies using GnT-V-deficient mice have di-
rectly demonstrated an essential role of GnT-V in tumor growth and
metastasis.9,10
NAcb1-2Mana1-3-(GlcNAcb1-2Mana1-6)Manb1-4GlcNAcb1-4Glc
NAc, is labeled with 2-aminopyridine to yield a fluorescent substrate
(Gn,Gn-bi-PA). This acceptor is obtained from the carbohydrate moi-
ety of human transferrin through laborious and time-consuming
processes, repeated digestion with various enzymes, chemical mod-
ifications, and purification.13–15 Moreover, the PA-oligosaccharide
could still be a substrate of GnT-III.
GnT-V catalyzes the transfer of an N-acetylglucosamine (GlcNAc)
residue from a donor, UDP-GlcNAc, to an acceptor, the mannose
Thus, we planned and produced a GnT-V-specific radioiodinated
substrate for use in assays of GnT-V activity in vitro. First, to ensure
⇑
Corresponding author. Tel.: +81 75 753 4566; fax: +81 75 753 4568.
E-mail address: hsaji@pharm.kyoto-u.ac.jp (H. Saji).
a high degree of specificity, GlcNAcb1-2Mana
1-6Glc12,16–18 was
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.047

T. Mukai et al. / Bioorg. Med. Chem. 19 (2011) 4312–4321
4313
employed as the saccharide moiety on the basis of previous find-
ings; (i) it could not be a substrate of GnT-I and GnT-IV because
of the lack of an acceptor site for GnT-I and GnT-IV, that is, a Man
group that afforded 12, anomerical deacetylation of 12 with piper-
idine, and reaction with CCl3CN in the presence of DBU that affor-
ded the imidate 13. (Scheme 3).
residue linked to the bisecting Man with an
a
1–3 linkage, (ii) it is
Glycosylation of 2-(4-bromophenyl)ethyl alcohol with imidate
13 led to 2-(4-bromophenyl)ethyl 2,3,4-tri-O-acetyl-6-O-allyloxy-
carbonyl-b-D-glucopyranoside (14) (72%), which was subjected to
palladium-catalyzed deallylation to afford 15 (94%). 15 was reacted
with ethyl vinyl ether in the presence of a catalytic amount of pyrid-
inium p-toluenesulfonate to form 16 (99%). At this point, replace-
ment of the bromo group with an iodo group was investigated
before developing the route to 1 and [125I]1. The intermediate,
2-[4-(tributylstannyl)phenyl]ethyl 2,3,4-tri-O-acetyl-6-O-(1-eth-
a poor substrate of GnT-III because of the replacement of the accep-
tor residue, that is, bisecting Man, with Glc, and (iii) it already car-
ries an GlcNAc residue that should be transferred by GnT-II. Second,
the 1-hydroxyl of the reducing terminal Glc, which was adopted as
a substitute of the bisecting Man, was conjugated with a hydropho-
bic phenethyl group without loss of activity.11,12,16–18 Finally, the
radioiodine 125I was considered to be incorporated into the para-
position of the phenyl ring immediately before the biological assay
because the para-substitution of iodine is effective in preventing
deiodination in vivo.19 The p-iodophenyl-derivatized trisaccharide,
oxyethyl)-b-
bromo-to-tributyltin exchange reaction catalyzed by Pd(0)
(47%). The tributyltin derivative 17 was readily converted to 2-(4-
iodophenyl)ethyl 2,3,4-tri-O-acetyl-6-O-(1-ethoxyethyl)-b-
D-glucopyranoside (17), was prepared from 16 using
a
2-(4-iodophenyl)ethyl 6-O-[2-O-(2-acetamido-2-deoxy-b-
D-gluco-
pyranosyl)- -mannopyranosyl]-b- -glucopyranoside, was ex-
a
-D
D
D-
pected to behave as a substrate for in vitro assays of GnT-V activity.
Herein, we report synthesis of an unlabeled IPGMG (1), a 125I-la-
beled IPGMG ([125I]1), and the N-acetylglucosaminylated product
b1-6GlcNAc-bearing IPGMG (IPGGMG, 2). The reaction kinetics of
glucopyranoside (18) by reaction with iodine (94%). Treatment of
17 and 18 with pyridinium p-toluenesulfonate in methanol yielded
19 (95%) and 4 (98%), respectively (Scheme 3).
The treatment of glycosyl donor 3 with glycosyl acceptor 4 in the
presence of silver carbonate and silver perchlorate in dichlorometh-
ane and toluene yielded 20 (67%), which was deacetylated to fur-
nish the target compound 1 (59%) (Scheme 4). Next, synthesis of
the precursor of [125I]1, 22, was attempted from which the tributyl-
tin group was expected to be facilely replaced with a radioactive
iodo group by iododestannylation to yield [125I]1. However, deacet-
ylation of 21, which was obtained by the condensation of 3 and 19
in the presence of silver perchlorate and collidine (43%), did not
yield 22. Therefore, the tributyltin derivative 21 was transformed
into the fully protected and radiolabeled trisaccharide [125I]20 by
an iododestannylation reaction using hydrogen peroxide as the oxi-
dant. Conventional deacetylation of [125I]20 afforded the desired
radioiodinated compound [125I]1 (Scheme 4). It was anticipated
that no-carrier-added preparation would result in a final product
bearing a theoretical specific activity similar to that of 125I. The
radiochemical identity of [125I]1 was verified by co-injection with
nonradioactive 1 and comparison of their HPLC profiles. The final
radioiodinated compound [125I]1 showed a single peak of radioac-
tivity at the same retention time as that of nonradioactive 1. The
radioiodinated product was obtained in 50% radiochemical yield
with a radiochemical purity of >95% after HPLC.
[
125I]1 were evaluated in GnT-V-expressing cells and its recogni-
tion by GnT-V was compared with that of Gn,Gn-bi-PA. From these
results, the usefulness of [125I]1 for the GnT-V assay was assessed
(Fig. 1).
2. Results and discussion
2.1. Chemistry
For the synthesis of 1 and [125I]1, glycosylation of 3,4,6-tri-O-
acetyl-2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b-
D-glucopyr-
anosyl)- -mannopyranosyl bromide (3) and 2-(4-iodophenyl)
a-D
ethyl-2,3,4-tri-O-acetyl-b-D-glucopyranoside (4) was used as a key
reaction (Scheme 1).
Synthesis of the bromide 3 began with the condensation, which
was promoted by silver trifluoromethanesulfonate and collidine, of
benzyl 3-O-benzyl-4,6-O-benzylidene-
and 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
a-D
-mannopyranoside (5)20
D
-glucopyranosyl
bromide (6)21 to afford disaccharide 7 (57%). Subsequent treatment
of 7 with hydrazine hydrate followed by acetylation yielded 8 (80%,
two steps). Debenzylidenation of 8 in 70% AcOH and subsequent
acetylation yielded intermediate 9 (80%, two steps), whose hydrog-
enolysis and acetylation afforded octaacetate 10 (86%, two steps).
Subsequent treatment of 10 with 30% HBr–AcOH afforded 3, which
was used as the glycosyl donor for the next reaction (Scheme 2).
Synthesis of glycosyl acceptor 4 was achieved by the coupling of
2-(4-bromophenyl)ethyl alcohol and 2,3,4-tri-O-acetyl-6-O-allyl-
Finally, 2-(4-iodophenyl)ethyl 6-O-[2,6-di-O-(2-acetamido-2-
deoxy-b-D-glucopyranosyl)-a-D-mannopyranosyl]-b-D-glucopy-
ranoside (IPGGMG, 2), the N-acetylglucosaminylated product of 1,
was synthesized starting with removal of the benzyliden group of 7
with 70% AcOH to afford 23 (83%). Selective silylation of 6-OH of
the mannose unit with tert-butyldimethylsilyl chloride yielded
24 (94%), the remaining hydroxyl group of which was acetylated
to afford 25 (99%). Treatment of 25 with pyridinium p-toluenesul-
fonate afforded 26 (75%), whose glycosylation with the bromide 6
oxycarbonyl-
pared from 1,2,3,4-tetra-O-acetyl-b-
a
-
D
-glucopyranosyl trichloroacetimidate (13) pre-
D
-glucopyranose (11)22 in
three steps: protection of the 6-OH group with an allyloxycarbonyl
OH
HO
OH
HO
HO
AcHN
O
OH
OH
OH
HO
HO
O
OH
HO
O
O
O
GnT-V
O
HO
O
O
HO
NHAc
O
O
NHAc
O
O
125I
HO
HO
125I
UDP-GlcNAc
UDP
HO
HO
O
O
OH
125I]1
OH
[
[
125I]2
Figure 1. Glycosylation of [125I]1 by GnT-V.

4314
T. Mukai et al. / Bioorg. Med. Chem. 19 (2011) 4312–4321
AcO
AcO
HO
HO
Br
O
O
OH
O
OAc
OAc
HO
O
O
OH
AcO
O
NHAc
OAc
HO
Glycosylation
3
NHAc
O
O
+
I
HO
HO
O
O
HO
AcO
O
OAc
I
OH
OAc
1
4
Scheme 1. Synthesis of trisaccharide 1.
AcO
O
Br
O
OBn
OH
AcO
AcO
BnO
O
O
AcO
AcO
a
b, c
O
O
O
+
NPhth
Ph
O
57 %
80 %
O
Ph
(2 steps)
OAc
6
OBn
NPht
OBn
5
7
AcO
AcO
AcO
BnO
O
e, c
AcO
AcO
BnO
O
d, c
O
O
O
OAc
80 %
(2 steps)
86 %
(2 steps)
AcO
O
O
Ph
O
OAc
NHAc
OBn
NHAc
Br
OBn
8
9
AcO
AcO
AcO
AcO
O
AcO
AcO
AcO
O
O
f
O
OAc
O
OAc
OAc
quant.
O
OAc
NHAc
OAc
NHAc
OAc
3
10
Scheme 2. Synthesis of disaccharide 3. Reagents and condition: (a) AgOTf, collidine, CH3NO2, (b) NH2NH2Á H2O, EtOH, (c) Ac2O, pyridine, (d) 70% AcOH, (e) 10% Pd-C, H2, EtOH,
(f) 30% HBr-AcOH.
promoted by silver trifluoromethanesulfonate and collidine
yielded 27 (40%). Removal of the N-phthalimido groups with
hydrazine hydrate and subsequent acetylation yielded 28 (93%,
two steps). Cleavage of the benzyl groups of 28 by hydrogenolysis
followed by acetylation yielded 29 (72%, two steps). Treatment of
29 in acetic anhydride with HBr yielded bromide 30. Glycosylation
of 4 with bromide 30 promoted by silver carbonate and silver per-
chlorate yielded 31 (7%, two steps), which was sequentially deacet-
ylated to furnish the target compound 2 (Scheme 5).
concentrations (Fig. 3A). The data satisfied the Michaelis–Menten
equation, originally developed for enzymatic kinetics. The kinetic
parameters were Km = 23.7
l
M and Vmax = 159 pmol/hÁ
lg protein,
determined from the double-reciprocal Lineweaver–Burk plot (Fig-
ure 3B). The Km value is one order of magnitude lower than that for
a fluorescent substrate, Gn,Gn-bi-PA.
The novel radioiodinated substrate [125I]1 was used to assay
GnT-V activity in crude extracts of various tissues in normal rats.
To compare the findings with previous results obtained using
Gn,Gn-bi-PA as a substrate, we assayed the GnT-V activity under
similar experimental conditions.13 The results are summarized in
Table 1. This enzyme was uniformly distributed in rat tissues with
relatively high activity levels in the small intestine and serum. A
similar tendency was observed in a previous investigation using
Gn,Gn-bi-PA.13 The reaction velocity using [125I]1 was 3- to 22-fold
greater than that using Gn,Gn-bi-PA. These findings demonstrate
that 1 serves as a better substrate for GnT-V than Gn,Gn-bi-PA.
2.2. Biological studies
A typical HPLC profile of the GnT-V-reaction products of [125I]1
is shown in Figure 2. The reaction products showed peaks at reten-
tion times of 21 and 30 min, respectively, identical to those of the
nonradioactive compounds 2 and 1.
This result indicates that GnT-V catalyzes the transfer of a Glc-
NAc residue from UDP-GlcNAc to [125I]1 to form a b1–6 linkage,
providing evidence that [125I]1 acts as a substrate for GnT-V.
The kinetics of the conversion of 1 to 2 by GnT-V were deter-
mined using the IPGMG solution containing [125I]1. This radioio-
dinated substrate exhibited saturation kinetics at high substrate
3. Conclusions
We successfully designed and synthesized a novel radioiodin-
ated trisaccharide derivative, [125I]1, as a synthetic substrate for

T. Mukai et al. / Bioorg. Med. Chem. 19 (2011) 4312–4321
4315
O
OAc
OAc
O
OAc
OAc
O
O
CCl3
HO
AcO
a
AllocO
AcO
b, c
AllocO
AcO
NH
OAc
87 %
64 %
(2 steps)
OAc
OAc
OAc
11
12
13
O
O
O
O
RO
e
d
AllocO
AcO
AcO
OAc
Br
OAc
Br
94 %
72 %
OAc
OAc
15: R = H
16: R = EE (99 %)
14
f
O
O
O
O
i
g
EEO
AcO
HO
47 %
OAc
X
AcO
OAc
X
OAc
OAc
17: X = SnBu3
18: X = I (94 %)
19: X = SnBu3 (95 %)
4: X = I (98 %)
h
Scheme 3. Synthesis of glucose derivative 4. Reagents and condition: (a) AllocCl, pyridine, THF, (b) piperidine, THF, (c) CCl3CN, DBU, CH2Cl2, (d) 2-(4-bromophenyl)ethyl
alcohol, BF3Á Et2O, CH2Cl2, (e) Pd(PPh3)4, PPh3, HCO2H, THF, (f) ethyl vinyl ether, PPTS, CH2Cl2, (g) Pd(PPh3)4, PPh3, (n-Bu3Sn)2, DMF, (h) I2, CH2Cl2, (i) PPTS, MeOH.
RO
OR
RO
RO
O
OR
a (for 4)
3
+
4 or 19
O
RO
O
b (for 19)
NHAc
O
O
RO
RO
X
O
OR
c
1: X = I, R = H (59 %)
22: X = SnBu3, R = H
20: X = I, R = Ac (67 %)
21: X = SnBu3, R = Ac (43 %)
d
c
[
125I]1: X=125I, R = H
[
125I]20: X = 125I, R = Ac
Scheme 4. Synthesis of 1 and [125I]1. Reagents and condition: (a) AgClO4, Ag2CO3, CH2Cl2, toluene, (b) AgClO4, collidine, CH2Cl2, toluene, (c) NaOMe, MeOH, (d): [125I]NaI, 5%
H2O2 aq, 0.1 % HCl aq.
GnT-V. [125I]1 exhibited higher affinity than a fluorescent sub-
strate, Gn,Gn-bi-PA, suggesting it to be the preferred acceptor for
this enzyme. The present findings contribute to the development
of sensitive in vitro GnT-V assays.
of 6 (426 mg, 0.86 mmol) at –20 °C. The reaction mixture was stir-
red at –20 °C for 10 min and room temperature for 1.5 h before
being filtered through CeliteÒ and concentrated in vacuo. The resi-
due was purified by silica gel chromatography (toluene/diethyl
ether = 3:1) to afford 7 (280 mg, 57%). 1H NMR (300 MHz, CDCl3)
d: 1.93, 2.08 and 2.09 (each s, 3H), 3.20 (t, J = 10.1 Hz, 1H), 3.52–
3.65 (m, 1H), 3.71–3.76 (m, 1H), 3.88–3.96 (m, 3H), 4.15–4.17
(m, 1H), 4.25 (d of ABd, JAB = 12.2 Hz, J = 2.3 Hz, 1H), 4.33 (ABd,
JAB = 11.7 Hz, 1H), 4.34 (d of ABd, JAB = 12.2 Hz, J = 4.7 Hz, 1H),
4.52 (ABd, JAB = 11.7 Hz, 1H), 4.53 (dd, J = 10.8 and 8.4 Hz, 1H),
4.64 (s, 1H), 4.67–4.76 (m, 2H), 5.22 (t, J = 9.6 Hz, 1H), 5.44 (d,
J = 8.4 Hz, 1H), 5.50 (s, 1H), 5.92 (dd, J = 10.7 and 9.1 Hz, 1H),
7.20–7.48 (m, 15H), 7.77–7.82 (m, 2H), 7.88–7.91 (m, 2H); MS
FAB(+) m/z 866 [(M+H)+, 26]; HR-MS calcd for C47H47NO15Na
[(M+Na)+] 888.2843, found: 888.2845.
4. Experimental sections
4.1. Synthetic methods
All reagents were commercial products and used without fur-
ther purification unless otherwise indicated. 1H NMR spectra were
obtained on a JEOL JNM EX-270, JNM AL-300, and Varian Unity
INOVA 400 spectrometer. Signals are given in ppm using tetra-
methylsilane as an internal standard. MS spectra were determined
on a JEOL JMX-SX102A QQ and Shimadzu LCMS-QP8000
spectrometer.
a mass
4.1.2. Benzyl 3-O-benzyl-4,6-O-benzylidiene-2-O-[3,4,6-tri-O-
acetyl-2-acetamido-2-deoxy-b-D-glucopyranosyl]-a-D-manno
4.1.1. Benzyl 3-O-benzyl-4,6-O-benzylidiene-2-O-[3,4,6-tri-O-
pyranoside (8)
acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl]-a-D-manno
To an ethanol (50 mL) solution of 7 (900 mg, 1.04 mmol) was
added hydrazineÁhydrate (80% in water, 1.5 mL). The reaction mix-
ture was heated at reflux for 4.5 h before being filtered and concen-
trated in vacuo. The residue was purified by silica gel chroma
pyranoside (7)
To a mixture of 5 (255 mg, 0.57 mmol), silver trifluoromethane-
sulfonate (263 mg, 1.02 mmol), and collidine (135 L, 1.02 mmol)
in nitromethane (1 mL) was added a nitromethane (2 mL) solution
l

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T. Mukai et al. / Bioorg. Med. Chem. 19 (2011) 4312–4321
OAc
O
AcO
AcO
AcO
PhthN
OAc
OAc
OAc
a
e
BnO
O
O
O
OR1
OR2
7
AcO
AcO
BnO
O
O
O
83 %
40 %
AcO
O
NPhth
OBn
NPhth
OBn
23: R1 = R2 = H
b
27
24: R1 =TBS, R2 = H (94%)
25: R1 =TBS, R2 = Ac (99%)
26: R1 =H, R2 = Ac (75%)
c
d
OR
RO
RO
AcHN
O
OR
OR
OR
OAc
RO
O
O
O
AcO
AcHN
O
OAc
OAc
OAc
O
RO
AcO
AcO
RO
O
f, c
i
O
O
NHAc
O
O
O
93 %
7 %
RO
RO
I
(2 steps)
(2 steps)
NHAc
OR
O
28: R = Bn
g, c
h
OR
29: R = Ac (72%, 2 steps)
30: R = Br
31: R = Ac
2: R = H
j
Scheme 5. Synthesis of tetrasaccharide 2. Reagents and condition: (a) 70% AcOH, (b) TBSCl, imidazole, DMF, (c) Ac2O, pyridine, (d) PPTS, CH2Cl2, (e) 6, AgOTf, collidine,
CH3NO2, (f) NH2NH2 H2O, EtOH, (g) 10% Pd-C, H2, EtOH, (h) 30% HBr-AcOH, Ac2O, (i) 4, AgClO4, Ag2CO3, CH2Cl2, toluene, (j) NaOMe, MeOH.
[
125I]2
200
150
100
50
A
B
[
125I]1
0
0
100
200
300
S (µM)
400
500
0
10
20
30
40
50
0.04
0.03
0.02
0.01
0
Retention Time (min)
Figure 2. Chromatogram of reaction products of [125I]1.
tography (chloroform/methanol = 3:1) to afford the dephthaloyl
product (500 mg, 95%). A mixture of the dephthaloyl product
(500 mg, 0.82 mmol) and acetic anhydride (2.5 mL) in pyridine
(5 mL) was stirred overnight at room temperature. The mixture was
poured into ice water, and extracted with ethyl acetate. The organic
layer was washed with water, dried over sodium sulfate, filtered,
and concentrated invacuo. The residue waspurified bysilicagel chro-
matography (chloroform/methanol = 3:1) to afford 8 (625 mg, 84%).
1H NMR (400 MHz, CDCl3) d: 1.73, 2.01, 2.02 and 2.02 (each s, 3H)
3.46 (ddd, J = 9.9, 8.3 and 7.6 Hz, 1H), 3.70–3.78 (m, 1H), 3.82–3.86
(m, 2H), 4.01–4.04 (m, 1H), 4.11–4.24 (m, 5H), 4.49 (ABd,
JAB = 11.7 Hz, 1H), 4.70 (ABd, JAB = 11.4 Hz, 1H), 4.71 (ABd,
JAB = 11.7 Hz, 1H), 4.80 (ABd, JAB = 11.4 Hz, 1H), 4.86 (d, J = 1.6 Hz,
1H), 5.00 (dd, J = 10.1 and 9.3 Hz, 1H), 5.12 (d, J = 8.4 Hz, 1H), 5.52
(d, J = 7.5 Hz, 1H), 5.64 (s, 1H), 5.67 (dd, J = 10.6 and 9.3 Hz, 1H),
7.27–7.40 (m, 13H), 7.49–7.52 (m, 2H); MS FAB(+) m/z 778 [(M+H)+,
5]; HR-MS calcd for C41H48NO14 [(M+H)+] 778.3075, found: 778.3070.
y = 0.1492x + 0.0063
R2 = 0.9845
0
0.05
0.1
0.15
0.2
1/S
Figure 3. (A) Michaelis–Menten’s curve of the GnT-V-catalyzed reaction: (B)
Lineweaver–Burk plot of the GnT-V-catalyzed reaction.
4.1.3. Benzyl 4,6-di-O-acetyl-3-O-benzyl-2-O-[3,4,6-tri-O-acetyl-
2-acetamido-2-deoxy-b-D-glucopyranosyl]-a-D-mannopyrano
side (9)
A solution of 8 (160 mg, 0.21 mmol) and 70% acetic acid
(2 mL) was heated to 55 °C for 6.5 h. The mixture was poured
into ice water, neutralized with sodium hydrogencarbonate,

T. Mukai et al. / Bioorg. Med. Chem. 19 (2011) 4312–4321
4317
Table 1
GnT-V activity in rat tissues determined with [125I]1 or Gn,Gn-bi-PA as a substrate.
4.1.5. 3,4,6-Tri-O-acetyl-2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-
deoxy-b- -glucopyrosyl)- -mannopyranosyl bromide (3)
D
a-D
To a solution of 10 (136 mg, 0.20 mmol) and acetic anhydride
(1.5 mL) was added hydrogen bromide (30% in acetic acid, 3 mL).
The reaction mixture was stirred at room temperature for 4 h un-
der a nitrogen atmosphere, and hydrogen bromide (30% in acetic
acid, 3 mL) was added. The mixture was stirred overnight at room
temperature, poured into ice water, and extracted with chloroform.
The organic layer was washed with water and a saturated sodium
hydrogencarbonate solution, dried over sodium sulfate, filtered,
and evaporated to give 3 (137 mg, quant.). 1H NMR (400 MHz,
CDCl3) d: 1.95, 2.02, 2.03, 2.05, 2.08, 2.09 and 2.11 (each s, 3H),
3.60 (dt, J = 10.8 and 8.2 Hz, 1H), 3.71–3.77 (m, 1H), 4.00–4.03
(m, 1H), 4.08–4.14 (m, 2H), 4.24–4.28 (m, 2H), 4.48 (dd, J = 3.3
and 1.7 Hz, 1H), 5.01 (dd, J = 9.9 and 9.3 Hz, 1H), 5.08 (d,
J = 8.2 Hz, 1H), 5.35–5.52 (m, 3H), 5.87 (d, J = 7.9 Hz, 1H), 6.29 (d,
J = 1.3 Hz, 1H); MS FAB(+) m/z 698 (M+, 7); HR-MS calcd for
Tissue
Activity (pmol/h mg protein)
a
b
[
125I]IPGMG
Gn,Gn-bi-PA
10
Heart
Liver
Stomach
Lung
Spleen
Brain
Kidney
Intestine
Serum
31
48
1
1
0.8
1
4
2
1
6
11
53
5
65
6
117
18
240
67
223
8.2
25
38
21
38
30
68
112
116
179
244
261
671
774
1284
a
Each value represents the mean SD for three samples.
Results were reported previously.13
b
C
26H36BrNO16 (M+) 698.1296, found: 698.1301.
and extracted with chloroform. The organic layer was washed
with a saturated sodium hydrogencarbonate solution, dried over
sodium sulfate, filtered, and concentrated in vacuo. The residue
was purified by silica gel chromatography (chloroform/metha-
nol = 10:1) to afford the diol compound (125 mg, 91%). A mixture
of the diol compound (138 mg, 0.20 mmol) and acetic anhydride
(0.5 mL) in pyridine (1 mL) was stirred overnight at room tem-
perature. The mixture was poured into ice water, neutralized
with sodium hydrogencarbonate, and extracted with chloroform.
The organic layer was washed with water, dried over sodium
sulfate, filtered, and concentrated in vacuo. The residue was
purified by silica gel chromatography (chloroform/metha-
nol = 20:1) to afford 9 (137 mg, 88%). 1H NMR (400 MHz, CDCl3)
d: 1.82 (s, 3H), 2.00 (s, 6H), 2.01, 2.05 and 2.08 (each s, 3H), 3.29
(ddd, J = 9.9, 8.2 and 7.5 Hz, 1H), 3.71–3.75 (m, 1H), 3.85–3.90
(m, 2H), 4.03 (d of ABd, JAB = 12.1 Hz, J = 2.4 Hz, 1H), 4.10–4.20
(m, 2H), 4.24 (d of ABd, JAB = 12.1 Hz, J = 6.0 Hz, 1H), 4.49 (ABd,
JAB = 11.4 Hz, 1H), 4.51 (ABd, JAB = 11.6 Hz, 1H), 4.68 (ABd,
JAB = 11.4 Hz, 1H), 4.71 (ABd, JAB = 11.6 Hz, 1H), 4.92 (d,
J = 2.0 Hz, 1H), 4.97 (dd, J = 10.1 and 9.2 Hz, 1H), 5.24 (t,
J = 9.6 Hz, 1H), 5.27 (d, J = 8.2 Hz, 1H), 5.57 (d, J = 7.1 Hz, 1H),
5.74 (dd, J = 10.5 and 9.2 Hz, 1H), 7.28–7.41 (m, 10H); MS
FAB(+) m/z 774 [(M+H)+, 9]; HR-MS calcd for C38H48NO16
[(M+H)+] 774.2973, found: 774.2979.
4.1.6. 1,2,3,4-Tetra-O-acetyl-6-O-allyloxycarbonyl-b-D-glucopy
ranose (12)
To a tetrahydrofuran (30 mL) solution of 1,2,3,4-tetra-O-acetyl-
D-glucopyranose (11) (1.38 g, 4.31 mmol) in an ice bath were
b-
added allyl chloroformate (1.37 mL, 12.9 mmol) and pyridine
(1.39 mL, 17.2 mmol). The reaction mixture was stirred at room
temperature for 2 h under a nitrogen atmosphere, poured into
aqueous ammonium chloride (15 mL), and extracted with ethyl
acetate. The organic layer was washed with brine, dried over so-
dium sulfate, filtered, and concentrated in vacuo. The residue
was purified by silica gel chromatography (hexane/ethyl ace-
tate = 2:1) to afford 12 (1.63 g, 87%). 1H NMR (300 MHz, CDCl3) d:
2.01, 2.03, 2.04 and 2.10 (each s, 3H), 3.84 (m, 1H), 4.22–4.32 (m,
2H), 4.61–4.63 (m, 2H), 5.06–5.16 (m, 2H), 5.23–5.39 (m, 3H),
5.74 (d, J = 7.7 Hz, 1H), 5.86–5.99 (m, 1H); MS FAB(+) m/z 455
[(M+Na)+, 100]; HR-MS calcd for C18H24O12Na [(M+Na)+]
455.1166, found: 455.1171.
4.1.7. 2,3,4-Tri-O-acetyl-6-O-allyloxycarbonyl-a-D-glucopyrano
syl trichloroacetimidate (13)
To a tetrahydrofuran (50 mL) solution of 12 (3.45 g, 11.4 mmol) was
added piperidine (2.26 mL, 22.8 mmol). The reaction mixture was stir-
red at room temperature for 20 h under a nitrogen atmosphere. After
the reaction was completed, the mixture was diluted with ethyl ace-
tate. The organic layer was washed with 10% hydrochloric acid and
brine, dried over sodium sulfate, filtered, and concentrated in vacuo.
The residue was purified by silica gel chromatography (hexane/ethyl
acetate = 2:1) to give a crude compound (2.41 g). To a solution of the
crude compound (2.41 g) in dichloromethane (30 mL) were added
trichloroacetonitrile (1.23 mL, 12.3 mmol) and 1,8-diazabicy-
4.1.4. 1,3,4,6-Tetra-O-acetyl-2-O-(3,4,6-tri-O-acetyl-2-acet
amido-2-deoxy-b-
D
-glucopyrosyl)-
D-mannopyranose (10)
A mixture of 9 (1.13 g, 1.46 mmol) and P
D
-C (10%, ca. 600 mg) in
a mixed solvent of ethanol (35 mL) and acetic acid (12 mL) was
stirred overnight at 50 °C under atmospheric pressure of H2. The
catalysts were filtered, and the filtrate was evaporated. The residue
was chromatographed on a silica gel column (chloroform/metha-
nol = 15:2) to give the diol compound (740 mg, quant.). A mixture
of the diol compound (225 mg, 0.38 mmol) and acetic anhydride
(2 mL) in pyridine (4 mL) was stirred overnight at room tempera-
ture. The solvent was removed, and the residue was purified by sil-
ica gel chromatography (chloroform/methanol = 10:1) to give 10
(257 mg, 86%). 1H NMR (400 MHz, CDCl3) d: 1.96, 2.01, 2.02, 2.04,
2.05, 2.08, 2.12 and 2.15 (each s, 3H), 3.28 (ddd, J = 7.1, 5.0 and
2.2 Hz, 1H), 3.87 (ddd, J = 10.6, 8.6 and 8.4 Hz, 1H), 3.96 (ddd,
J = 7.3, 4.9 and 2.4 Hz, 1H), 4.02 (d of ABd, JAB = 12.3 Hz, J = 2.2 Hz,
1H), 4.07 (d of ABd, JAB = 12.5 Hz, J = 2.4 Hz, 1H), 4.20 (dd, J = 3.3
and 2.4 Hz, 1H), 4.22 (d of ABd, JAB = 12.5 Hz, J = 5.0 Hz, 1H), 4.25
(d of ABd, JAB = 12.3 Hz, J = 5.0 Hz, 1H), 4.80 (d, J = 8.4 Hz, 1H),
5.04 (dd, J = 10.3 and 3.8 Hz, 1H), 5.06 (dd, J = 10.1 and 1.8 Hz,
1H), 5.33 (dd, J = 10.8 and 4.2 Hz, 1H), 5.36 (dd, J = 10.8 and
4.0 Hz, 1H), 5.79 (d, J = 8.6 Hz, 1H), 5.98 (d, J = 2.0 Hz, 1H); MS
FAB(+) m/z 678 [(M+H)+, 6]; HR-MS calcd for C28H40NO18
[(M+H)+] 678.2245, found: 678.2241.
clo[5.4.0]undec-7-ene (46 lL, 0.31 mmol). The reaction mixture was
stirred at room temperature for 2 h. The solvent was removed, and
the residue was purified by silica gel chromatography (hexane/ethyl
acetate = 3:1) to afford 13 (2.73 g, 64%, 2 steps). 1H NMR (300 MHz,
CDCl3) d: 2.01, 2.04 and 2.06 (each s, 3H), 4.22–4.33 (m, 3H), 4.62 (dt,
J = 1.3 and 5.9 Hz, 2H), 5.12 (dd, J = 3.7 and 10.2 Hz, 1H), 5.19 (t,
J = 9.6 Hz, 1H), 5.28 (qd, J = 1.3 and 10.5 Hz, 1H), 5.36 (qd, J = 1.5 and
17.2 Hz, 1H), 5.58 (t, J = 9.6 Hz, 1H), 5.92 (ddt, J = 5.9, 10.5 and
17.2 Hz, 1H), 6.57 (d, J = 3.7 Hz, 1H), 8.69 (s, 1H); MS FAB(+) m/z 556
[(M+Na)+, 11]; HR-MS calcd for C18H22O11Cl3NNa [(M+Na)+]
556.0156, found: 556.0152.
4.1.8. 2-(4-Bromophenyl)ethyl 2,3,4-tri-O-acetyl-6-O-allyloxycar
bonyl-b-
D
-glucopyranoside (14)
To
a
dichloromethane (20 mL) solution of 13 (904 mg,
1.69 mmol) and 2-(4-bromophenyl)ethyl alcohol (197
1.41 mmol) was added boron trifluoride diethyl etherate (36
l
l
L,
L,

4318
T. Mukai et al. / Bioorg. Med. Chem. 19 (2011) 4312–4321
0.28 mmol). The reaction mixture was stirred at –20 °C for 2 h, and
a saturated sodium hydrogencarbonate solution was added. Fol-
lowing extraction with chloroform, the organic phase was dried
over sodium sulfate and filtered. The solvent was removed, and
the residue was purified by silica gel chromatography (toluene/
diethyl ether = 3:1) to afford 14 (584 mg, 72%). 1H NMR
(270 MHz, CDCl3) d: 1.89, 1.99 and 2.03 (each s, 3H), 2.80–2.86
(m, 2H), 3.58–3.75 (m, 2H), 4.06–4.29 (m, 3H), 4.46 (d, J = 7.9 Hz,
1H), 4.62 (dt, J = 1.3 and 5.9 Hz, 2H), 4.93–5.05 (m, 2H), 5.14–
5.40 (m, 3H), 5.85–5.99 (m, 1H), 7.04–7.08 (m, 2H), 7.37–7.40
(m, 2H); MS FAB(+) m/z 595 [(M+Na)+, 54]; HR-MS calcd for
7.13–7.16 (m, 2H), 7.35–7.37 (m, 2H); MS FAB(+) m/z 795
[(M+Na)+, 43]; HR-MS calcd for C36H60O10SnNa [(M+Na)+]
795.3106, found: 795.3111.
4.1.12. 2-(4-Iodophenyl)ethyl 2,3,4-tri-O-acetyl-6-O-(1-ethoxy
ethyl)-b-
D
-glucopyranoside (18)
To
a
dichloromethane (10 mL) solution of 17 (305 mg,
0.40 mmol) was added iodine (201 mg, 0.79 mmol). The reaction
mixture was stirred at 0 °C for 2 h under a nitrogen atmosphere,
and a saturated sodium hydrogencarbonate and sodium thiosulfate
solution was added. Following extraction with chloroform, the or-
ganic phase was dried over sodium sulfate and filtered. The solvent
was removed, and the residue was purified by silica gel chromatog-
raphy (toluene/diethyl ether = 3:1) to afford 18 (226 mg, 94%). 1H
NMR (300 MHz, CDCl3) d: 1.17 (t, J = 7.0 Hz, 1.5H), 1.17 (t,
J = 7.0 Hz, 1.5H), 1.27 (d, J = 5.5 Hz, 1.5H), 1.29 (d, J = 5.1 Hz,
1.5H), 1.89 (s, 3H), 1.99 (s, 3H), 2.01 (s, 3H), 2.79–2.84 (m, 2H),
3.42–3.69 (m, 6H), 4.10 (dt, J = 9.5 and 5.9 Hz, 1H), 4.45 (d,
J = 7.7 Hz, 1H), 4.70 (q, J = 5.1 Hz, 0.5H), 4.71 (q, J = 5.1 Hz, 0.5H),
4.95 (dd, J = 9.7 and 7.7 Hz, 1H), 5.03 (dd, J = 9.7 and 9.5 Hz, 1H),
5.16 (t, J = 9.5 Hz, 1H), 6.92–6.95 (m, 2H), 7.57–7.59 (m, 2H); MS
FAB(+) m/z 631 [(M+Na)+, 16]; HR-MS calcd for C24H33O10INa
[(M+Na)+] 631.1016, found: 631.1011.
C
24H29O11BrNa [(M+Na)+] 595.0791, found: 595.0787.
4.1.9. 2-(4-Bromophenyl)ethyl 2,3,4-tri-O-acetyl-b-D-glucopyran
oside (15)
To a tetrahydrofuran (30 mL) solution of 14 (2.12 g, 3.70 mmol)
were added tetrakis(triphenylphosphine)palladium (0) (214 mg,
0.18 mmol), triphenylphosphine (291 mg, 1.11 mmol), and formic
acid (279 mL, 7.39 mmol). The reaction mixture was stirred at
room temperature for 6 h under a nitrogen atmosphere. After the
reaction was completed, the mixture was diluted with ethyl ace-
tate. The organic layer was washed with water and brine, dried
over sodium sulfate, filtered, and concentrated in vacuo. The resi-
due was purified by silica gel chromatography (hexane/ethyl ace-
tate = 1:1) to afford 15 (1.70 g, 94%). 1H NMR (300 MHz, CDCl3) d:
1.89, 2.00 and 2.05 (each s, 3H), 2.81–2.86 (m, 2H), 3.46–3.52 (m,
2H), 3.55–3.67 (m, 3H), 3.71–3.75 (m, 1H), 4.13 (ddd, J = 9.5, 6.1
and 5.9 Hz, 1H), 4.49 (d, J = 7.9 Hz, 1H), 4.96 (dd, J = 9.7 and
7.9 Hz, 1H), 5.01 (dd, J = 9.7 and 9.5 Hz, 1H), 5.21 (t, J = 9.5 Hz,
1H), 7.05–7.08 (m, 2H), 7.38–7.41 (m, 2H); MS FAB(+) m/z 511
[(M+Na)+, 99]; HR-MS calcd for C20H25O9BrNa [(M+Na)+]
511.0580, found: 511.0573.
4.1.13. 2-[4-(Tributylstannyl)phenyl]ethyl 2,3,4-tri-O-acetyl-b-D-
glucopyranoside (19)
A mixture of 17 (310 mg, 0.40 mmol), pyridinium p-toluenesul-
fonate (10 mg, 0.040 mmol), and methanol (10 mL) was stirred at
room temperature for 2 h. The solvent was removed, and the resi-
due was purified by silica gel chromatography (hexane/ethyl ace-
tate = 3:1 to 2:1) to afford 19 (268 mg, 95%). 1H NMR (300 MHz,
CDCl3) d: 0.88 (t, J = 7.2 Hz, 9H), 1.02 (t, J = 8.1 Hz, 6H), 1.33 (sextet,
J = 7.2 Hz, 6H), 1.53 (tt, J = 8.1 and 7.2 Hz, 6H), 1.89 (s, 3H), 2.00 (s,
3H), 2.04 (s, 3H), 2.12 (t, J = 6.1 Hz, 1H), 2.86 (dd, J = 7.2 and 6.6 Hz,
2H), 3.46–3.52 (m, 1H), 3.55–3.61 (m, 1H), 3.63–3.76 (m, 2H), 4.13
(dt, J = 9.5 and 6.1 Hz, 1H), 4.53 (d, J = 8.1 Hz, 1H), 4.98 (dd, J = 9.7
and 8.1 Hz, 1H), 5.02 (dd, J = 9.7 and 9.5 Hz, 1H), 5.22 (t, J = 9.5 Hz,
1H), 7.14–7.16 (m, 2H), 7.35–7.38 (m, 2H); MS FAB(+) m/z 723
[(M+Na)+, 100]; HR-MS calcd for C32H52O9NaSn [(M+Na)+]
723.2531, found: 723.2533.
4.1.10. 2-(4-Bromophenyl)ethyl 2,3,4-tri-O-acetyl-6-O-(1-ethoxy
ethyl)-b-
To
D
-glucopyranoside (16)
dichloromethane (3 mL) solution of 15 (173 mg,
a
0.35 mmol) were added ethyl vinyl ether (51 lL, 0.53 mmol), and
pyridinium p-toluenesulfonate (9 mg, 0.035 mmol). The reaction
mixture was stirred at room temperature for 1 h under a nitrogen
atmosphere. The solvent was removed, and the residue was puri-
fied by silica gel chromatography (hexane/ethyl acetate = 2:1) to
afford 16 (197 mg, 99%). 1H NMR (300 MHz, CDCl3) d: 1.17 (t,
J = 7.1 Hz, 1.5H), 1.18 (t, J = 7.1 Hz, 1.5H), 1.28 (d, J = 5.3 Hz,
1.5H), 1.29 (d, J = 5.3 Hz, 1.5H), 1.89 (s, 1.5H), 1.89 (s, 1.5H), 1.99
(s, 3H), 2.02 (s, 3H), 2.80–2.85 (m, 2H), 3.40–3.70 (m, 6H), 4.06–
4.16 (m, 1H), 4.46 (d, J = 7.9 Hz, 1H), 4.70 (q, J = 5.3 Hz, 0.5H),
4.72 (q, J = 5.3 Hz, 0.5H), 4.96 (dd, J = 9.7 and 7.9 Hz, 1H), 5.03
(dd, J = 9.7 and 9.5 Hz, 1H), 5.16 (t, J = 9.5 Hz, 1H), 7.05–7.07 (m,
2H), 7.37–7.40 (m, 2H); MS FAB(+) m/z 583 [(M+Na)+, 34]; HR-
MS calcd for C24H33O10BrNa [(M+Na)+] 583.1155, found: 583.1151.
4.1.14. 2-(4-Iodophenyl)ethyl 2,3,4-tri-O-acetyl-b-D-glucopyrano
side (4)
To a methanol (10 mL) solution of 18 (222 mg, 0.41 mmol) was
added pyridinium p-toluenesulfonate (10 mg, 0.036 mmol). The
reaction mixture was stirred at 55 °C for 2 h. The solvent was
removed, and the residue was purified by silica gel chromatogra-
phy (hexane/ethyl acetate = 1:1) to afford 4 (192 mg, 98%). 1H
NMR (300 MHz, CDCl3) d: 1.89, 2.00 and 2.05 (each s, 3H), 2.20
(br.t, J = Hz, 1H), 2.80–2.84 (m, 2H), 3.46–3.52 (m, 1H), 3.55–3.77
(m, 3H), 4.12 (dt, J = 9.5 and 5.9 Hz, 1H), 4.49 (d, J = 7.7 Hz, 1H),
4.96 (dd, J = 9.5 and 7.7 Hz, 1H), 5.01 (t, J = 9.5 Hz, 1H), 5.21 (t,
J = 9.5 Hz, 1H), 6.93–6.95 (m, 2H), 7.58–7.60 (m, 2H); MS FAB(+)
m/z 559 [(M+Na)+, 100]; HR-MS calcd for C20H25O9NaI [(M+Na)+]
559.0441, found: 559.0447.
4.1.11. 2-[4-(Tributylstannyl)phenyl]ethyl 2,3,4-tri-O-acetyl-6-O
-(1-ethoxyethyl)-b-D-glucopyranoside (17)
To a N,N-dimethylformamide (5 mL) solution of 16 (161 mg,
0.29 mmol) were added tetrakis(triphenylphosphine)palladium
(0) (33 mg, 0.029 mmol), triphenylphosphine (15 mg, 0.057 mmol),
and bis(tributyltin) (289 lL, 0.57 mmol). The reaction mixture was
4.1.15. 2-(4-Iodophenyl)ethyl 6-O-[2-O-(3,4,6-tri-O-acetyl-2-
stirred at 100 °C for 4 h under a nitrogen atmosphere. The solvent
was removed, and the residue was purified by silica gel chromatog-
raphy (hexane/ethyl acetate = 5:1 to 2:1) to afford 17 (103 mg,
47%). 1H NMR (300 MHz, CDCl3) d: 0.88 (t, J = 7.3 Hz, 9H), 1.02 (t,
J = 8.1 Hz, 6H), 1.17 (t, J = 7.2 Hz, 1.5H), 1.18 (t, J = 7.2 Hz, 1.5H),
1.27–1.36 (m, 9H), 1.47–1.57 (m, 6H), 1.89 (s, 3H), 1.99 (s, 3H),
2.02 (s, 3H), 2.86 (t, J = 6.8 Hz, 2H), 3.43–3.71 (m, 6H), 4.08–4.15
(m, 1H), 4.48 (d, J = 8.1 Hz, 1H), 4.70–4.72 (m, 1H), 4.98 (t,
J = 8.1 Hz, 1H), 5.04 (t, J = 9.5 Hz, 1H), 5.17 (t, J = 9.5 Hz, 1H),
acetamido-2-deoxy-b-
mannopyranosyl]-2,3,4-tri-O-acetyl-b-
D
-glucopyranosyl)-3,4,6-tri-O-acetyl-
a-D-
D
-glucopyranoside (20)
To a solution of 4 (214 mg, 0.40 mmol), dichloromethane
(10 mL), and toluene (10 mL) was added silver carbonate (121 mg,
0.44 mmol). The reaction mixture was stirred at room temperature
for 1 h under a nitrogen atmosphere, and silver perchlorate
(12 mg, 0.060 mmol) was added. After stirring for 20 min at room
temperature, a solution of 3 (334 mg, 0.48 mmol), dichloromethane
(10 mL), and toluene (10 mL) was added and the mixture was stirred

T. Mukai et al. / Bioorg. Med. Chem. 19 (2011) 4312–4321
4319
for another 15 h. After the reaction was completed, the mixture was
filtered. The filtrate was concentrated in vacuo, and the residue was
purified by silica gel chromatography (hexane/ethyl acetate = 4:1 to
ethyl acetate only) to afford 20 (310 mg, 67%). 1H NMR (300 MHz,
CDCl3) d: 1.87, 1.93, 1.94, 2.00, 2.01, 2.01, 2.03, 2.05, 2.09 and 2.09
(each s, 3H), 2.82–2.88 (m, 2H), 3.36–3.45 (m, 1H), 3.51–3.54 (m,
1H), 3.61–3.78 (m, 4H), 3.87–3.92 (m, 1H), 4.00–4.09 (m, 2H),
4.13–4.20 (m, 3H), 4.26–4.32 (m, 1H), 4.47 (d, J = 7.7 Hz, 1H), 4.70
(s, 1H), 4.89–5.05 (m, 4H), 5.08 (d, J = 8.1 Hz, 1H), 5.18 (t,
J = 9.5 Hz, 1H), 5.21 (t, J = 9.9 Hz, 1H), 5.60 (t, J = 9.2 Hz, 1H), 5.61
(d, J = 9.2 Hz, 1H), 6.94–6.97 (m, 2H), 7.56–7.59 (m, 2H); MS
FAB(+) m/z 1154 [(M+H)+, 3]; HR-MS calcd for C46H61O25NI
[(M+H)+] 1154.2578, found: 1154.2588.
and concentrated in vacuo. The residue was purified by silica gel
chromatography (chloroform/methanol = 20:1) to afford 23
(1.07 g, 83%). 1H NMR (300 MHz, CDCl3) d: 1.26 (br.t, J = 6.6 Hz,
1H), 1.89 (s, 3H), 2.01 (s, 3H), 2.04 (s, 3H), 2.28 (br.s, 1H), 3.12–
3.16 (m, 1H), 3.37–3.46 (m, 2H), 3.68–3.69 (m, 2H), 3.84–3.90
(m, 1H), 4.13 (br.s, 1H), 4.20 (d of ABd, JAB = 12.4 Hz, J = 2.4 Hz,
1H), 4.27 (d of ABd, JAB = 12.4 Hz, J = 4.8 Hz, 1H), 4.30 (ABd,
JAB = 11.7 Hz, 1H), 4.38 (ABd, JAB = 11.0 Hz, 1H), 4.41 (dd, J = 10.6
and 8.4 Hz, 1H), 4.49 (ABd, JAB = 11.7 Hz, 1H), 4.62 (d, J = 1.5 Hz,
1H), 4.75 (ABd, JAB = 11.0 Hz, 1H), 5.17 (t, J = 9.5 Hz, 1H), 5.36 (d,
J = 8.4 Hz, 1H), 5.87 (dd, J = 11.0 and 9.2 Hz, 1H), 7.18–7.21 (m,
2H), 7.28–7.34 (m, 8H), 7.76–7.80 (m, 2H), 7.85–7.88 (m, 2H);
MS FAB(+) m/z 800 [(M+Na)+, 53]; HR-MS calcd for C40H43O15NNa
[(M+Na)+] 800.2530, found: 800.2527.
4.1.16. 2-[4-(Tributylstannyl)phenyl]ethyl 6-O-[2-O-(3,4,6-tri-O-
4.1.19. Benzyl 3-O-benzyl-6-O-tert-butyldimethylsilyl-2-O-
acetyl-2-acetamido-2-deoxy-b-
etyl- -mannopyranosyl]-2,3,4-tri-O-acetyl-b-
oside (21)
To a solution of 19 (30 mg, 0.043 mmol), dichloromethane
(1 mL), and toluene (1 mL) was added collidine (14 L, 0.10 mmol).
D
-glucopyranosyl)-3,4,6-tri-O-ac
[3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
-mannopyranoside (24)
To a N,N-dimethylformamide (10 mL) solution of 23 (1.07 g,
1.38 mmol) were added tert-butyldimethylsilyl chloride
D-glucopyranosyl]-
a-
D
D-glucopyran
a-D
l
(311 mg, 2.06 mmol) and imidazole (281 mg, 4.13 mmol). The
reaction mixture was stirred at room temperature for 16 h under
a nitrogen atmosphere, poured into aqueous ammonium chloride
(20 mL), and extracted with ethyl acetate. The organic layer was
washed with water, dried over sodium sulfate, filtered, and con-
centrated in vacuo. The residue was purified by silica gel chro-
matography (hexane/ethyl acetate = 2:1) to afford 24 (1.15 g,
94%). 1H NMR (300 MHz, CDCl3) d: -0.13 (s, 3H), -0.12 (s, 3H),
0.78 (s, 9H), 1.86 (s, 3H), 2.00 (s, 3H), 2.03 (s, 3H), 2.64 (br.s,
1H), 3.14–3.20 (m, 1H), 3.41–3.49 (m, 2H), 3.60–3.71 (m, 2H),
3.82–3.87 (m, 1H), 4.13–4.26 (m, 3H), 4.31 (ABd, JAB = 11.4 Hz,
1H), 4.40 (d, J = 9.5 Hz, 1H), 4.45 (ABd, JAB = 11.2 Hz, 1H), 4.56
(ABd, JAB = 11.4 Hz, 1H), 4.64 (s, 1H), 4.78 (ABd, JAB = 11.2 Hz,
1H), 5.16 (t, J = 9.7 Hz, 1H), 5.44 (d, J = 8.1 Hz, 1H), 5.78 (dd,
J = 10.6 and 9.2 Hz, 1H), 7.21–7.38 (m, 10H), 7.71–7.74 (m,
2H), 7.82–7.84 (m, 2H); MS FAB(+) m/z 914 [(M+Na)+, 62]; HR-
The reaction mixture was stirred at room temperature for 20 min
under a nitrogen atmosphere, and silver perchlorate (11 mg,
0.052 mmol) was added. After stirring for 20 min at room temper-
ature, a solution of 3 (36 mg, 0.052 mmol), dichloromethane
(2 mL), and toluene (2 mL) was added and the mixture was stirred
for another 5 h. After the reaction was completed, the mixture was
filtered. The filtrate was washed with 1% hydrochloric acid, dried
over sodium sulfate, filtered, and concentrated in vacuo. The resi-
due was purified by silica gel chromatography (ethyl acetate) to af-
ford 21 (24 mg, 43%). 1H NMR (300 MHz, CDCl3) d: 0.88 (t,
J = 7.3 Hz, 9H), 1.01 (t, J = 8.2 Hz, 6H), 1.32 (sextet, J = 7.3 Hz, 6H),
1.52 (tt, J = 8.2 and 7.3 Hz, 6H), 1.85 (s, 3H), 1.90 (s, 3H), 1.93 (s,
3H), 2.00 (s, 3H), 2.00 (s, 3H), 2.01 (s, 3H), 2.03 (s, 3H), 2.05 (s,
3H), 2.09 (s, 3H), 2.09 (s, 3H), 2.85–2.91 (m, 2H), 3.38–3.48 (m,
1H), 3.52–3.55 (m, 1H), 3.66–3.80 (m, 4H), 3.90–3.95 (m, 1H),
4.00–4.04 (m, 2H), 4.13–4.21 (m, 3H), 4.26–4.32 (m, 1H), 4.49 (d,
J = 7.9 Hz, 1H), 4.69 (s, 1H), 4.91–5.07 (m, 5H), 5.16–5.25 (m, 2H),
5.51–5.62 (m, 2H), 7.13–7.16 (m, 2H), 7.33–7.36 (m, 2H); MS
FAB(+) m/z 1318 [(M+H)+, 18]; HR-MS calcd for C58H88O25NSn
[(M+H)+] 1318.4668, found: 1318.4672.
MS calcd for
914.3398.
C
46H57O15NSiNa [(M+Na)+] 914.3395, found:
4.1.20. Benzyl 4-O-acetyl-3-O-benzyl-6-O-tert-butyldimeth
ylsilyl-2-O-[3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
glucopyranosyl]- -mannopyranoside (25)
D
-
a
-D
4.1.17. 2-(4-Iodophenyl)ethyl 6-O-[2-O-(2-acetamido-2-deoxy-
A mixture of 24 (1.14 g, 1.28 mmol) and acetic anhydride (5 mL)
in pyridine (5 mL) was stirred at room temperature for 2 h and
methanol (20 mL) was added. The solvent was removed, and the
residue was purified by silica gel chromatography (hexane/ethyl
acetate = 2:1) to give 25 (1.18 g, 99%). 1H NMR (300 MHz, CDCl3)
d: -0.19 (s, 3H), -0.18 (s, 3H), 0.77 (s, 9H), 1.85 (s, 3H), 1.95 (s,
3H), 2.01 (s, 6H), 3.22 (d of ABd, JAB = 11.3 Hz, J = 7.2 Hz, 1H), 3.40
(d of ABd, JAB = 11.3 Hz, J = 2.6 Hz, 1H), 3.55–3.62 (m, 1H), 3.75–
3.81 (m, 2H), 4.11–4.24 (m, 3H), 4.32 (ABd, JAB = 11.6 Hz, 1H),
4.44 (ABd, JAB = 11.7 Hz, 1H), 4.44 (dd, J = 10.8 and 8.6 Hz, 1H),
4.60 (ABd, JAB = 11.6 Hz, 1H), 4.69 (d, J = 2.4 Hz, 1H), 4.70 (ABd,
JAB = 11.7 Hz, 1H), 4.88 (t, J = 9.1 Hz, 1H), 5.15 (t, J = 9.5 Hz, 1H),
5.53 (d, J = 8.4 Hz, 1H), 5.74 (dd, J = 10.7 and 9.1 Hz, 1H), 7.22–
7.35 (m, 10H), 7.68–7.71 (m, 2H), 7.80–7.83 (m, 2H); MS FAB(+)
m/z 956 [(M+Na)+, 45]; HR-MS calcd for C48H59O16NSiNa
[(M+Na)+] 956.3501, found: 956.3503.
b-
side (1)
To a methanol (10 mL) solution of 20 (188 mg, 0.16 mmol) was
added sodium methoxide (28% in methanol, 332 L, 1.63 mmol)
D-glucopyranosyl)-a-D-mannopyranosyl]-b-D-glucopyrano
l
and the mixture was stirred at room temperature for 2 h. After
the reaction was completed, the mixture was neutralized with
AmberlystÒ 15 (ca. 1.0 g) and filtered. The filtrate was concentrated
in vacuo and the residue was purified by a recycle HPLC (LC-908,
Japan Analytical Industry Co. Ltd. Tokyo, Japan) on a reversed phase
column (methanol/distilled water = 7:3) to afford 1 (74 mg, 59%).
1H NMR (300 MHz, CD3OD) d: 1.99 (s, 3H), 2.89–2.92 (m, 2H),
3.16–4.04 (m, 21H), 4.28 (d, J = 7.3 Hz, 1H), 4.46 (d, J = 7.7 Hz,1H),
7.06–7.09 (m, 2H), 7.59–7.62 (m, 2H); MS FAB(+) m/z 776
[(M+H)+, 10]; HR-MS calcd for C28H43O16NI [(M+H)+] 776.1627,
found: 776.1635.
4.1.18. Benzyl 3-O-benzyl-2-O-[3,4,6-tri-O-acetyl-2-deoxy-2-
4.1.21. Benzyl 4-O-acetyl-3-O-benzyl-2-O-[3,4,6-tri-O-acetyl-2-
phthalimido-b-D-glucopyranosyl]-a-D-mannopyranoside (23)
deoxy-2-phthalimido-b-D-glucopyranosyl]-a-D-mannopyrano
A mixture of 7 (1.45 g, 1.67 mmol) and 70% acetic acid (10 mL)
was stirred at 55 °C for 2 h. The mixture was poured into ice water,
neutralized with sodium hydrogencarbonate, and extracted with
chloroform. The organic layer was washed with a saturated sodium
hydrogencarbonate solution, dried over sodium sulfate, filtered,
side (26)
A mixture of 25 (1.17 g, 1.25 mmol), pyridinium p-toluenesulfo-
nate (32 mg, 0.13 mmol), and methanol (10 mL) was stirred at
50 °C for 2 h. The solvent was removed, and the residue was puri-
fied by silica gel chromatography (hexane/ethyl acetate = 1:1) to

4320
T. Mukai et al. / Bioorg. Med. Chem. 19 (2011) 4312–4321
afford 26 (765 mg, 75%). 1H NMR (300 MHz, CDCl3) d: 1.44 (br.dd,
J = 9.4 and 4.4 Hz, 1H), 1.88(s, 3H), 1.95 (s, 3H), 2.03 (s, 3H), 2.04 (s,
3H), 3.03–3.10 (m, 1H), 3.17–3.25 (m, 1H), 3.39–3.46 (m, 1H),
3.80–3.84 (m, 2H), 4.10–4.11 (m, 1H), 4.16–4.21 (m, 1H), 4.22–
4.28 (m, 1H), 4.35 (ABd, JAB = 11.7 Hz, 1H), 4.42–4.48 (m, 1H),
4.47 (ABd, JAB = 11.9 Hz, 1H), 4.51 (ABd, JAB = 11.7 Hz, 1H), 4.68
(ABd, JAB = 11.9 Hz, 1H), 4.71 (d, J = 2.0 Hz, 1H), 4.93 (t, J = 9.6 Hz,
1H), 5.17 (t, J = 9.6 Hz, 1H), 5.45 (d, J = 8.4 Hz, 1H), 5.82 (dd,
J = 10.7 and 9.1 Hz, 1H), 7.17–7.21 (m, 2H), 7.26–7.35 (m, 8H),
7.71–7.74 (m, 2H), 7.83–7.86 (m, 2H); MS FAB(+) m/z 842
[(M+Na)+, 100]; HR-MS calcd for C42H45O16NNa [(M+Na)+]
842.2636, found: 842.2639.
through CeliteÒ and concentrated in vacuo. The residue was dis-
solved in pyridine (2 mL) and acetic anhydride (2 mL) was
added. The mixture was stirred overnight at room temperature
and methanol (20 mL) was added. The solvent was removed,
and the residue was purified by silica gel chromatography (chlo-
roform/methanol = 20:1) to give 29 (78 mg, 72%, 2 steps). 1H
NMR (300 MHz, CDCl3) d: 1.97, 2.00, 2.01, 2.02, 2.05, 2.06,
2.06, 2.09, 2.11 and 2.12 (each s, 3H), 2.97–3.05 (m, 2H), 3.13–
3.16 (m, 1H), 3.62–3.66 (m, 1H), 3.75–3.79 (m, 2H), 3.96–4.01
(m, 1H), 4.10–4.29 (m, 6H), 4.92 (t, J = 9.7 Hz, 1H), 5.06–5.10
(m, 4H), 5.45 (t, J = 9.7 Hz, 1H), 5.46 (d, J = 9.2 Hz, 1H), 5.90
(dd, J = 10.6 and 9.2 Hz, 1H, 6.05 (d, J = 1.8 Hz, 1H), 6.41 (d,
J = 9.5 Hz, 1H), 7.23 (d, J = 3.9 Hz, 1H); MS FAB(+) m/z 965
[(M+H)+, 5]; HR-MS calcd for C40H57N2O25 [(M+H)+] 965.3250,
found: 965.3255.
4.1.22. Benzyl 2,6-di-O-[3,4,6-tri-O-acetyl-2-deoxy-2-phthalimi
do-b-
ranoside (27)
To a mixture of 26 (564 mg, 0.69 mmol), silver trifluorometh-
anesulfonate (318 mg, 1.24 mmol), and collidine (164 L,
D-glucopyranosyl]-4-O-acetyl-3-O-benzyl-a-D-mannopy
4.1.25. 2-(4-Iodophenyl)ethyl 6-O-[2,6-di-O-(3,4,6-tri-O-acetyl-
l
2-acetamido-2-deoxy-b-
D
-glucopyranosyl)-3,4-di-O-acetyl-
a-D-
1.24 mmol) in nitromethane (5 mL) was added a nitromethane
(5 mL) solution of 6 (514 mg, 1.03 mmol) at –20 °C. The reaction
mixture was stirred at –20 °C for 30 min and room temperature
for 2 h before being filtered through CeliteÒ and concentrated in va-
cuo. The residue was purified by silica gel chromatography (1st;
hexane/ethyl acetate = 1:1, 2nd; chloroform/methanol = 20:1) to
afford 27 (337 mg, 40%). 1H NMR (300 MHz, CDCl3) d: 1.83, 1.86,
1.91, 2.00, 2.01, 2.03 and 2.07 (each s, 3H), 2.87–2.94 (m, 1H),
3.55–3.74 (m, 6H), 4.00–4.01 (m, 1H), 4.07–4.41 (m, 7H), 4.35
(ABd, JAB = 12.0 Hz, 1H), 4.47 (d, J = 2.0 Hz, 1H), 4.59 (ABd,
JAB = 12.0 Hz, 1H), 4.73 (t, J = 9.4 Hz, 1H), 5.00 (d, J = 8.3 Hz, 1H),
5.06 (t, J = 9.4 Hz, 1H), 5.12 (t, J = 9.4 Hz, 1H), 5.42 (d, J = 8.6 Hz,
1H), 5.71 (dd, J = 10.7 and 9.1 Hz, 1H), 7.01–7.04 (m, 2H), 7.18–
7.29 (m, 8H), 7.61–7.76 (m, 6H), 7.83–7.86 (m, 2H); MS FAB(+)
m/z 1259 [(M+Na)+, 20]; HR-MS calcd for C62H64N2O25Na
[(M+Na)+] 1259.3696, found: 1259.3704.
mannopyranosyl]-2,3,4-tri-O-acetyl-b-
D
-glucopyranoside (31)
To a solution of 29 (70 mg, 0.073 mmol) and acetic anhydride
(1.5 mL) was added hydrogen bromide (30% in acetic acid, 3 mL).
The mixture was stirred overnight at room temperature, poured
into ice water, and extracted with chloroform. The organic layer
was washed with water and a saturated sodium hydrogencarbon-
ate solution, dried over sodium sulfate, filtered, and evaporated
to give 30 (67 mg). To a solution of 4 (30 mg, 0.055 mmol),
dichloromethane (1 mL), and toluene (1 mL) was added silver
carbonate (17 mg, 0.06 mmol). The reaction mixture was stirred
at room temperature for 1 h under a nitrogen atmosphere, and
silver perchlorate (2 mg, 0.0082 mmol) was added. After stirring
for 30 min at room temperature,
a solution of 30 (65 mg,
0.066 mmol), dichloromethane (2 mL), and toluene (2 mL) was
added and the mixture was stirred for another 15 h. After the
reaction was completed, the mixture was filtered. The filtrate
was concentrated in vacuo, and the residue was purified by silica
gel chromatography (chloroform/methanol = 20:1) to afford 31
(5 mg, 7%, 2 steps). 1H NMR (400 MHz, CDCl3) d: 1.87, 1.94,
1.96, 1.97, 1.99, 2.00, 2.03, 2.04, 2.05, 2.06, 2.08, 2.09, and 2.09
(each s, 3H), 2.82–2.96 (m, 3H), 3.06–3.09 (m, 1H), 3.55–3.58
(m, 1H), 3.63–3.73 (m, 5H), 3.77–3.82 (m, 1H), 3.98–4.01 (m,
1H), 4.09–4.32 (m, 8H), 4.45 (d, J = 8.1 Hz, 1H), 4.82 (d,
J = 1.5 Hz, 1H), 4.88–4.98 (m, 3H), 5.02–5.10 (m, 3H), 5.17 (t,
J = 9.5 Hz, 1H), 5.35 (t, J = 9.6 Hz, 1H), 5.38 (d, J = 8.2 Hz, 1H),
5.92 (dd, J = 10.8 and 9.2 Hz, 1H), 6.43 (d, J = 9.9 Hz, 1H), 6.95–
6.97 (m, 2H), 7.30 (d, J = 7.0 Hz, 1H), 7.56–7.59 (m, 2H); MS
4.1.23. Benzyl 2,6-di-O-[3,4,6-tri-O-acetyl-2-acetamido-2-deoxy
-b-
side (28)
To an ethanol (10 mL) solution of 27 (157 mg, 0.13 mmol) was
added hydrazineÁhydrate (80% in water, 200 L). The reaction mix-
D-glucopyranosyl]-4-O-acetyl-3-O-benzyl-a-D-mannopyrano
l
ture was heated at reflux for 4 h before being concentrated in va-
cuo. The residue was dissolved in pyridine (2 mL) and acetic
anhydride (2 mL) was added. The mixture was stirred overnight
at room temperature and methanol (20 mL) was added. The sol-
vent was removed, and the residue was purified by silica gel chro-
matography (chloroform/methanol = 20:1) to give 28 (125 mg,
93%, 2 steps). 1H NMR (300 MHz, CDCl3) d: 1.94, 1.98, 2.02, 2.02,
2.04 and 2.06 (each s, 3H), 2.07 (s, 6H), 2.07 (s, 3H), 2.98 (br dt,
J = 7.1 and 9.5 Hz, 1H), 3.13 (br d, J = 10.6 Hz, 1H), 3.56 (br d,
J = 9.8 Hz, 1H), 3.64 (ddd, J = 2.6, 4.7 and 9.5 Hz, 1H), 3.79 (br dt,
J = 3.0 and 10.1 Hz, 1H), 3.87 (dd, J = 3.7 and 9.8 Hz, 1H), 4.09–
4.15 (m, 5H), 4.21–4.32 (m, 4H), 4.51 (ABd, JAB = 11.8 Hz, 1H),
4.65 (ABd, JAB = 11.8 Hz, 1H), 4.73 (d, J = 11.4 Hz, 1H), 4.96 (t,
J = 9.2 Hz, 1H), 5.00 (t, J = 9.3 Hz,1H), 5.03–5.11 (m, 2H), 5.30 (t,
J = 9.8 Hz, 1H), 5.54 (d, J = 8.4 Hz, 1H), 5.98 (dd, J = 9.2 and
10.7 Hz, 1H), 6.63 (d, J = 9.8 Hz, 1H), 7.24–7.39 (m, 11H); MS
FAB(+) m/z 1061 [(M+H)+, 5]; HR-MS calcd for C50H65N2O23Na
[(M+H)+] 1061.3978, found: 1061.3981.
FAB(+) m/z 1463 [(M+Na)+, 5]; HR-MS calcd for C58H77N2O32
I
[(M+Na)+] 1463.3402, found: 1463.3394.
4.1.26. 2-(4-Iodophenyl)ethyl 6-O-[2,6-di-O-(2-acetamido-2-
deoxy-b-
ranoside (IPGGMG, 2)
To a methanol (1 mL) solution of 31 (13 mg, 0.009 mmol) was
added sodium methoxide (28% in methanol, 10 L, 0.050 mmol)
D-glucopyranosyl)-a-D-mannopyranosyl]-b-D-glucopy
l
and the mixture was stirred at room temperature for 30 min. After
the reaction was completed, the mixture was neutralized with
AmberlystÒ 15 (ca. 100 mg) and filtered. The filtrate was concen-
trated in vacuo to afford 2 (5 mg, 57%). 1H NMR (400 MHz, CD3OD)
d: 2.02, 2.06 (each s, 3H), 2.85–2.93 (m, 2H), 3.12–3.19 (m, 1H),
3.24–3.38 (m, 8H), 3.45–3.76 (m, 11H), 3.84–3.89 (m, 3H), 3.96
(br s, 1H), 4.01–4.10 (m, 2H), 4.28 (d, J = 7.7 Hz, 1H), 4.45 (d,
J = 8.2 Hz, 1H), 4.58 (d, J = 7.7 Hz, 1H), 4.80 (s, 1H), 7.06–7.09
(m, 2H), 7.59–7.61 (m, 2H); MS FAB(+) m/z 1001 [(M+Na)+, 2];
HR-MS calcd for C36H55O21N2INa [(M+Na)+] 1001.2240, found:
1001.2245.
4.1.24. 2,6-Di-O-[3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-b-
D-
glucopyranosyl]-1,3,4-tri-O-acetyl- -mannopyranose (29)
a-D
To an ethanol (10 mL) solution of 28 (118 mg, 0.11 mmol)
was added Pd-C (10%, ca. 100 mg) and ammonium formate
(140 mg). The reaction mixture was refluxed overnight, filtered

T. Mukai et al. / Bioorg. Med. Chem. 19 (2011) 4312–4321
4321
4.2. Iododestannylation Reaction
supernatants were collected and used as crude enzyme prepara-
tions. The GnT-V activity and protein concentration were deter-
mined according to the procedures described above.
The radiolabeled compound [125I]20 was prepared from the cor-
responding tributyltin derivative 21 by iododestannylation. Briefly,
to initiate the reaction, 10
of a tributyltin derivative (100
125I]iodide, and 20
L of 0.1 N HCl in a sealed vial. The reaction
was allowed to proceed at room temperature for 30 min. The reac-
tion mixture was neutralized with sodium bicarbonate, and
extracted with ethyl acetate. The extract was evaporated and
l
L of H2O2 (5%) was added to a mixture
References
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[
l
methanol (100
lL) was added. To the solution was added sodium
methoxide (28% in methanol, 1.6
lL). The reaction mixture was
stirred at room temperature for 45 min, and neutralized with
AmberlystÒ 15. The residue was purified by HPLC on a Cosmosil
5C18 AR-300 column (4.6Â150 mm) with an isocratic solvent of
H2O/acetonitrile (83/17) at a flow rate of 1.0 mL/min.
4.3. Enzymatic Reactions of Radioiodinated IPGMG
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GnT-V-transfected WiDr cells were used as the enzyme source.
The protein concentration was measured by the Lowry method.23
Lysate of GnT-V-overexpressing WiDr cells was incubated with
MES buffer (125 mM, pH 6.25) containing 40 mM UDP-GlcNAc,
200 mM GlcNAc, 0.5% Triton X-100, and 10 mM EDTA for 10 min
at 37 °C. [125I]IPGMG was then added, and the incubation contin-
ued for an additional 4 h. The reaction was stopped by heating at
100 °C for 1 min. Enzymatic products were analyzed by HPLC on
a TSK-GEL ODS-80TM column (4.6Â150 mm) with an isocratic sol-
vent of H2O/acetonitrile (85/15) at a flow rate of 1.0 mL/min.
4.4. GnT-V Activities in Rat Tissues
Animal experiments were conducted in accordance with our
institutional guidelines, and the experimental procedures were ap-
proved by the Kyoto University Animal Care Committee. Various
tissues from male Wistar rats weighing 240–260 g were homoge-
nized in 4 volumes of 10 mM Tris-HCl buffer (pH 7.4) containing
0.25 M sucrose. After centrifugation at 900 Â g for 10 min, the
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