Synthesis of neutral glycosphingolipids from Zygomycetes

Article abstract of DOI:10.1016/j.carres.2008.04.019

Novel neutral glycosphingolipids (NGSLs) containing Gal-α1→6Gal, previously found in the Zygomycetes species Mucor hiemalis, were synthesized. The structures of these compounds are different from those of other fungal GSLs, and they are expected to be involved in host-parasite interactions. A key step in their synthesis is direct 1,2-cis α-selective galactosylation of 4,6-diol tri- and tetrasaccharide acceptors with a galactosyl donor in the presence of N-iodosuccinimide (NIS)/trifluoromethanesulfonic acid (TfOH). The fully protected glycosides were deprotected to give the two target glycosphingolipids.

Full text of DOI:10.1016/j.carres.2008.04.019

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 2315–2324
Note
Synthesis of neutral glycosphingolipids from Zygomycetes
Noriyasu Hada,a Junko Oka,a Kyoko Hakamata,a Kenji Yamamotob
and Tadahiro Takedaa,*
aKyoritsu University of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
bGraduated School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
Received 16 October 2007; received in revised form 9 April 2008; accepted 14 April 2008
Available online 22 April 2008
Abstract—Novel neutral glycosphingolipids (NGSLs) containing Gal-a1?6Gal, previously found in the Zygomycetes species
Mucor hiemalis, were synthesized. The structures of these compounds are different from those of other fungal GSLs, and they
are expected to be involved in host–parasite interactions. A key step in their synthesis is direct 1,2-cis a-selective galactosylation
of 4,6-diol tri- and tetrasaccharide acceptors with a galactosyl donor in the presence of N-iodosuccinimide (NIS)/trifluoromethane-
sulfonic acid (TfOH). The fully protected glycosides were deprotected to give the two target glycosphingolipids.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: a-Galactosylation; 1,2-cis-Glycoside; Di-tert-butylsilylene group; Zygomycetes species; Mucor hiemalis
In our continuing systematic studies on the role and
biological functions of glycosphingolipids, we have syn-
thesized glycolipids found in various lower animal spe-
cies.1–5 Recently, Aoki et al. reported the discovery of
one of the novel class of neutral glycosphingolipids
(NGSLs), D-Gal-(a1?6)-D-Gal-(a1?6)-D-Gal-(a1?6)-
D-Gal-(b1?6)-D-Gal-b1-Cer, and their precursor tri-
and tetrasaccharides, in Mucor hiemalis, a typical
Zygomycetes species (Fig. 1).6 The structures of these
materials are different from those of other fungal
NGSLs, and their biosynthetic pathway does not exist
in human beings. NGSLs can be considered as a selec-
tive target for zygomycosis toxicity, which may lead to
the development of new antifungal agents featuring both
superior growth inhibition of Zygomycetes and destruc-
tion of the pathogenetic factor. In our previous paper,
we reported the synthesis of trisaccharide glyco-
sphingolipids (1).7 The key reaction in that synthesis
was 1,2-cis a-selective galactosylation using a 4,6-O-di-
tert-butylsilylene (DTBS) group, which was reported
by Ando and Kiso.8 Highly a-selective galactosylation
was carried out due to the DTBS group, and the yield
was further improved due to the influence of a 2-O-ben-
zylated donor (a 2-O-benzoylated donor was previously
used). In this work, we report the synthesis of tetra- and
pentasaccharide glycolipids (2, 3).
It is thought that almost complete regioselective gly-
cosylation can be achieved for a galactoside acceptor
with two unprotected hydroxyl groups by making effec-
tive use of the difference in their reactivities due to their
primary and secondary nature; additionally, 2,3-O-benz-
ylated protecting groups could be transformed into the
corresponding 2,3-O-benzoylated protecting groups in
order to decrease the reactivity of the 4-OH group.
Thus, for the synthesis of the target compounds, NGSLs
2 and 3, core oligosaccharides 6 and 18 were selected as
glycosyl acceptors, because compounds 6 and 18 each
contains two free hydroxyl groups at C-4 and C-6 of
the nonreducing end of the galactose residue for
a-galactosylation. This greatly simplifies the preparation
CDS
Galβ1-6Galβ1-Cer
CTS
CTeS
CPS
Galα1-6Galβ1-6Galβ1-Cer
Galα1-6Galα1-6Galβ1-6Galβ1-Cer
Galα1-6Galα1-6Galα1-6Galβ1-6Galβ1-Cer
1
2
3
Figure 1.
*
Corresponding author. Tel.: +81 3 54002696; fax: +81 3 54002556;
e-mail: takeda-td@kyoritsu-ph.ac.jp
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.04.019

2316
N. Hada et al. / Carbohydrate Research 343 (2008) 2315–2324
t-Bu
t-Bu
t-Bu
t-Bu
Si
Si
O
O
OH
OH
O
O
O
O
c
O
c
BzO
c
BzO
BnO
BzO
BzO
OBz
BnO
OBz
O
O
a
O
b
OBz
b
O
O
O
b
b
O
BzO
O
BzO
O
BzO
OBz
OBz
OBz
OBz
OBz
OBz
O
O
a
O
a
O
BzO
a
O
BzO
O
BzO
TMS
OBz
TMS
OBz
TMS
OBz
6
4
5
Scheme 1. Reagents and conditions: (a) (i) Pd–C, H2, MeOH/THF, (ii) BzCl, Pyr., 86% (two steps); (b) TBAF, AcOH/THF, 95%.
of galactoside acceptors for 6-O-glycosylation without
the repetition of protection and deprotection. Removal
of the benzyl groups from 47 by catalytic hydrogenolysis
over 10% Pd–C in THF/MeOH (1:2) and subsequent
benzoylation gave 5. Selective removal of the DTBS
group using tetrabutylammonium fluoride (TBAF) gave
the corresponding 4,6-diol trisaccharide acceptor 6
(Scheme 1). Several examples of the conditions used
for glycosylation of 6 with 77 are given in Table 1. The
glycosylation reactions were carried out under an Ar
atmosphere in CH2Cl2 using 1.0–1.5 equiv of N-iodosuc-
cinimide (NIS) and 0.05–0.30 equiv of trifluoromethane-
sulfonic acid (TfOH)9 with respect to the donor. The
donor was used in excess (1.1–1.5 equiv). The desired
a-galactoside was not obtained with complete regiose-
lectivity, although the yield was satisfactory in all the
cases (entries 1–5). The best result was obtained under
conditions in which the concentration had been diluted
to 1.5 equiv with respect to the donor (entry 5). In this
reaction, selective glycosylation of acceptor 6 with
phenyl 1-thioglycoside derivative 7 gave the expected
tetrasaccharide 8 (76.6%) and by-product 9 (15.7%);
acetylation of 8 and 9 gave compounds 11 and 12,
signal at d 4.93 (d, 1H, J1,2 = 3.1 Hz, H-1d), indicating
that the newly formed glycosidic linkage was a-type.
In addition, compounds 11 and 12 could be distin-
guished by the presence of either a doublet peak at
5.97 ppm assigned to H-4 of Gal c of 11, or one at
4.57 ppm assigned to H-4 of Gal c of 12. Removal of
the DTBS and benzyl groups from 11 by TBAF, cata-
lytic hydrogenolysis over 10% Pd–C, and subsequent
acetylation gave 13. Selective removal of the 2-(tri-
methylsilyl)ethyl (SE) group with TFA in dichlorometh-
ane and treatment10 with trichloroacetonitrile in the
presence of DBU gave the corresponding a-trichloro-
acetimidate 14. Glycosylation of (2S,3R)-3-O-benz-
oyl-2-hexadecanamido-4-octadecane-1,3-diol 152 with
glycosyl donor 14 was carried out in the presence of
˚
TMSOTf and 4 A MS to afford the tetrasaccharide
derivative 16 (52.2%). Finally, removal of the acyl
´
groups of 16 under Zemplen conditions and column
chromatography using a Sephadex LH-20 furnished
the target glycolipid 2 (Scheme 3). The structure and
purity of 2 were demonstrated by 1H NMR and
HR-FABMS data.
Next, the tetrasaccharide derivative 11 was converted
into 4,6-diol acceptor 18 via debenzylation and benzoyl-
ation, followed by removal of the DTBS group. Glyc-
osylation of 18 with 7 in the presence of NIS/TfOH
gave the desired pentasaccharide 19, along with a by-
product 20 (mixed yield: 74.2%). As these compounds
could not be separated at this stage. Therefore, their
structures were confirmed after removal of the DTBS
1
respectively (Scheme 2). The H NMR spectrum of 11
showed 9H signals at d 5.97–5.59 (H-2, 3, 4 of Gal a,
b, c) along with a signal at d 4.87 (d, 1H, J1,2
=
3.7 Hz, H-1d), indicating that the newly formed
1
glycosidic linkage was a-type. Similarly, the H NMR
spectrum of 12 showed 8H signals at d 6.10–5.66
(H-2,3,4 of Gal a,b and H-2,3 of Gal c) along with a
Table 1. Galactosylation under various conditions
Entry
1
Donor 7
Acceptor 6 (70 lmol)
Time
Promoter (equiv)
Solvent
Temp. (°C)
ꢀ20
Product: yield (%)
1.1 equiv
1.0 equiv
30 min
NIS(1.0)–TfOH(0.05)
CH2Cl2 (1 mL)
8: 59
9: 19
8: 56
9: 12
10: 4
8: 53
9: 16
8: 51
9: 17
10: 14
2
1.1 equiv
1.0 equiv
10 min
NIS(1.5)–TfOH(0.20)
CH2Cl2 (1 mL)
ꢀ20
3
4
1.1 equiv
1.5 equiv
1.0 equiv
1.0 equiv
1 h
NIS(1.5)–TfOH(0.30)
NIS(1.5)–TfOH(0.30)
CH2Cl2 (3 mL)
CH2Cl2 (1 mL)
ꢀ60
ꢀ60
30 min

N. Hada et al. / Carbohydrate Research 343 (2008) 2315–2324
2317
OH
O
OH
c
BzO
OBz
t-Bu
OBz
O
Si
t-Bu
O
O
b
O
O
BzO
a
+
OBz
OBz
O
8 + 9 +10
SPh
BnO
O
OBn
a
O
BzO
7
TMS
OBz
6
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
Si
Si
Si
t-Bu
Si
t-Bu
t-Bu
O
d
O
d
O
O
O
O
O
O
O
O
O
O
BnO
BnO
O
BnO
BnO
BnO
BnO
BnO
OBn
O
OR
O
O
OR
O
O
O
O
BzO
c
c
BzO
BzO
OBz
OBz
OBz
OBz
O
OBz
OBz
O
O
O
O
BzO
b
O
O
b
BzO
BzO
O
OBz
OBz
OBz
OBz
OBz
OBz
O
O
O
O
BzO
a
BzO
a
O
TMS
BzO
OBz
O
TMS
OBz
TMS
OBz
10
8 R=H
11 R=Ac
9 R=H
12 R=Ac
b
b
˚
Scheme 2. Reagents and conditions: (a) NIS/TfOH, CH2Cl2, MS4 A; (b) Ac2O, Pyr.
O
OR4
O
OR4
R3O
OR2
O
OR2
HN
C15H31
(CH2)12CH3
d
R2O
HO
OR3
O
OR2
OR2
OAc
OBz
O
O
O
15
BzO
c
R1O
OBz
OBz
OR1
OR1
c
O
O
O
O
b
BzO
R1O
O
O
O
OR1
OR1
OBz
OBz
HN
O
O
a
O
BzO
R1O
R1
OR1
BzO
R2
R1
11 OSE
13 OSE
R2
H
R3 R4
OR1
Bn DTBS
a
16
R1=Bz, R2=Ac
H
Ac
Ac
d
b
CTeS (2) R1, R2=H
14
H OC(NH)CCl3 Ac Ac
Scheme 3. Reagents and conditions: (a) (i) TBAF, AcOH/THF, 99%, (ii) Pd–C, H2, MeOH/THF, (iii) Ac2O, Pyr, 93% (two steps); (b) (i) TFA,
˚
CH2Cl2, (ii) CCl3CN, DBU, CH2Cl2, 94% (two steps); (c) TMSOTf, MS4 A, CH2Cl2, 52%; (d) NaOMe, 1,4-dioxane/MeOH, 88%.
group (21:22 = 1.5:1). After removal of the DTBS
group, debenzylation and acetylation of 19 gave 23. Fur-
thermore, 23 was distinguishable from the debenzylated
and acetylated compound 22 based on the difference in
chemical shifts of their 1H NMR H-4 signals. The 2-(tri-
methylsilyl)ethyl group was removed with trifluoroacetic
acid in dichloromethane and the 1-hydroxy compound
was converted to imidate 24. Glycosylation of 15 with
glycosyl donor 24 was carried out in the presence of
TMSOTf, giving the desired pentasaccharide derivative
25 (52.0%). Finally, removal of the acyl groups of 25
furnished the target glycolipid 3 (Scheme 4). The struc-
1. Experimental
1.1. General
Optical rotations were measured with a Jasco P-1020 dig-
ital polarimeter. H NMR and 13C NMR spectra were
1
recorded with a JMN A500 FT NMR spectrometer with
Me4Si as the internal standard for solutions in CDCl3.
MALDI-TOFMS was recorded on a Perseptive Voyager
RP mass spectrometer. High-resolution mass spectra
were recorded on a JEOL JMS-700 under FAB condi-
tions. TLC was performed on Silica Gel 60 F254 (E.
Merck) with detection by quenching of UV fluorescence
and by charring with 10% H2SO4. Column chromato-
graphy was carried out on Silica Gel 60 (E. Merck). Phen-
yl 2,3-di-O-benzyl-4,6-O-di-tert-butylsilylene-1-thio-b-
D-galactopyranoside (7) and 2-(trimethylsilyl)ethyl
1
ture and purity of 3 were demonstrated by H NMR
and HR-FABMS data. In conclusion, a highly efficient
and systematic synthesis of novel neutral glyco-
sphingolipids from the Zygomycetes species Mucor hie-
malis has been achieved.

2318
N. Hada et al. / Carbohydrate Research 343 (2008) 2315–2324
OR
OR
BnO
OR
OR
BnO
OH
BzO
OH
t-Bu
O
O
O
Si
t-Bu
OBn
O
OBn
O
OH
O
OBz
OAc
O
O
O
OH
BzO
O
O
O
SPh
BnO
BzO
BzO
OBn
OBz
7
OBz
OAc
O
OBz
OBz
OAc
O
b
O
a
O
O
O
+
11
BzO
BzO
BzO
O
OBz
OBz
OBz
OBz
OBz
O
O
20 R = DTBS
22 R = H
OBz
74 %
TMS
19 R = DTBS
21 R = H
c
O
O
O
c
O
BzO
O
BzO
O
BzO
OBz
OBz
OBz
OBz
OBz
O
O
18
O
O
BzO
BzO
TMS
TMS
OBz
OBz
OR2
O
OR2
OAc
OAc
O
OAc
OAc
O
e
R2O
AcO
AcO
O
OAc
OR2
OAc
O
OAc
O
C15H31
OR2
O
OAc
NH
O
O
O
HO
(CH2)12CH3
d
BzO
R1O
BzO
OBz
OAc
OBz
15
f
OR1
OR2
OBz
OAc
O
O
O
O
O
O
d
e
c
BzO
BzO
R1O
OBz
OBz
21
OR1
OBz
OBz
O
OR1
O
O
O
O
O
O
b
BzO
O
O
BzO
R1O
OBz
OBz
OBz
OBz
OR1
OR1
O
O
O
HN
O
a
BzO
O
O
R1O
BzO
23
TMS
OBz
OBz
OR1
O
OR1
CCl3
NH
25
R1=Bz, R2=Ac
g
24
CPS (3) R1, R2=H
Scheme 4. Reagents and conditions: (a) (i) Pd–C, H2, MeOH/THF, (ii) BzCl, Pyr, (iii) TBAF, AcOH/THF, 81%, (three steps); (b) NIS/TfOH,
˚
MS4 A,CH2Cl2, 19, 20 mix.,74%; (C) TBAF, AcOH/THF, 21: 54%, 22: 36%; (d) (i) Pd–C, H2, MeOH/THF, (ii) Ac2O, Pyr, 96%, (two steps); (e) (i)
˚
TFA, CH2Cl2, (ii) CCl3CN, DBU, CH2Cl2, 91% (two steps); (f) TMSOTf, MS4 A, CH2Cl2, 52%; (g) NaOMe, 1,4-dioxane/MeOH, 92%.
2,3-di-O-benzyl-4,6-O-di-tert-butylsilylene-a-D-galacto-
pyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyrano-
syl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyranoside (4)
were prepared as reported in our previous paper.7
5.00 (d, 1H, J3,4 = 3.1 Hz, H-4 of Gal c), 4.90 (d, 1H,
J1,2 = 7.9 Hz, H-1 of Gal b), 4.78 (d, 1H, J1,2
7.9 Hz, H-1 of Gal a), 4.44 (d, 1H, J6a,6b = 12.2 Hz,
H-6a of Gal c), 4.33 (d, 1H, H-6b of Gal c), 4.23–4.17
(m, 3H, H-5 of Gal a, b, H-6a of Gal b), 4.05 (s, 1H,
H-5 of Gal c), 4.00–3.90 (m, 2H, H-6b of Gal b,
=
1.2. 2-(Trimethylsilyl)ethyl 2,3-di-O-benzoyl-4,6-O-di-
tert-butylsilylene-a-D-galactopyranosyl-(1?6)-2,3,4-tri-
O-benzoyl-b-D-galactopyranosyl-(1?6)-2,3,4-tri-O-benz-
oyl-b-D-galactopyranoside (5)
CH2CH2O), 3.73 (dd, 1H, J5,6a = 6.1 Hz, J6a,6b
=
11.0 Hz, H-6a of Gal a), 3.58–3.52 (m, 2H, H-6b of
Gal a, CH2CH2O), 1.20 and 1.04 (2s, 18H, 2 t-Bu),
0.94–0.76 (m, 2H, CH2CH2O), ꢀ0.15 (s, 9H, Si(CH3)3).
13C NMR (125 MHz, CDCl3) d 166.1, 165.9, 165.4,
165.2, 133.4, 133.1, 130.0, 129.8, 129.7, 129.5, 129.4,
129.0, 128.9, 128.5, 128.4, 128.3, 128.25, 128.20, 101.0
(C-1 of Gal b), 100.9 (C-1 of Gal a), 97.7 (C-1 of Gal
c), 72.9, 72.1, 72.0, 71.8, 71.1, 70.9, 69.9, 69.8, 68.6,
68.3, 67.9, 67.54, 67.46, 67.4, 66.9, 66.6, 27.7, 27.5,
27.3, 23.2, 20.7, 17.7 (–OCH2CH2), ꢀ1.5 (Si(CH3)3).
A solution of 4 (530 mg, 0.34 mmol) in THF (1.0 mL)
and MeOH (2.0 mL) was stirred in the presence of
10% Pd–C (550 mg) for 1 h at room temperature under
an H2 atmosphere, then filtered and concentrated. The
residue was benzoylated with benzoyl chloride
(151 lL, 1.37 mmol) in pyridine (3 mL). The reaction
mixture was poured into ice-water and extracted with
CHCl3. The extract was washed sequentially with 5%
HCl, aq NaHCO3 and water, dried (MgSO4), and con-
centrated. The product was purified by silica gel column
chromatography using 20:1 toluene/acetone as the elu-
MALDI-TOFMS:
calcd
for
C87H92O24Si2Na
[M+Na]+: m/z 1599.5. Found: m/z 1599.9.
1.3. 2-(Trimethylsilyl)ethyl 2,3-di-O-benzoyl-a-D-galac-
topyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyr-
anosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyranoside
(6)
24
ent to give 5 (496 mg, 85.5%): ½aꢁD +160.2 (c 1.0,
1
CHCl3). H NMR (500 MHz, CDCl3) d 8.16–7.24 (m,
40H, 8Ph), 5.97 (d, 1H, J3,4 = 3.7 Hz, H-4 of Gal b),
5.89 (d, 1H, J3,4 = 3.7 Hz, H-4 of Gal a), 5.86–5.77
(m, 3H, H-2 of Gal a, b, c), 5.61–5.56 (m, 3H, H-3 of
Gal a, b, c), 5.21 (d, 1H, J1,2 = 3.7 Hz, H-1 of Gal c),
A solution of 5 (371 mg, 0.24 mmol) and acetic acid
(61 lL, 1.1 mmol) in THF (2 mL) was treated with

N. Hada et al. / Carbohydrate Research 343 (2008) 2315–2324
2319
1 M TBAF in THF (590 lL, 0.56 mmol) at room tem-
perature and then was stirred for 12 h. After concentra-
tion, the residue was added to the water, extracted with
ethyl acetate, and the organic layer was proceeded as
usual. The product was purified by silica gel column
chromatography using 3:1 toluene/acetone as the eluent
b), 4.68 (d, 1H, J1,2 = 9.8 Hz, H-1 of Gal a). 13C
NMR (125 MHz, CDCl3) d 166.1, 165.8, 165.5, 165.4,
165.3, 165.2, 165.11, 165.08, 138.9, 138.1, 133.4, 133.3,
133.1, 133.03, 133.00, 132.8, 130.04, 129.96, 129.84,
129.78, 129.7, 129.6, 129.4, 129.2, 129.0, 128.8, 128.6,
128.5, 128.4, 128.3, 128.20, 128.15, 128.1, 127.7, 127.6,
127.4, 101.0 (C-1 of Gal b), 100.8 (C-1 of Gal a), 99.2
(C-1 of Gal d), 97.4 (C-1 of Gal c), 77.7, 73.9, 72.8,
72.2, 71.9, 71.7, 71.0, 70.9, 69.9, 69.8, 68.9, 68.8, 68.7,
68.5, 68.1, 67.7, 67.44, 67.38, 67.2, 66.5, 29.6, 27.6,
27.3, 23.4, 20.6, 17.7 (–OCH2CH2), ꢀ1.5 (Si(CH3)3).
24
to give 6 (322 mg, 95.3%): ½aꢁD +152.9 (c 0.8, CHCl3).
1H NMR (500 MHz, CDCl3) d 8.13–7.26 (m, 40H, 8
Ph), 6.00 (d, 1H, J3,4 = 3.7 Hz, H-4 of Gal a), 5.93 (d,
1H, J3,4 = 3.7 Hz, H-4 of Gal b), 5.81–5.67 (m, 4H,
H-2 of Gal a, b, c, H-3 of Gal c), 5.59–5.50 (m, 2H,
H-3 of Gal a, b), 5.27 (d, 1H, J1,2 = 3.7 Hz, H-1 of
Gal c), 4.91 (d, 1H, J1,2 = 7.9 Hz, H-1 of Gal b), 4.79
(d, 1H, J1,2 = 7.9 Hz, H-1 of Gal a), 4.79 (s, 1H, H-4
of Gal c), 4.28–4.19 (m, 4H, H-5 of Gal a, b, H-6a of
Gal c, H-6b of Gal c), 4.02 (d, 1H, J5,6a = 4.9 Hz, H-5
of Gal c), 3.98–3.93 (m, 3H, H-6a of Gal a, H-6b of
Gal a, CH2CH2O), 3.74–3.67 (m, 2H, H-6a of Gal b,
H-6b of Gal b), 3.57 (dt, 1H, CH2CH2O), 0.95–0.74
(m, 2H, CH2CH2O), ꢀ0.17 (s, 9H, Si(CH3)3).13C
NMR (125 MHz, CDCl3) d 166.1, 165.6, 165.5, 165.4,
165.2, 133.4, 133.2, 133.0, 130.0, 129.8, 129.74, 129.71,
129.5, 129.4, 129.2, 129.0, 128.9, 128.8, 128.54, 128.48,
128.34, 128.29, 128.2, 101.1 (C-1 of Gal b), 100.8 (C-1
of Gal a), 97.9 (C-1 of Gal c), 73.1, 72.3, 72.1, 72.0,
70.9, 70.3, 69.9, 69.7, 69.2, 68.8, 68.7, 68.1, 68.0, 67.6,
67.4, 62.7, 17.7 (–OCH2CH2), ꢀ1.5 (Si(CH3)3). MAL-
DI-TOFMS: calcd for C79H76O24SiNa [M+Na]+: m/z
1459.4. Found: m/z 1459.5.
MALDI-TOFMS:
calcd
for
C107H114O29Si2Na
[M+Na]+: m/z 1941.7. Found: m/z 1941.8. Compound
24
9: ½aꢁD +122.2 (c 1.0, CHCl3). 1H NMR (500 MHz,
CDCl3) d 4.92 (d, 1H, J1,2 = 3.7 Hz, H-1 of Gal c),
4.83 (d, 1H, J1,2 = 7.97 Hz, H-1 of Gal b), 4.71 (d, 1H,
J1,2 = 2.4 Hz, H-1 of Gal d), 4.70 (d, 1H,
J1,2 = 9.8 Hz, H-1 of Gal a). 13C NMR (125 MHz,
CDCl3) d 165.9, 165.7, 165.4, 165.2, 138.7, 137.2,
133.3, 133.1, 132.9, 130.0, 129.74, 129.68, 129.4, 129.3,
129.2, 129.0, 128.8, 128.5, 128.4, 128.3, 128.2, 128.11,
128.05, 127.6, 101.0 (C-1 of Gal b), 100.7 (C-1 of Gal
a), 100.4 (C-1 of Gal d), 97.4 (C-1 of Gal c), 77.5,
76.9, 74.73, 74.68, 73.0, 72.3, 72.0, 71.8, 70.8, 70.5,
70.02, 69.99, 69.8, 68.8, 68.6, 68.1, 67.8, 67.3, 67.1,
66.2, 60.1, 29.7, 27.6, 27.3, 23.4, 20.6, 17.7 (–OCH2-
CH2), ꢀ1.5 (Si(CH3)3). MALDI-TOFMS: calcd for
C107H114O29Si2Na [M+Na]+: m/z 1941.7. Found: m/z
1941.8.
1.4. 2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-4,6-O-di-tert-
butylsilylene-a-D-galactopyranosyl-(1?6)-2,3-di-O-benz-
oyl-a-D-galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galacto-
pyranoside (8), 2-(trimethylsilyl)ethyl 2,3-di-O-benzyl-
4,6-O-di-tert-butylsilylene-a-D-galactopyranosyl-(1?4)-
2,3-di-O-benzoyl-a-D-galactopyranosyl-(1?6)-2,3,4-tri-
O-benzoyl-b-D-galactopyranosyl-(1?6)-2,3,4-tri-O-benz-
oyl-b-D-galactopyranoside (9)
1.5. 2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-4,6-O-di-tert-
butylsilylene-a-D-galactopyranosyl-(1?6)-4-O-acetyl-
2,3-di-O-benzoyl-a-D-galactopyranosyl-(1?6)-2,3,4-tri-
O-benzoyl-b-D-galactopyranosyl-(1?6)-2,3,4-tri-O-ben-
zoyl-b-D-galactopyranoside (11)
A solution of 8 (622 mg, 0.32 mmol) in pyridine (3 mL)
and Ac2O (2 mL) was stirred for 12 h at room tempera-
ture. Toluene was added and evaporated, then extracted
with CHCl3, washed with 5% HCl, aq NaHCO3 and
water, dried (MgSO4), and concentrated. The product
was purified by silica gel column chromatography using
15:1 toluene/EtOAc as the eluent to give 11 (607 mg,
A solution of compound 7 (61.8 mg, 0.10 mmol) and 6
˚
(100 mg, 69.6 lmol) containing activated 4 A MS
(300 mg) in a dry CH2Cl2 (3 mL) was stirred under an
atmosphere of argon for 2 h at room temperature. After
cooling to ꢀ60 °C, successively NIS (35 mg, 0.85 mmol)
and TfOH (2.8 lL, 0.17 mmol) were added and stirring
was continued at ꢀ60 °C for 30 min, then neutralized
with Et3N. The reaction mixture was filtered, and the fil-
trate was washed with aq sodium thiosulfate, dried
(MgSO4), and concentrated. The product was purified
by silica gel column chromatography using 15:1 hex-
ane/EtOAc as the eluent to give 8 (102 mg, 76.6%) and
24
95.6%): ½aꢁD +126.9 (c 1.0, CHCl3). 1H NMR
(500 MHz, CDCl3) d 8.12–7.26 (m, 50H, 10Ph), 5.97
(d, 1H, J3,4 = 3.7 Hz, H-4 of Gal c), 5.90 (d, 1H,
J3,4 = 3.7 Hz, H-4 of Gal b), 5.87–5.78 (m, 4H, H-2 of
Gal a, 2 of Gal b, 3 of Gal c, 4 of Gal a), 5.69 (dd,
1H, J1,2 = 3.7 Hz, J2,3 = 10.4 Hz, H-2 of Gal c), 5.63
(dd, 1H, J2,3 = 10.4 Hz, H-3 of Gal b), 5.59 (dd, 1H,
J2,3 = 10.4 Hz, J3,4 = 3.1 Hz, H-3 of Gal a), 5.21 (d,
1H, H-1 of Gal c), 4.95–4.81 (m, 4H, 2 benzylmethyl-
ene), 4.91 (d, 1H, J1,2 = 8.6 Hz, H-1 of Gal b), 4.87 (d,
1H, J1,2 = 3.7 Hz, H-1 of Gal d), 4.74 (d, 1H,
J1,2 = 7.9 Hz, H-1 of Gal a), 4.59 (d, 1H,
J3,4 = 3.1 Hz, H-4 of Gal d), 4.43 (t, 1H,
24
9 (21.0 mg, 15.7%). Compound 8: ½aꢁD +131.9 (c 1.0,
1
CHCl3). H NMR (500 MHz, CDCl3) d 5.13 (d, 1H,
J1,2 = 3.7 Hz, H-1 of Gal c), 4.91 (d, 1H, J1,2 = 3.7 Hz,
H-1 of Gal d), 4.79 (d, 1H, J1,2 = 7.9 Hz, H-1 of Gal

2320
N. Hada et al. / Carbohydrate Research 343 (2008) 2315–2324
J5,6a = J5,6b = 6.1 Hz, H-5 of Gal a), 4.32–4.24 (m, 3H,
H-6a of Gal b, c, H-6b of Gal c), 4.21–4.17 (m, 2H,
H-6a of Gal d, H-6b of Gal d), 4.11 (dd, 1H,
J2,3 = 10.4 Hz, H-2 of Gal d), 4.02–3.97 (dd, 1H,
CH2CH2O), 3.93 (dd, 1H, H-3 of Gal d), 3.88 (dd,
1H, J5,6b = 6.7 Hz, J6a,6b = 9.8 Hz, H-6b of Gal b),
3.83–3.76 (m, 4H, H-5 of Gal b, c, d, H-6a of Gal a),
3.59–3.52 (m, 2H, H-6b of Gal a, CH2CH2O), 2.18 (s,
3H, Ac), 1.16 and 1.12 (2s, 18H, 2 t-Bu), 0.94–0.76 (m,
2H, CH2CH2O), ꢀ0.12 (s, 9H, Si(CH3)3). 13C NMR
(125 MHz, CDCl3) d 169.9, 166.1, 165.4, 165.2, 165.1,
139.1, 138.6, 137.8, 133.3, 132.0, 130.0, 129.8, 129.74,
129.68, 129.6, 129.5, 129.4, 129.3, 129.0, 128.8, 128.5,
128.43, 128.38, 128.3, 128.2, 128.2, 128.11, 128.08,
127.7, 127.6, 127.34, 127.25, 100.9 (C-1 of Gal b),
100.8 (C-1 of Gal a), 99.0 (C-1 of Gal d), 97.2 (C-1 of
Gal c), 77.6, 74.4, 73.6, 72.7, 72.0, 71.9, 71.8, 71.14,
71.06, 70.0, 69.8, 68.7, 68.5, 68.2, 68.0, 67.7, 67.3, 67.1,
66.9, 66.3, 27.7, 27.3, 23.3, 21.4, 20.62, 20.59, 17.7
(–OCH2CH2), ꢀ1.5 (Si(CH3)3). MALDI-TOFMS: calcd
for C109H116O30Si2Na [M+Na]+: m/z 1983.7. Found:
m/z 1983.4.
1.7. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-a-D-
galactopyranosyl-(1?6)-4-O-acetyl-2,3-di-O-benzoyl-a-
D-galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galac-
topyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyr-
anoside (13)
A solution of 11 (400 mg, 0.20 mmol) and acetic acid
(53 lL, 0.31 mmol) in THF (2 mL) was treated with
1 M TBAF in THF (408 lL, 0.41 mmol) at room tem-
perature and then was stirred for 12 h. After concentra-
tion, the residue was added to the water, extracted with
ethyl acetate, and the organic layer was proceeded as
usual. The product was purified by silica gel column
chromatography using 5:1 toluene/acetone as the eluent
to give the diol compound (366 mg, 98.5%). To a solu-
tion of the compound (366 mg, 0.29 mmol) in THF
(1.5 mL) and MeOH (3.0 mL) was stirred in the pres-
ence of 10% Pd–C (400 mg) for 1 h at room temperature
under an H2 atmosphere, then filtered and concentrated.
The residue was treated with Ac2O (2 mL) in pyridine
(3 mL). The reaction mixture was poured into ice-water
and extracted with CHCl3. The extract was washed
sequentially with 5% HCl, aq NaHCO3 and water, dried
(MgSO4), and concentrated. The product was purified
by silica gel column chromatography using 10:1 tolu-
ene/acetone as the eluent to give 13 (356 mg, 93.0%).
1.6. 2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-4,6-O-di-tert-
butylsilylene-a-D-galactopyranosyl-(1?4)-6-O-acetyl-
2,3-di-O-benzoyl-a-D-galactopyranosyl-(1?6)-2,3,4-tri-
O-benzoyl-b-D-galactopyranosyl-(1?6)-2,3,4-tri-O-benz-
oyl-b-D-galactopyranoside (12)
24
½aꢁD +125.3 (c 1.2, CHCl3). 1H NMR (500 MHz,
CDCl3) d 8.12–7.26 (m, 40H, 8Ph), 5.19 (d, 1H,
J1,2 = 3.1 Hz, H-1 of Gal c), 5.04 (d, 1H, J1,2 = 3.1 Hz,
H-1 of Gal d), 4.97 (d, 1H, J1,2 = 7.9 Hz, H-1 of Gal
b), 4.82 (d, 1H, J1,2 = 7.9 Hz, H-1 of Gal a), 2.25,
2.21, 2.20, 2.11 and 2.10 (each s, 15H, 5Ac), 0.94–0.77
(m, 2H, CH2CH2O), ꢀ0.12 (s, 9H, Si(CH3)3). 13C
NMR (125 MHz, CDCl3) d 170.8, 170.4, 170.2, 169.9,
169.6, 166.1, 165.4, 165.3, 165.2, 165.1, 133.33, 133.26,
133.1, 133.0, 130.1, 130.0, 129.8, 129.73, 129.68, 129.5,
129.4, 129.2, 129.0, 128.9, 128.5, 128.29, 128.26, 128.2,
128.13, 101.0 (C-1 of Gal b), 100.8 (C-1 of Gal a),
97.5 (C-1 of Gal c), 96.9 (C-1 of Gal d), 72.7, 71.9,
71.8, 70.0, 69.9, 68.6, 68.5, 68.1, 67.9, 67.5, 67.4, 67.3,
66.5, 66.1, 65.7, 61.7, 20.6, 20.5, 17.7 (–OCH2CH2),
ꢀ1.5 (Si(CH3)3). MALDI-TOFMS: calcd for
C95H96O34SiNa [M+Na]+: m/z 1831.5. Found: m/z
1831.7.
Compound 12 was derived according to the procedure
described for the treatment of 11. Compound 9
24
(275 mg, 0.14 mmol)?12 (255 mg, 90.7%): ½aꢁD +139.9
1
(c 1.0, CHCl3). H NMR (500 MHz, CDCl3) d 8.18–
7.22 (m, 50H, 10 Ph), 6.10 (d, 1H, J3,4 = 3.7 Hz, H-4
of Gal b), 5.97 (d, 1H, J3,4 = 3.7 Hz, H-4 of Gal a),
5.90–5.84 (m, 2H, H-2 of Gal a, 2 of Gal b), 5.79–5.66
(m, 4H, H-2 of Gal c, H-3 of Gal a, b, c), 5.17 (d, 1H,
J1,2 = 3.1 Hz, H-1 of Gal c), 4.99 (d, 1H, J1,2 = 7.9 Hz,
H-1 of Gal b), 4.98–4.79 (m, 4H, 2 benzylmethylene),
4.93 (d, 1H, J1,2 = 3.1 Hz, H-1 of Gal d), 4.87 (d, 1H,
J1,2 = 7.9 Hz, H-1 of Gal a), 4.66 (s, 1H, H-4 of Gal
d), 4.57 (br d, H-4 of Gal c), 2.21 (s, 3H, Ac), 1.13
and 1.06 (2s, 18H, 2 t-Bu), 0.91–0.72 (m, 2H,
CH2CH2O), ꢀ0.12 (s, 9H, Si(CH3)3). 13C NMR
(125 MHz, CDCl3) d 166.1, 165.7, 165.6, 165.2, 189.0,
138.2, 133.3, 133.0, 130.1, 130.0, 129.82, 129.76, 129.7,
129.4, 129.2, 129.0, 128.8, 128.6, 128.5, 128.4, 128.34,
128.26, 128.2, 128.1, 127.6, 127.5, 127.3, 100.9 (C-1 of
Gal b), 100.7 (C-1 of Gal a), 100.4 (C-1 of Gal d),
96.8 (C-1 of Gal c), 77.8, 76.4, 74.0, 73.8, 73.2, 72.4,
71.9, 71.7, 70.8, 70.6, 70.3, 70.1, 69.9, 69.7, 69.0, 68.4,
68.2, 68.1, 67.3, 66.9, 66.1, 62.9, 27.6, 27.2, 23.3, 20.9,
20.7, 17.6 (–OCH2CH2), ꢀ1.5 (Si(CH3)3). MALDI-
TOFMS: calcd for C109H116O30Si2Na [M+Na]+: m/z
1983.7. Found: m/z 1983.5.
1.8. 2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl-(1?6)-
4-O-acetyl-2,3-di-O-benzoyl-a-D-galactopyranosyl-
(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyranosyl-(1?6)-
2,3,4-tri-O-benzoyl-a-D-galactopyranosyl trichloro-
acetimidate (14)
To a solution of 13 (283 mg, 0.16 mmol) in CH2Cl2
(2.0 mL), cooled to 0 °C, was added trifluoroacetic acid
(4.0 mL) and the mixture was stirred for 1.5 h at room
temperature and concentrated. Ethyl acetate and
toluene (1:2) were added and evaporated to give the

N. Hada et al. / Carbohydrate Research 343 (2008) 2315–2324
2321
1-hydroxy compound. To a solution of the residue in
CH2Cl2 (2.0 mL) cooled at 0 °C were added trichloro-
acetonitrile (157 lL, 1.56 mmol) and DBU (23 lL,
0.16 mmol). The reaction mixture was stirred for 1.5 h
at 0 °C. After completion of the reaction, the mixture
was concentrated. Column chromatography of the resi-
due on silica gel (1:2 hexane/ethyl acetate) gave 14
(272 mg, 94.0%): ½aꢁD +145.7 (c 1.0, CHCl3). H NMR
(500 MHz, CDCl3) d 8.51–7.17 (m, 41H, 8Ph, NH),
6.76 (d, 1H, J1,2 = 3.7 Hz, H-1 of Gal a), 5.06 (d, 1H,
J1,2 = 3.7 Hz, H-1 of Gal c), 4.93 (d, 1H, J1,2 = 3.1 Hz,
H-1 of Gal d), 4.83 (d, 1H, J1,2 = 7.9 Hz, H-1 of Gal
b), 2.13, 2.090, 2.086, 1.99 and 1.97 (each s, 15H,
5Ac). 13C NMR (125 MHz, CDCl3) d 170.7, 170.3,
170.1, 169.8, 169.6, 166.1, 165.6, 165.3, 165.1, 160.4,
133.4, 133.2, 133.1, 133.0, 130.0, 129.9, 129.8, 129.7,
129.5, 129.3, 129.0, 128.8, 128.6, 128.4, 128.3, 128.2,
128.1, 101.0 (C-1 of Gal b), 97.4 (C-1 of Gal c), 96.9
(C-1 of Gal d), 93.6 (C-1 of Gal a), 90.7 (CCl3), 71.8,
71.0, 69.7, 68.8, 68.5, 68.3, 68.2, 68.1, 67.9, 67.6, 67.41,
67.38, 66.5, 66.2, 65.7, 61.70, 61.65, 20.6, 20.5. MAL-
DI-TOFMS: calcd for C92H84Cl3NO34Na [M+Na]+:
m/z 1874.4. Found: m/z 1875.8.
128.31, 128.26, 128.18, 128.15, 125.8, 101.1 (C-1 of
Gal b), 100.5 (C-1 of Gal a), 97.6 (C-1 of Gal c), 97.0
(C-1 of Gal d), 73.4, 72.8, 71.8, 71.7, 71.44, 71.36,
70.6, 69.8, 68.5, 68.4, 68.3, 68.1, 67.9, 67.8, 67.6, 67.43,
67.39, 66.9, 66.5, 66.1, 65.8, 61.7, 50.1, 36.9, 36.5, 31.9,
31.5, 29.7, 29.6, 29.5, 29.3, 29.1, 25.7, 25.6, 25.5,
22.7, 20.6, 20.5, 14.1. MALDI-TOFMS: calcd for
C131H155NO37Na [M+Na]+: m/z 2357.0. Found: m/z
2357.1.
24
1
1.10. a-D-Galactopyranosyl-(1?6)-a-D-galactopyran-
osyl-(1?6)-b-D-galactopyranosyl-(1?6)-b-D-galac-
topyranosiyl-(1?1)-(2S,3R)-2-hexadecanamido-
octadecane-1,3-diol (2)
To a solution of 16 (28.5 mg, 12.2 lmol) in MeOH
(1.0 mL) and 1,4-dioxane (1.0 mL) was added NaOMe
(25 mg) at room temperature and the mixture was stir-
red for 12 h, then neutralized with Amberlite IR
120[H+]. The mixture was filtered off and concentrated.
The product was purified by sephadex LH-20 column
chromatography (1:1 CHCl3/MeOH) to give
2
24
(12.7 mg, 87.6%): ½aꢁD +54.4 (c 0.3, CHCl3/
1
MeOH = 1:1). H NMR (500 MHz, DMSO-d6–D2O) d
4.72 (d, 1H, J1,2 = 3.7 Hz, H-1 of Gal c), 4.69 (d, 1H,
J1,2 = 3.1 Hz, H-1 of Gal d), 4.17 (d, 1H,
J1,2 = 7.3 Hz, H-1 of Gal b), 4.07 (br d, 1H, H-1 of
Gal a), 1.58–1.08 (m, 54H, 27 –CH2–), 0.82 (t, 6H, 2
CH3). HR-FABMS: calcd for C58H109NO23Na
[M+Na]+: m/z 1210.7288. Found: m/z 1210.7238.
1.9. 2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl-(1?6)-
4-O-acetyl-2,3-di-O-benzoyl-a-D-galactopyranosyl-
(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyranosyl-(1?6)-
2,3,4-tri-O-benzoyl-b-D-galactopyranosyl-(1?1)-
(2S,3R)-3-O-benzoyl-2-hexadecanamidooctadecane-1,3-
diol (16)
To a solution of 14 (68 mg, 36.7 lmol) and (2S,3R)-3-
O-benzoyl-2-hexadecanamido-4-octadecane-1,3-diol 15
(47 mg, 73.4 lmol) in dry CH2Cl2 (1.0 mL) was added
1.11. 2-(Trimethylsilyl)ethyl 2,3-di-O-benzoyl-a-D-galac-
topyranosyl-(1?6)-4-O-acetyl-2,3-di-O-benzoyl-a-D-
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galacto-
pyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyran-
oside (18)
˚
4 A MS (500 mg), and the mixture was stirred for 2 h
at room temperature, then cooled to 0 °C. TMSOTf
(6 lL, 33.0 lmol) was added, and the mixture was stir-
red for 1.5 h at 0 °C, then neutralized with Et3N. The
solids were filtrated off and washed with CHCl3. The
combined filtrate and washings were successively
washed with water, dried (MgSO4), and concentrated.
The product was purified by silica gel column chroma-
tography using 3:1 toluene/EtOAc as the eluent to give
To a solution of 11 (220 mg, 0.11 mmol) in THF
(1.5 mL) and MeOH (3.0 mL) was added 10% Pd–C
(250 mg), and the reaction mixture was stirred in a H2
atmosphere for 40 min. at room temperature, then fil-
tered and concentrated. The residue was benzoylated
with benzoyl chloride (52 lL, 0.45 mmol) in pyridine
(3 mL) for 3 h. The reaction mixture was poured into
ice-water and extracted with CHCl3. The extract was
washed sequentially with 5% HCl, aq NaHCO3 and
water, dried (MgSO4), and concentrated. The product
was purified by silica gel column chromatography using
10:1 toluene/acetone as the eluent to give the compound
(212 mg, 94.8%). A solution of this compound (221 mg,
0.22 mmol) and acetic acid(29 lL, 0.50 mmol)in THF
(2 mL) was treated with 1M TBAF in THF (222 lL,
0.22 mmol) at room temperature and then was stirred
for 12 h. After concentration, the residue was added to
the water, extracted with ethyl acetate, and the organic
24
16 (44.7 mg, 52.2%). ½aꢁD +96.8 (c 0.4, CHCl3). 1H
NMR (500 MHz, CDCl3) d 8.11–7.19 (m, 45H, 9Ph),
5.17 (d, 1H, J1,2 = 3.7 Hz, H-1 of Gal c), 4.92 (d, 1H,
J1,2 = 3.7 Hz, H-1 of Gal d), 4.56 (d, 1H, J1,2 = 7.3Hz,
H-1 of Gal b), 4.36 (d, 1H, J1,2 = 7.9Hz, H-1 of Gal
a), 2.12, 2.09, 2.07, 1.98 and 1.95 (each s, 15H, 5Ac),
1.75–1.09 (m, 54H, 27 ꢀCH2–), 0.88 (t, 6H, 2CH3).
13C NMR (125 MHz, CDCl3) d 172.4, 170.7, 170.3,
170.1, 169.8, 169.6, 166.1, 165.4, 165.3, 165.2, 165.1,
165.0, 133.5, 133.4, 133.3, 133.1, 133.0, 132.8, 130.7,
130.2, 130.0, 129.9, 129.8, 129.73, 129.66, 129.6,
129.5, 129.4, 129.3, 129.0, 128.8, 128.6, 128.5, 128.4,

2322
N. Hada et al. / Carbohydrate Research 343 (2008) 2315–2324
layer was proceeded as usual. The product was purified
by silica gel column chromatography using 5:1 toluene/
NMR (500 MHz, CDCl3) d 8.00–7.15 (m, 60H, 12Ph),
5.32 (d, 1H, J1,2 = 3.7 Hz, H-1 of Gal c), 5.13 (d, 1H,
J1,2 = 3.7 Hz, H-1 of Gal e), 5.00 (d, 1H, J1,2 = 3.7 Hz,
H-1 of Gal d), 4.87 (d, 1H, J1,2 = 7.9 Hz, H-1 of Gal
b), 4.69 (d, 1H, J1,2 = 7.9 Hz, H-1 of Gal a), 2.06 (s,
3H, Ac), 0.83–0.66 (m, 2H, CH2CH2O), ꢀ0.13 (s, 9H,
Si(CH3)3). 13C NMR (125 MHz, CDCl3) d 169.7,
166.1, 166.0, 165.8, 165.5, 165.31, 165.26, 165.2, 165.1,
138.1, 133.3, 133.2, 133.0, 130.0, 129.9, 129.8, 129.7,
129.6, 129.5, 129.4, 129.2, 129.0, 128.9, 128.8, 128.44,
128.39, 128.3, 128.23, 128.15, 127.7, 100.9 (C-1 of Gal
b), 100.7 (C-1 of Gal a), 98.4 (C-1 of Gal e), 97.6 (C-1
of Gal c), 97.3 (C-1 of Gal d), 77.5, 75.3, 73.4, 72.7,
72.6, 72.0, 71.8, 71.7, 71.0, 70.02, 69.98, 69.8, 68.9,
68.8, 68.5, 68.3, 67.9, 67.6, 67.34, 67.28, 67.2, 66.9,
66.4, 62.8, 29.6, 20.5, 17.7 (–OCH2CH2), ꢀ1.5
(Si(CH3)3). MALDI-TOFMS: calcd for C121H118O37Si-
Na [M+Na]+: m/z 2213.7. Found: m/z 2213.3. Com-
24
acetone as the eluent to give 18 (177 mg, 85.8%): ½aꢁD
1
+138.1 (c 2.0, CHCl3). H NMR (500 MHz, CDCl3) d
8.19–7.28 (m, 50H, 10Ph), 5.47 (d, 1H, J1,2 = 3.7 Hz,
H-1 of Gal c), 5.22 (d, 1H, J1,2 = 3.7 Hz, H-1 of Gal
d), 5.05 (d, 1H, J1,2 = 7.9 Hz, H-1 of Gal b), 4.86 (d,
1H, J1,2 = 7.9 Hz, H-1 of Gal a), 2.16 (s, 3H, Ac),
0.97–0.81 (m, 2H, CH2CH2O), ꢀ0.12 (s, 9H, Si(CH3)3).
13C NMR (125 MHz, CDCl3) d 169.6, 166.1, 166.0,
165.7, 165.6, 165.44, 165.41, 165.2, 165.13, 165.10,
133.3, 133.0, 130.0, 129.9, 129.8, 129.69, 129.66, 129.5,
129.31, 129.27, 129.1, 129.0, 128.8, 128.7, 128.43,
128.36, 128.3, 128.21, 128.18, 128.15, 128.1, 101.0 (C-1
of Gal b), 100.7 (C-1 of Gal a), 98.0 (C-1 of Gal c),
97.6 (C-1 of Gal d), 72.8, 72.0, 71.8, 71.0, 70.2, 69.9,
69.8, 69.2, 68.8, 68.6, 68.5, 68.0, 67.8, 67.54, 67.51,
67.3, 66.7, 62.6, 20.4, 17.7 (–OCH2CH2), ꢀ1.5
(Si(CH3)3). MALDI-TOFMS: calcd for C101H96O32Si-
Na [M+Na]+: m/z 1871.6. Found: m/z 1871.3.
24
pound 22: ½aꢁD +132.5 (c 0.6, CHCl3). 1H NMR
(500 MHz, CDCl3) d 8.00–7.16 (m, 60H, 12 Ph), 5.06
(d, 1H, J1,2 = 3.7 Hz, H-1 of Gal c), 5.04 (d, 1H,
J1,2 = 3.7 Hz, H-1 of Gal d), 4.82 (d, 1H,
J1,2 = 3.7 Hz, H-1 of Gal e), 4.67 (d, 1H, J1,2 = 7.9 Hz,
H-1 of Gal b), 4.66 (d, 1H, J1,2 = 8.5Hz, H-1 of Gal
a), 4.47 (br d, 1H, H-4 of Gal d), 2.04 (s, 3H, Ac),
0.89–0.67 (m, 2H, CH2CH2O), ꢀ0.12 (s, 9H, Si(CH3)3).
13C NMR (125 MHz, CDCl3) d 166.4, 166.1, 166.0,
165.4, 165.3, 165.18, 165.15, 165.1, 137.7, 137.1, 133.3,
133.2, 133.1, 133.0, 130.1, 130.04, 129.96, 129.9, 129.8,
129.7, 129.6, 129.4, 129.33, 129.28, 129.2, 129.1, 128.6,
128.53, 128.46, 128.4, 128.3, 128.2, 128.13, 128.10,
128.0, 127.9, 127.8, 100.8 (C-1 of Gal a, b), 100.3 (C-1
of Gal e), 97.9 (C-1 of Gal c), 97.4 (C-1 of Gal d),
77.6, 77.5, 76.3, 74.8, 72.7, 72.3, 72.02, 71.97, 71.8,
70.8, 70.7, 70.0, 69.9, 68.6, 68.51, 68.45, 68.3, 68.1,
67.8, 67.4, 67.0, 66.5, 66.4, 62.9, 60.5, 27.2, 20.5, 17.7
(–OCH2CH2), ꢀ1.5 (Si(CH3)3). MALDI-TOFMS: calcd
for C121H118O37SiNa [M+Na]+: m/z 2213.7. Found: m/z
2213.6.
1.12. 2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-4,6-O-di-
tert-butylsilylene-a-D-galactopyranosyl-(1?6)-2,3-di-O-
benzoyl-a-D-galactopyranosyl-(1?6)-4-O-acetyl-2,3-di-
O-benzoyl-a-D-galactopyranosyl-(1?6)-2,3,4-tri-O-benz-
oyl-b-D-galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-
galactopyranoside (19), 2-(trimethylsilyl)ethyl 2,3-di-O-
benzyl-4,6-O-di-tert-butylsilylene-a-D-galactopyranosyl-
(1?4)-2,3-di-O-benzoyl-a-D-galactopyranosyl-(1?6)-4-
O-acetyl-2,3-di-O-benzoyl-a-D-galactopyranosyl-(1?6)-
2,3,4-tri-O-benzoyl-b-D-galactopyranosyl-(1?6)-2,3,4-
tri-O-benzoyl-b-D-galactopyranoside (20)
Compound 20 was derived according to the procedure
described for the treatment of 8. Compound 18
(146 mg, 78.7 lmol)?20 (136 mg, 74.2%): MALDI-
TOFMS: calcd for C129H134O37Si2Na [M+Na]+: m/z
2353.8. Found: m/z 2353.2.
1.13. 2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-a-D-galac-
topyranosyl-(1?6)-2,3-di-O-benzoyl-a-D-galactopyran-
osyl-(1?6)-4-O-acetyl-2,3-di-O-benzoyl-a-D-galacto-
pyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyrano-
syl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyranoside
(21), 2-(trimethylsilyl)ethyl 2,3-di-O-benzyl-a-D-galacto-
pyranosyl-(1?4)-2,3-di-O-benzoyl-a-D-galactopyranosyl-
(1?6)-4-O-acetyl-2,3-di-O-benzoyl-a-D-galactopyrano-
syl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyranosyl-
(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyranoside (22)
1.14. 2-(Trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-a-D-
galactopyranosyl-(1?6)-4-O-acetyl-2,3-di-O-benzoyl-a-
D-galactopyranosyl-(1?6)-4-O-acetyl-2,3-di-O-benzoyl-
a-D-galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galacto-
pyranoside (23)
To a solution of 21 (69.3 mg, 31.6 lmol) in THF
(0.4 mL) and MeOH (0.8 mL) was added 10% Pd–C
(80 mg), and the reaction mixture was stirred in a H2
atmosphere for 3 h at room temperature, then filtered
and concentrated. The residue was acetylated with
Ac2O (1 mL) in pyridine (1.5 mL). The reaction mixture
was poured into ice-water and extracted with CHCl3.
The extract was washed sequentially with 5% HCl, aq
Compounds 21 and 22 were derived according to the
procedure described for the treatment of 6. Compounds
19 and 20 (136 mg, 58.4 lmol)?21 (69.3 mg, 54.1%) and
24
22 (46.1 mg, 36.0%): ½aꢁD +135.1 (c 0.9, CHCl3). 1H

N. Hada et al. / Carbohydrate Research 343 (2008) 2315–2324
2323
NaHCO3 and water, dried (MgSO4), and concentrated.
1.16. 2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl-
The product was purified by silica gel column chroma-
tography using 10:1 toluene/acetone as the eluent to give
(1?6)-4-O-acetyl-2,3-di-O-benzoyl-a-D-galactopyran-
osyl-(1?6)-4-O-acetyl-2,3-di-O-benzoyl-a-D-galactopyr-
anosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyranosyl-
(1?6)-2,3,4-tri-O-benzoyl-b-D-galacto-pyranosyl-(1?1)-
(2S,3R)-3-O-benzoyl-2-hexadecanamidooctadecane-1,3-
diol (25)
24
23 (67.5 mg, 96.1%). ½aꢁD +172.3 (c 0.5, CHCl3). 1H
NMR (500 MHz, CDCl3) d 8.00–7.17 (m, 50H, 10Ph),
5.86–5.16 (m, 15H, H-2 of Gal a, b, c, d, e, H-3 of
Gal a, b, c, d, e, H-4 of Gal a, b, c, d, e), 5.21 (d, 1H,
J1,2 = 3.6 Hz, H-1 of Gal c), 5.07 (d, 1H, J1,2 = 3.7 Hz,
H-1 of Gal e), 4.99 (d, 1H, J1,2 = 3.1 Hz, H-1 of Gal
d), 4.81 (d, 1H, J1,2 = 7.9 Hz, H-1 of Gal b), 4.67 (d,
1H, J1,2 = 7.9Hz, H-1 of Gal a), 2.35, 2.134, 2.128,
2.12, 2.05 and 2.04 (each s, 18H, 6Ac), 0.81–0.71 (m,
2H, CH2CH2O), ꢀ0.12 (s, 9H, Si(CH3)3). 13C NMR
(125 MHz, CDCl3) d 170.7, 170.4, 170.2, 169.9, 169.8,
166.2, 166.0, 165.4, 165.23, 165.16, 165.0, 153.4, 138.5,
133.2, 133.0, 130.1, 130.0, 129.8, 129.7, 129.5, 129.4,
129.3, 129.1, 129.0, 128.9, 128.5, 128.44, 128.38, 128.3,
128.2, 128.1, 125.3, 124.9, 100.8 (C-1 of Gal b), 100.7
(C-1 of Gal a), 97.40 (C-1 of Gal c), 97.37 (C-1 of Gal
e), 96.5 (C-1 of Gal d), 72.7, 71.9, 71.8, 70.0, 69.9,
68.8, 68.7, 68.6, 68.4, 68.3, 68.1, 67.8, 67.7, 67.6, 67.5,
67.3, 67.0, 66.8, 66.6, 66.2, 61.9, 29.7, 21.4, 20.7, 20.6,
20.5, 17.7 (–OCH2CH2), ꢀ1.5 (Si(CH3)3). MALDI-
TOFMS: calcd for C117H116O42SiNa [M+Na]+: m/z
2243.7. Found: m/z 2243.9.
Compound 25 was derived according to the procedure
described for the treatment of 16. Compound 24
24
(50.0 mg, 22.1 lmol)?25 (31.5 mg, 52.0%). ½aꢁD +117.2
1
(c 0.8, CHCl3). H NMR (500 MHz, CDCl3) d 8.08–
7.17 (m, 55H, 11 Ph), 5.20 (d, 1H, J1,2 = 3.7 Hz, H-1 of
Gal c), 5.06 (d, 1H, J1,2 = 3.1 Hz, H-1 of Gal e), 4.95
(d, 1H, J1,2 = 3.7 Hz, H-1 of Gal d), 4.52 (d, 1H,
J1,2 = 7.3 Hz, H-1 of Gal b), 4.36 (d, 1H, J1,2 = 7.3 Hz,
H-1 of Gal a), 2.14, 2.12, 2.11, 2.04, 2.03 and 1.94 (each
s, 18H, 6Ac), 1.75–1.07 (m, 54H, 27 –CH2–), 0.88 (t, 6H,
2CH3). 13C NMR (125 MHz, CDCl3) d 172.4, 170.7,
170.4, 170.2, 169.9, 169.7, 166.1, 165.9, 165.5, 165.3,
165.2, 165.0, 133.4, 133.3, 133.1, 133.0, 132.8, 131.0,
130.6, 130.1, 130.0, 129.74, 129.68, 129.6, 129.41,
129.36, 129.3, 129.2, 129.0, 128.9, 128.6, 128.5, 128.4,
128.34, 128.25, 128.2, 128.1, 101.0 (C-1 of Gal b), 100.7
(C-1 of Gal a), 97.6 (C-1 of Gal c), 97.4 (C-1 of Gal e),
96.6 (C-1 of Gal d), 75.7, 73.6, 72.9, 71.8, 71.4, 71.3,
70.6, 69.8, 68.8, 68.6, 68.49, 68.45, 68.4, 68.3, 68.1,
67.7, 67.6, 67.5, 67.1, 67.0, 66.6, 66.1, 61.9, 56.8, 50.2,
36.9, 36.5, 31.9, 31.5, 29.7, 29.6, 29.3, 29.1, 28.9, 27.9,
25.5, 25.2, 24.1, 22.7, 20.7, 20.6, 14.1, 1.0. MALDI-TOF-
MS: calcd for C153H175NO45Na [M+Na]+: m/z 2769.1.
Found: m/z 2769.1.
1.15. 2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl-
(1?6)-4-O-acetyl-2,3-di-O-benzoyl-a-D-galactopyran-
osyl-(1?6)-4-O-acetyl-2,3-di-O-benzoyl-a-D-galactopyr-
anosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyranosyl-
2,3,4-tri-O-benzoyl-a-D-galactopyranosyl trichloroace-
timidate (24)
1.17. a-D-Galactopyranosyl-(1?6)-a-D-galactopyran-
osyl-(1?6)-a-D-galactopyranosyl-(1?6)-b-D-galactopyr-
anosyl-(1?6)-b-D-galactopyranosyl-(1?1)-(2S,3R)-2-
hexadecanamidooctadecane-1,3-diol (3)
Compound 24 was derived according to the procedure
described for the treatment of 14. Compound 23
24
(102 mg, 45.5 lmol)?24 (95.2 mg, 91.3%): ½aꢁD +155.3
1
(c 0.8, CHCl3). H NMR (500 MHz, CDCl3) d 8.65–
7.16 (m, 50H, 11 Ph, NH), 6.77 (d, 1H, J1,2 = 3.7 Hz,
H-1 of Gal a), 5.20 (d, 1H, J1,2 = 3.1Hz, H-1 of Gal
c), 5.04 (d, 1H, J1,2 = 3.7 Hz, H-1 of Gal e), 4.99 (d,
1H, J1,2 = 3.7 Hz, H-1 of Gal d), 4.78 (d, 1H,
J1,2 = 7.9 Hz, H-1 of Gal b), 2.16, 2.134, 2.128, 2.12,
2.05 and 1.97 (each s, 18H, 6Ac). 13C NMR
(125 MHz, CDCl3) d 170.7, 170.6, 170.4, 170.2, 168.9,
169.8, 166.1, 166.0, 165.6, 165.3, 165.24, 165.21,
165.16, 165.03, 165.00, 160.5, 133.4, 133.2, 133.0,
130.1, 129.9, 129.7, 129.5, 129.4, 129.3, 129.0, 128.9,
128.8, 128.6, 128.5, 128.3, 128.2, 128.1, 100.7 (C-1 of
Gal b), 97.4 (C-1 of Gal c), 97.3 (C-1 of Gal e), 96.5
(C-1 of Gal d), 93.7 (C-1 of Gal a), 90.7 (CCl3), 71.8,
71.7, 70.8, 69.8, 68.8, 68.7, 69.6, 68.4, 68.3, 68.1, 67.8,
67.7, 67.6, 67.5, 67.0, 66.64, 66.59, 66.5, 66.2,
61.9, 29.7, 20.6. MALDI-TOFMS: calcd for
Compound 3 was derived according to the procedure
described for the treatment of 2. Compound 25
24
(23.0 mg, 8.37 lmol)?2 (10.4 mg, 92.0%). ½aꢁD +20.9
(c 0.04, CHCl3/MeOH = 1:1). 1H NMR (500 MHz,
DMSO-d6/D2O) d 4.59 (br d, 3H, H-1 of Gal c, d, e),
4.17 (d, 1H, J1,2 = 6.7 Hz, H-1 of Gal b), 4.07 (d, 1H,
J1,2 = 7.3 Hz, H-1 of Gal a), 1.60–1.10 (m, 54H, 27
–CH2–), 0.83 (t, 6H, 2CH3). HR-FABMS: calcd for
C64H119NO28Na [M+Na]+: m/z 1372.7816. Found:
m/z 1372.7858.
References
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C
114H104Cl3NO42Na [M+Na]+: m/z 2286.5. Found:
m/z 2287.4.

2324
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