Synthesis of a new glycosphingolipid, neurosporaside, from Neurospora crassa

Article abstract of DOI:10.1016/j.carres.2014.11.018

The glycosphingolipid neurosporaside (α-D-Glcp-(1→2)-β-D-Galp-(1→6)-β-D-Galp-(1→6)-β-D-Galp-(1→)-Cer) occurs in Neurospora crassa. We attempted to synthesize neurosporaside by block synthesis (route A) and linear synthesis (route B). Oligosaccharide derivatives were synthesized using trimethylsilyltrifluoromethanesulfonate and N-iodosuccinimide/trifluoromethane sulfonic acid as promoters. The target tetrasaccharide could not be attained via route A, but route B showed potential: glycosidic bonds (β-D-Galp-(1→6)-β-D-Galp-(1→6)-β-D-Galp) were formed stereoselectively, leading to the synthesis of glycosphingolipid 2.

Full text of DOI:10.1016/j.carres.2014.11.018

Carbohydrate Research 404 (2015) 9–16
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of a new glycosphingolipid, neurosporaside, from
Neurospora crassa
b
a
a
a
Isao Ohtsuka a, , Noriyasu Hada , Misaki Kanemaru , Takanari Fujii , Toshiyuki Atsumi ,
⇑
Nobuko Kakiuchi a
a School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino-cho, Nobeoka City, Miyazaki 882-8508, Japan
b Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The glycosphingolipid neurosporaside (a-D-Glcp-(1?2)-b-D-Galp-(1?6)-b-D-Galp-(1?6)-b-D-Galp-(1?)-
Received 29 September 2014
Received in revised form 18 November 2014
Accepted 23 November 2014
Available online 3 December 2014
Cer) occurs in Neurospora crassa. We attempted to synthesize neurosporaside by block synthesis (route A)
and linear synthesis (route B). Oligosaccharide derivatives were synthesized using trimethylsilyltrifluo-
romethanesulfonate and N-iodosuccinimide/trifluoromethane sulfonic acid as promoters. The target
tetrasaccharide could not be attained via route A, but route B showed potential: glycosidic bonds
(b-
D
-Galp-(1?6)-b-
D
-Galp-(1?6)-b-
D
-Galp) were formed stereoselectively, leading to the synthesis of
Keywords:
Neurospora crassa
Neurosporaside
glycosphingolipid 2.
Ó 2014 Elsevier Ltd. All rights reserved.
Glycosphingolipid
Oligosaccharide synthesis
1. Introduction
glycosphingolipid,
neurosporaside
a-D-Glcp-(1?2)-b-D-Galp-
(1?6)-b- -Galp-(1?6)-b-
D
D
-Galp-(1?)-Cer (1, Fig. 1), from N.
Glycosphingolipids have attracted attention recently due to
their biological activities. Sialic acid-containing glycosphingolipids
such as gangliosides are especially well known for their involve-
ment in extracellular recognition, cell–cell interactions, differenti-
ation, oncogenesis, and the immune system.1,2 Many carbohydrate
researchers have focused on glycosphingolipids containing sialic
acid from higher animals. Systematic studies of their structure
and function at the molecular level were undertaken via synthesis
of analogs. The biological functions in invertebrates of glycolipids
lacking sialic acid, however, are unknown.5 Thus, it is hoped that
synthesis and investigation of the functions of such glycolipids
may help to elucidate disease mechanisms and facilitate the devel-
opment of new drugs.6,7 Organic synthesis is a powerful method
for exploring structure–activity relationships, providing access to
large numbers of homogeneous and structurally defined oligosac-
charides, including both natural and unnatural compounds. We
are interested in the synthesis of novel naturally occurring
glycosphingolipids.
crassa.8 A previous study of N. crassa reported only one neutral
glycosphingolipid, b-glucopyranosylceramide. The structure of
neurosporaside is unique, however, in that the sugar moiety linked
to the ceramide is formed by a chain of three b-galactopyranoside
residues, with a glucopyranoside
a-linked to the outer galacto-
pyranoside. The biological activities of these compounds cannot
be compared with those of glycosphingolipids from other
animal species because those molecules contain different cera-
mide structures. Therefore, ceramide structures must also be
considered in functional studies of the carbohydrate moieties
in glycosphingolipids. The ceramides of the major natural
glycosphingolipids comprise a fatty acid and a saturated or unsat-
urated sphingosine.4,9 In this paper, we have attempted to synthe-
size structures containing general ceramides (2, Fig. 1).
2. Results and discussion
The aim of this study was an efficient and stereoselective syn-
thesis of compound 2. We considered two retrosynthetic plans:
block glycosylation with two types of disaccharide derivatives
(route A), and linear glycosylation with monosaccharide deriva-
tives coupled one by one from the reducing end of sugar residues
(route B; Fig. 2).10 Route A is very efficient from the perspective
of total yield, but this route faces a critical obstacle: glycosylation
Neurospora crassa is prominent in the long history of genetics,
biochemistry, and molecular biology, having been employed as a
convenient model for studies of fungal growth and development.
In 2011, Costantina et al. isolated and characterized a novel
⇑
Corresponding author. Tel.: +81 982 23 5701; fax: +81 982 23 5702.
E-mail address: ohtsuka@phoenix.ac.jp (I. Ohtsuka).
by Gal (II) and Gal (III) is
a-/b-non-selective. In contrast, route B
http://dx.doi.org/10.1016/j.carres.2014.11.018
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

10
I. Ohtsuka et al. / Carbohydrate Research 404 (2015) 9–16
OH
2.1. Monosaccharide derivatives 7, 8 and disaccharide
derivative 12
*DOꢀ,,,ꢁ
HO
O
O
HO
O
*DOꢀ,,ꢁ
HO
Compound 3 was protected at the 3,4-di-OH groups with an iso-
propylidene group, and at 6-OH with a t-butyl dimethylsilyl group
(TBS), resulting in 2-OH derivative 5.11 These protecting groups in
5 were removed with TsOH after protection at 2-OH with a
levulinoyl group (Lev), followed by benzoylation at 3-, 4-, and 6-
OH, giving monosaccharide derivative 6 (81% over three steps).
Compound 7 was obtained when the Lev group of compound 6
was selectively deprotected by treatment with hydrazine acetate
(NH2NH2–AcOH) (92%).12 Trichloroacetimidate derivative 8 was
synthesized by selective removal of the p-methoxyphenyl group
with ceric ammonium nitrate (CAN) in CH3CN/H2O (6:1)13 and
treatment with trichloroacetonitrile and 1,8-diazabicyclo[5,4,0]-
7-undecane (DBU; 90% over two steps; Scheme 1).
O
O
OH
HO
O
HO
HO
*DOꢀ,ꢁ
*OF
HO
OH
O
HO
OR
HO
OH
R
OH
O
C21H43
1
2
HN
OH
C13H27
The synthesis of disaccharide derivative 11 using acceptor 914
and donor 1015 was achieved with N-iodosuccinimide (NIS) and
trifluoromethane sulfonic acid (TfOH)16 in 72% yield. The structure
of 11 was confirmed by 1H and 13C NMR spectroscopy. Selective
removal of the TBS group of 11 was achieved with TsOH; we thus
obtained glycosyl acceptor 12 for synthesis of the tetrasaccharide
via route A and for synthesis of the trisaccharide via route B
(Scheme 2).
OH
O
HN
C13H27
C13H27
OH
OH
Figure 1. Structure of glycosphingolipids neurosporaside (1) from Neurospora
crassa and its motif 2.
2.2. Tetrasaccharide derivative 19 via routes A and B
We expected that tetrasaccharide derivative 19 could be
obtained by block synthesis using disaccharide donor 15 and
acceptor 12 (route A). Synthesis of disaccharide derivative 14 by
coupling of monosaccharide acceptor 7 and donor 13 was achieved
relies on glycosylation, which is expected to proceed with high ste-
reoselectivity; however, we expect a lower total yield than route A
given the number of additional reaction steps. We attempted to
synthesize compound 2 via both routes A and B.
OH
HO
O
O
HO
O
HO
O
O
O
OH
HO
O
HO
HO
HN
C13H27
C13H27
HO
OH
OH
O
HO
O
HO
2
OH
OH
OBz
Route A
BzO
OBz
BzO
7
O
O
O
BzO
OMP
OH
OBn
OBz
BzO
O
BzO
BzO
O
O
O
O
OBn
BnO
BzO
O
BzO
O
CCl3
NH
BzO
O
BzO
BzO
O
BnO
BnO
OBn
SPh
OBn
O
BnO
BnO
BnO
19
OMP
OBn
15
OBz
13
OBn
OTBS
BzO
Route B
OH
BzO
O
BzO
SPh
O
OBz
10
O
BzO
BzO
OH
BzO
OBz
BzO
BzO
12 BzO
O
9
OMP
O
OBn
O
BzO
OMP
O
BzO
OBz
BzO
OBz
O
HO
BzO
18
BnO
BnO
SPh
O
OBz
BzO
O
OBn
BzO
BzO
BzO
13
O
O
BzO
OMP
O
CCl3
NH
LevO
OBz
8
Figure 2. Retrosynthesis of 2 by route A and route B.

I. Ohtsuka et al. / Carbohydrate Research 404 (2015) 9–16
11
OBz
BzO
d
e
O
BzO
OMP
HO
7
OH
HO
OH
OTBS
OBz
BzO
O
O
a
b
c
O
O
O
O
HO
OMP
O
OMP
O
OMP
BzO
OMP
OBz
BzO
HO
HO
HO
LevO
6
3
4
5
O
BzO
LevO
CCl3
NH
O
8
Scheme 1. Reagents and conditions: (a) (MeO)2C(CH3)2, CSA, DMF, 80%; (b) TBS-Cl, pyr., 95%; (c) (1) Lev-OH, DCC, DMAP, CH2Cl2; (2) TsOH, CH2Cl2–MeOH; (3) BzCl, pyr., 81%
(three steps); (d) NH2NH2–AcOH, MeOH–THF, 92%; (e) (1) CAN, CH3CN–H2O; (2) CCl3CN, DBU, CH2Cl2, 90% (two steps).
OH
BzO
OTBS
BzO
the resulting hemiacetal into an imidate (58% over two steps). Gly-
cosylation of disaccharide donor 15 with acceptor 12 using trim-
ethylsilyltrifluoromethanesulfonate (TMSOTf)19 failed to give
tetrasaccharide derivative 19; only its stereoisomer 16 was
obtained. The structure of 16 was confirmed by 1H NMR
(d = 5.16 ppm, d, 1H, J1,2 = 3.6 Hz, H-100) and 13C NMR
(d = 97.1 ppm, C-100) spectroscopy.20 Synthesis of tetrasaccharide
derivative 19 was thus not achieved by route A (Scheme 3). Disac-
O
O
BzO
OMP
BzO
SPh
OBz
OBz
10
9
a
charide donor 15 contains an
no neighboring group effect.21 Thus, tetrasaccharide synthesis via
route A was -/b-non-selective. We had hoped that steric hin-
a-(1?2) glycosidic bond, which has
OR
BzO
a
O
drance by a bulky group (D
-glucose), the solvent effect of EtCN,22
O
BzO
BzO
and low temperature (ꢀ78 °C)23 would lead selectively to the b-
BzO
O
OBz
BzO
OMP
isomer. These effects were weak, however. The
a-stereodirecting
R
effect of benzoyl groups at O-3, O-4, and O-6 led instead to stereo-
isomer 16.24
11
12
TBS
H
Total yield was lower from route B than from route A, but route
B allowed for stereoselective formation of glycosidic bonds. Trisac-
charide derivative 17 was synthesized from donor 8 and acceptor
12 using TMSOTf in 83% yield. NH2NH2–AcOH treatment of 17
selectively removed the Lev group, giving 2-OH derivative 18 in
85% yield. Having obtained trisaccharide acceptor 18, we per-
formed NIS and TfOH-promoted glycosylation of 18 with glycopyr-
anosyl donor 13 in dichloromethane/diethyl ether (1:1) at 0 °C,
giving the desired tetrasaccharide derivative 19 (82%), as evi-
denced by 1H NMR (d = 5.11 ppm, d, 1H, J1,2 = 7.8 Hz, H-100) and
13C NMR (d = 102.3 ppm, C-100) (Scheme 4).26
b
Scheme 2. Reagents and conditions: (a) NIS, TfOH, CH2Cl2, 72%; (b) TsOH, CH2Cl2–
MeOH, 97%.
with NIS and TfOH (96%). The a
-linkage was confirmed by 1H and
13C NMR spectrometry. The anomeric protons of Glcp appeared as a
doublet (d 5.79 ppm, d, J = 3.8 Hz, 1H, H–1000).18 Disaccharide donor
15 was prepared from protected disaccharide 14 by cleavage of the
anomeric p-methoxyphenyl protecting group and conversion of
12
OBz
BzO
O
OBn
O
BzO
O
c
BzO
O
O
OBz
BzO
O
OBn
BzO
BnO
BnO
BnO
SPh
O
BzO
OBn
BzO
O
O
13
R1
OBn
BzO
O
19
BnO
BzO
OMP
a
O
OBz
OBn
R2
BnO
BnO
OBz
BzO
OBz
BzO
OBn
R1
O
O
O
BzO
O
BzO
OMP
R2
OH
OBn
BzO
7
O
BnO
14
15
OMP
H
O
b
OBn
BnO
OC(NH)CCl3
H
O
BzO
BzO
BzO
BzO
O
OMP
16
OBz
Scheme 3. Reagents and conditions: (a) NIS, TfOH–Et2O, CH2Cl2, 96%; (b) (1) CAN, CH3CN–H2O; (2) CCl3CN, DBU, CH2Cl2, 58% (two steps); (c) TMSOTf, EtCN, 76%.

12
I. Ohtsuka et al. / Carbohydrate Research 404 (2015) 9–16
12
OBz
BzO
O
O
BzO
a
BzO
RO
BzO
OBz
BzO
O
O
BzO
BzO
O
O
R
O
BzO
O
BzO
BzO
OMP
8
O
Lev
H
O
OBn
BzO
17
18
BnO
BnO
O
BzO
b
c
BzO
BzO
OBn
O
R1
BzO
BzO
R1
OMP
R2
R2
13
H
19
20
d
OC(NH)CCl3
H
Scheme 4. Reagents and conditions: (a) TMSOTf, CH2Cl2, 83%; (b) NH2NH2–AcOH, MeOH–THF, 85%; (c) NIS, TfOH–Et2O, CH2Cl2, 82%; (d) (1) CAN, CH3CN–H2O; (2) CCl3CN,
DBU, CH2Cl2, 61% (two steps).
OR1
R1O
20
O
O
O
R1O
O
R1O
O
O
OR2
a
R2O
O
R1O
R1O
HN
C13H27
C13H27
R1O
OR2
OR2
O
O
R2O
O
R1O
R1O
HN
C13H27
C13H27
OBn
R1
R2
Bn
H
OR2
HO
22
Bz
H
b
21
OBn
2
Scheme 5. Reagents and conditions: (a) TMSOTf, CH2Cl2, 40%; (b) (1) H2, Pd–C, AcOH, MeOH; (2) NaOMe, MeOH–1,4-dioxane, quant. (two steps).
2.3. Glycosphingolipid 2
3. Experimental
3.1. General
Deprotection of 19 with CAN in CH3CN/H2O (6:1) afforded the
hemiacetal, which was converted to -trichloroacetimidate
a
derivative 20 by treatment with trichloroacetonitrile and DBU.
Glycosylation of (2S,3S,4R)-3,4-di-O-benzyl-2-hexadecanamido-
octadecane-3,4-diol (21)25 with glycosyl donor 20 was carried
out in the presence of TMSOTf and AW-300 molecular sieves to
afford tetrasaccharide derivative 22 (40%). The Bn group was
removed from 22 by catalytic hydrogenolysis over 10% Pd/C in
CH2Cl2, MeOH, and AcOH (1:1:1). Finally, removal of the benzoyl
(Bz) group of 22 under Zemplén conditions, and column chroma-
tography over Sephadex LH-20 afforded the target glycosphingo-
Optical rotations were measured using a Jasco P-1020 digital
polarimeter. 1H NMR and 13C NMR spectra were recorded using a
JNM AL 400 FT NMR spectrometer (JEOL Ltd) and JNM-EC 400 FT
NMR spectrometer (JEOL Ltd) with Me4Si as the internal standard
for samples in CDCl3 and CD3OD. NMR measurements were carried
out at 24 1 °C. High-resolution mass spectra were recorded on a
JMS-T100 (JEOL Ltd). Ion trap-LC–MS data were recorded on an
LCO Advantage instrument (Thermo-quest Ltd). TLC was per-
formed on silica gel 60 F254 (E. Merck), and detection was based
on quenched UV fluorescence and staining with 10% H2SO4. Col-
umn chromatography was performed using silica gel 60 (E. Merck).
Molecular sieves were stored in an oven at 350 °C. CH2Cl2 and EtCN
which were used by glycosylations were distilled by CaH2. Recrys-
tallization of NIS was completed by 1,4-dioxane–CCl4.
lipid
2 (Scheme 5). The structure and purity of 2 were
determined by 1H and 13C NMR spectroscopy, along with HR-
FABMS data.
2.4. Conclusion
In summary, we achieved the first synthesis of the glycosphin-
3.2. 4-Methoxyphenyl 2-O-levulinoyl-3,4,6-tri-O-benzoyl-b-D-
golipid neurosporaside
(a-D-Glcp-(1?2)-b-D-Galp-(1?6)-b-D-
galactopyranoside (6)
Galp-(1?6)-b- -Galp-(1?)Cer) from N. crassa. Using route A,
D
synthesis of tetrasaccharide derivative 19 using two types of disac-
charide derivative, 12 and 15, failed. Only stereoisomer 16 was
obtained. Linear synthesis (route B), using stepwise coupling of
monosaccharide derivatives from the reducing end of sugar resi-
Lev-OH (7.35 mL, 71.8 mmol), DCC (15.0 g, 71.8 mmol), and
DMAP (877 mg, 7.20 mmol) were added to a solution of 5 (15.8 g,
35.9 mmol) in CH2Cl2 (90 mL), and the reaction mixture was stirred
for 4 h at room temperature. The mixture was filtered, diluted with
CH2Cl2, washed with 5% HCl, aqueous NaHCO3, and then dried,
concentrated, and redissolved in CH2Cl2 (50 mL) and MeOH
(50 mL). TsOH (20.4 g, 108 mmol) was added to the solution, and
dues, however, showed potential. Glycosidic bonds (b-
(1?6)-b- -Galp-(1?6)-b- -Galp) were formed stereoselectively,
yielding glycosphingolipid 2.
D-Galp-
D
D

I. Ohtsuka et al. / Carbohydrate Research 404 (2015) 9–16
13
the mixture was stirred for 2 h at room temperature. The reaction
was neutralized with Et3N, diluted with CH2Cl2, washed with aque-
ous NaHCO3, and then dried and concentrated. The residue was
protected with benzoyl chloride (25.0 mL, 215.5 mmol) in pyridine
(90 mL) for 3 h. The reaction mixture was quenched with MeOH at
0 °C and extracted with CHCl3. The extract was washed with 5% HCl
and aqueous NaHCO3, and then dried and concentrated. The crude
product was purified by silica gel column chromatography (hex-
(s, 3H, –COCH3); 13C NMR (100 MHz, CDCl3): d 205.6, 171.8,
165.8, 165.4, 165.3, 160.8, 137.8, 133.7, 133.4, 129.8, 129.7,
129.6, 128.9, 128.8, 128.7, 128.6, 128.3, 125.2, 93.6 (C-1), 69.6,
68.4, 68.3, 67.4, 62.137.6, 29.5, 27.7; LC–MS (ESI) calcd for C34H31-
Cl3NO+11 (M+H)+ m/z 735.09; measured m/z 735.09.
3.5. 4-Methoxyphenyl 2,3,4-tri-O-benzoyl-6-O-tert-
butyldimethylsilyl-b-
D-galactopyranosyl (1?6)-2,3,4-tri-O-
ane/AcOEt = 5:1 to 1:1) to give 6 (20.3 g, 81%—three steps) as col-
benzoyl-b- -galactopyranoside (11)
D
25
orless needle-like crystals. Mp 150–151 °C; [
a]
+6.6 (c 1.0,
D
CHCl3); Rf 0.25 (hexane/AcOEt = 2:1); 1H NMR (400 MHz, CDCl3):
d 8.08–6.74 (m, 19H, Ph ꢁ4), 5.95 (d, 1H, H-4), 5.78 (t, 1H, H-2),
5.49 (dd, 1H, H-3), 5.10 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.64 (dd, 1H, H-
6a), 4.49 (dd, 1H, H-6b), 4.37 (t, 1H, H-5), 3.76 (s, 3H, –OMe),
2.63–2.50 (m, 4H, –CH2CH2–), 2.03 (s, 3H, –COCH3); 13C NMR
(100 MHz, CDCl3): d 205.7, 171.5, 165.9, 165.1, 165.4, 155.7,
151.3, 133.6, 133.4, 133.3, 130.0, 129.9, 129.8, 129.4, 128.8,
128.6, 128.5, 128.3, 118.8, 114.5, 100.9 (C-1), 71.7 (C-3), 71.6 (C-
5), 69.2 (C-2), 67.9 (C-4), 62.2 (C-6), 55.6 (–OMe), 37.8, 29.6,
27.8; LC–MS (ESI) calcd for C39H37O+12 (M+H)+ m/z 697.22; mea-
sured m/z 697.22; HR-MS C39H36NaO+12 Calcd 719.2110, Found
719.2127 [M+Na]+.
A solution of 9 (5.80 g, 9.60 mmol) and 10 (10.2 g, 14.5 mmol)
containing activated type AW-300 molecular sieves (10 g) in dry
CH2Cl2 (20 mL) was stirred under a nitrogen atmosphere for 2 h
at room temperature. After cooling to ꢀ15 °C, NIS (6.49 g,
29.0 mmol) and TfOH (268 lL, 2.9 mmol) were added sequentially,
and the mixture was stirred for 1.5 h at 0 °C. The reaction was
neutralized with Et3N, filtered, and extracted with CHCl3. The
organic solvent was washed with aqueous sodium thiosulfate,
5% HCl and aqueous NaHCO3, and then dried and concentrated
to give a clear needle-like crystal. The crude product was purified
by silica gel column chromatography (toluene/AcOEt = 150:1 to
100:1) to give 11 (8.28 g, 72%) as colorless needle-like crystals.
25
[a
]
+7.2 (c 1.0, CHCl3); Rf 0.58 (hexane/AcOEt = 2:1); 1H NMR
D
3.3. 4-Methoxyphenyl 3,4,6-tri-O-benzoyl-b-
galactopyranoside (7)
D
-
(400 MHz, CDCl3): d 8.10–6.68 (m, 34H, Ph ꢁ7), 5.99–5.95 (m,
2H, H-4, H-2), 5.90 (d, 1H, H-40), 5.73 (t, 1H, H-20), 5.58–5.53 (m,
2H, H-3, H-30), 5.14 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.90 (d, 1H,
J1,2 = 7.8 Hz, H-10), 4.27 (t, 1H, H-50), 4.12 (dd, 1H, H-6a0), 4.01
(dd, 1H, H-6b0), 3.95 (t, 1H, H-5), 3.70 (s, 3H, –OMe), 3.67 (t, 1H,
H-6a), 3.59 (t, 1H, H-6b); 13C NMR (100 MHz, CDCl3): d 165.5,
165.4, 165.3, 165.3, 165.2, 165.2, 155.8, 151.3, 133.5, 133.2,
130.0, 129.9, 129.8, 129.7, 129.7, 129.6, 129.5, 129.3, 129.2,
129.1, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 125.3,
118.9, 114.6, 101.5 (C-1), 101.0 (C-10), 73.9 (C-5), 73.8 (C-50),
72.0 (C-30), 71.6 (C-3), 70.0 (C-20), 69.6 (C-2), 68.5 (C-4), 67.6 (C-
40), 67.5 (C-6), 60.3 (C-60), 55.5 (–OMe), 25.7, 18.0, ꢀ5.7, ꢀ5.8;
LC–MS (ESI) calcd for C67H67O18Si+ (M+H)+ m/z 1187.4; measured
NH2NH2–AcOH (10.5 g, 11.4 mmol) was added to a solution of 6
(4.00 g, 5.70 mmol) in THF (20 mL) and MeOH (2.0 mL), and the
reaction mixture was stirred for 2 h at room temperature. The reac-
tion mixture was diluted with AcOEt, washed with 5% HCl and
aqueous NaHCO3, and then dried and concentrated. The crude
product was purified by silica gel column chromatography (hex-
ane/AcOEt = 5:1 to 3:2) to give 7 (3.15 g, 92%) as a white solid crys-
25
tals. Mp 136–139 °C; [
a
]
+5.3 (c 1.0, CHCl3); Rf 0.22 (hexane/
D
AcOEt = 1:1); 1H NMR (400 MHz, CDCl3): d 8.10–6.71 (m, 19H, Ph
ꢁ4), 5.93 (d, 1H, H-4), 5.48 (dd, 1H, H-3), 5.01 (d, 1H,
J1,2 = 7.9 Hz, H-1), 4.62 (dd, 1H, H-6a), 4.46 (dd, 1H, H-6b), 4.38
(t, 1H, H-2), 4.35 (q, 1H, H-5), 3.74 (s, 3H, –OMe); 13C NMR
(100 MHz, CDCl3): d 165.9, 165.9, 165.5, 155.6, 150.9, 133.6,
133.3, 129.9, 129.8, 129.7, 129.4, 129.1, 129.0, 128.6, 128.4,
128.3, 118.6, 114.5, 102.2 (C-1), 73.2 (C-3), 71.6 (C-5), 69.6 (C-2),
68.1 (C-4), 62.3 (C-6), 55.6 (–OMe); LC–MS (ESI) calcd for
m/z 1187.4; HR-MS
C
67H66NaO18Si+ Calcd 1209.3916, Found
1209.4017 [M+Na]+.
3.6. 4-Methoxyphenyl 2,3,4-tri-O-benzoyl-b-
D-galactopyranosyl-
(1?6)-2,3,4-tri-O-benzoyl-b- -galactopyranoside (12)
D
C
34H31O+10 (M+H)+ m/z 599.18; measured m/z 599.18; HR-MS C34-
H30NaO+10 Calcd 621.1736, Found 621.1733 [M+Na]+.
TsOH (1.51 g, 8.00 mmol) was added to a solution of 11 (4.66 g,
4.00 mmol) in CH2Cl2 (20 mL) and MeOH (20 mL), and the reaction
mixture was stirred for 2 h at room temperature. The reaction was
neutralized with Et3N, diluted with CH2Cl2, washed with aqueous
NaHCO3, and then dried and concentrated. The crude product was
purified by silica gel column chromatography (toluene/
3.4. 3,4,6-Tri-O-benzoyl-2-O-levulinoyl-b-D-galactopyranosyl
trichloroacetimidate (8)
CAN (4.55 g, 8.50 mmol) was added to a solution of 6 (1.16 g,
1.70 mmol) in CH3CN (32 mL) and H2O (5.0 mL), and the reaction
mixture was stirred for 1 h at room temperature. The reaction mix-
ture was extracted with AcOEt, washed with aqueous NaHCO3, and
then dried and concentrated. The resulting crude product was puri-
fied by silica gel column chromatography (toluene/acetone = 30:1
AcOEt = 15:1 to 2:1) to give 12 (4.07 g, 97%) as a white solid crys-
25
tals. Mp 145–147 °C; [
a]
+10.6 (c 1.0, CHCl3); Rf 0.35 (hexane/
D
AcOEt = 2:1); 1H NMR (400 MHz, CDCl3): d 8.13–6.67 (m, 34H,
Ph ꢁ7), 6.01 (d, 1H, H-4), 5.96 (t, 1H, H-2), 5.81 (t, 1H, H-20),
5.78 (d, 1H, H-40), 5.58 (dd, 1H, H-3), 5.53 (dd, 1H, H-30), 5.16
(d, 1H, J1,2 = 7.8 Hz, H-1), 4.89 (d, 1H, J1,2 = 7.8 Hz, H-10), 4.26 (t,
1H, H-5), 4.10 (dd, 1H, H-6a0), 4.03 (dd, 1H, H-6b0), 3.93 (t, 1H,
H-50), 3.71 (s, 3H, –OMe), 3.63 (dd, 1H, H-6a), 3.45 (dd, 1H, H-
6b); 13C NMR (100 MHz, CDCl3): d 166.7, 165.5, 165.4, 165.3,
165.2, 155.8, 151.2, 137.8, 133.8, 133.3, 133.2, 133.1, 129.9,
129.8, 129.5, 129.3, 129.2, 128.8, 128.7, 128.6, 128.6, 128.4,
128.3, 128.3, 128.2, 128.2, 114.5, 101.5 (C-1), 101.1 (C-10), 74.1
(C-50), 73.4 (C-5), 71.8 (C-30), 71.7 (C-3), 69.9 (C-20), 69.6 (C-2),
68.8 (C-40), 68.3 (C-4), 67.6 (C-6), 60.6 (C-60), 55.6 (–OMe), 21.4;
LC–MS (ESI) calcd for C61H53O+18 (M+H)+ m/z 1073.31; measured
to 10:1) to give the hemiacetal as a yellow oil. Cl3CCN (321
lL,
3.21 mmol) and DBU (72 L, 0.48 mmol) were added to a solution
l
of the hemiacetal in CH2Cl2 (3.0 mL) at 0 °C and stirred for 2 h at
0 °C under a nitrogen atmosphere. After completion of the reaction,
the mixture was purified by silica gel column chromatography (tol-
uene/AcOEt = 120:1 to 15:1) to give 8 (1.12 g, 90%—two steps) as a
25
yellow oil. [
a
]
D
ꢀ6.4 (c 1.0, CHCl3); Rf 0.52 (toluene/AcOEt = 6:1);
1H NMR (400 MHz, CDCl3): d 7.73 (s, 1H, NHCCl3), 8.05–7.13 (m,
15H, Ph ꢁ3), 6.78 (d, 1H, J1,2 = 3.7 Hz, H-1), 6.11 (d, 1H, H-4),
5.87 (dd, 1H, H-3), 5.73 (dd, 1H, H-2), 4.81 (t, 1H, H-5), 4.58 (dd,
1H, H-6a), 4.40 (t, 1H, H-6b), 2.67–2.50 (m, 4H, –CH2CH2–), 2.03
m/z 1073.31; HR-MS
C
61H52NaO+18 Calcd 1095.3052, Found
1095.3122 [M+Na]+.

14
I. Ohtsuka et al. / Carbohydrate Research 404 (2015) 9–16
3.7. 4-Methoxyphenyl 2,3,4,6-tetra-O-benzyl-
a
-
D
-glucopyranosyl-
oil. The crude product was purified by silica gel column chroma-
tography (hexane/AcOEt = 8:1 to 2:1) to give 16 (1.01 g, 76%
(1?2)-3,4,6-tri-O-benzoyl-b-D-galactopyranoside (14)
a
25
only) as a colorless oil. [
a]
+8.2 (c 1.0, CHCl3); Rf 0.38 (hexane/
D
A solution of 7 (2.00 g, 3.30 mmol) and 13 (4.22 g, 6.60 mmol)
containing activated type AW-300 molecular sieves (20 g) in dry
CH2Cl2 (30 mL) and dry Et2O (10 mL) was stirred under a nitrogen
atmosphere for 2 h at room temperature. After cooling to 0 °C, NIS
AcOEt = 1:1); 1H NMR (400 MHz, CDCl3): d 8.21–6.63 (m, 69H, Ph
ꢁ14), 6.10 (d, 1H, H-400), 5.91 (t, 1H, H-20), 5.68 (dd, 1H, H-300),
5.84 (t, 1H, H-2), 5.76 (d, 1H, H-40), 5.68 (d, 1H, H-4), 5.47 (dd,
1H, H-3), 5.35 (dd, 1H, H-30), 5.16 (d, 1H, J1,2 = 3.6 Hz, H-100), 5.01
(d, 1H, J1,2 = 3.8 Hz, H-1000), 4.88 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.86 (d,
1H, J1,2 = 7.8 Hz, H-10), 4.78–.61 (m, 8H), 4.40 (dd, 1H, H-200),
4.31–4.28 (m, 2H), 4.16 (q, 1H, H-5000), 4.08–3.66 (m, 11H), 3.51
(m, 2H, H-2000, H-3000), 3.27 (dd, 1H, H-6a000), 3.04 (dd, 1H, H-6b);
13C NMR (100 MHz, CDCl3): d 166.2, 165.7, 165.5, 165.4, 165.3,
165.1, 155.4, 151.4, 138.7, 138.5, 137.8, 133.5, 133.3, 133.0,
132.8, 130.3, 130.0, 129.9, 129.8, 129.7, 129.6, 129.6, 129.3,
128.8, 128.7, 128.6, 128.5, 128.5, 128.3, 128.2, 127.9, 127.8,
127.6, 127.4, 127.2, 118.5, 114.5, 101.4 (C-1), 100.5 (C-197.1 (C-
100), 96.7 (C-1000), 81.5, 79.2, 77.2, 75.4, 74.5, 73.8, 73.3, 73.0, 72.8,
72.3, 71.7, 71.4, 70.6, 69.9, 69.8, 69.7, 69.2, 68.8, 68.6, 68.1, 67.9,
67.7, 63.9, 55.4; LC–MS (ESI) calcd for C122H109O+31 (M+H)+ m/z
2069.69; measured m/z 2069.69; HR-MS C122H108NaO+31 Calcd
2091.6772, Found 2091.6472 [M+Na]+.
(2.98 g 13.2 mmol) and TfOH (123 lL, 1.33 mmol) were added
sequentially, and the mixture was stirred for 1.5 h at 0 °C. The reac-
tion was neutralized with Et3N, filtered, and extracted with CHCl3.
The organic solvent was washed with aqueous sodium thiosulfate,
5% HCl and aqueous NaHCO3, dried, and concentrated to give a
clear oil. The crude product was purified by silica gel column chro-
matography (toluene/acetone = 40:1 to 10:1) to give 14 (3.59 g,
96%
a only) as a colorless oil. [a]
25 +8.8 (c 1.0, CHCl3); Rf 0.42 (tol-
D
uene/AcOEt = 8:1); 1H NMR (400 MHz, CDCl3): d 8.12–6.69 (m,
39H, Ph ꢁ8), 5.93 (d, 1H, H-400), 5.79 (d, 1H, J1,2 = 3.8 Hz, H-1000),
5.65 (dd, 1H, H-300), 3.83 (d, 1H, J1,2 = 7.8 Hz, H-100), 4.84, 4.66,
4.60, 4.58, 4.42, 4.39, 4.22, 4.17 (each d, 8H, (PhCH2O ꢁ8), 4.56
(t, 1H, H-200), 4.52 (dd, 1H, H-6a00), 4.35 (t, 1H, H-6b00), 3.81 (t, 1H,
H-4000), 3.76 (s, 3H, –OMe), 3.71 (t, 1H, H-3000), 3.56–3.52 (m, 2H,
H-2000, H-500), 3.22 (dd, 1H, H-6a000), 3.06 (dd, 1H, H-6b000); 13C NMR
(100 MHz, CDCl3): d 165.9, 165.5, 165.4, 155.4, 150.3, 138.7,
138.5, 137.7, 133.6, 133.3, 133.1, 129.8, 129.7, 129.5, 129.2,
129.1, 128.7, 128.4, 128.2, 128.1, 128.0, 127.8, 127.6, 127.5,
127.1, 117.4, 114.7, 101.5 (C-100), 96.2 (C-1000), 81.3 (C-4000), 79.5
(C-2000), 77.3 (C-500), 75.5 (PhCH2O), 74.2 (PhCH2O), 73.4 (C-200),
72.8 (PhCH2O), 72.1 (C-300), 71.8 (PhCH2O), 71.2 (C-3000), 70.4 (C-
400), 68.1 (C-600), 62.2 (C-6000), 55.6 (–OMe); LC–MS (ESI) calcd for
3.10. 4-Methoxyphenyl 3,4,6-tri-O-benzoyl-2-O-levulinoyl-b-
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b- -galactopyr-
anosyl-(1?6)-2,3,4-tri-O-benzoyl-b- -galactopyranoside (17)
D-
D
D
A solution of 12 (1.20 g, 1.12 mmol) and 8 (1.12 g, 1.52 mmol)
containing activated type AW-300 molecular sieves (6.0 g) in dry
CH2Cl2 (12 mL) was stirred under a nitrogen atmosphere for 2 h
C
C
68H65O+15 (M+H)+ m/z 1121.42; measured m/z 1121.42; HR-MS
at room temperature. After cooling to 0 °C, TMSOTf (55 lL,
68H64NaO+15 Calcd 1143.4143, Found 1143.4257 [M+Na]+.
0.31 mmol) was added, and the mixture was stirred for 2 h at
0 °C. The reaction was neutralized with Et3N, filtered, and
extracted with CHCl3. The organic solvent was washed with 5%
HCl and aqueous NaHCO3, and then dried and concentrated to give
a clear oil. The crude product was purified by silica gel column
3.8. 2,3,4,6-Tetra-O-benzyl-a-D-glucopyranosyl-(1?2)-3,4,6-tri-
O-benzoyl-b- -galactopyranosyl trichloroacetimidate (15)
D
CAN (5.18 g, 9.46 mmol) was added to a solution of 14 (2.12 g,
1.89 mmol) in CH3CN (30 mL), CH2Cl2 (12 mL) and H2O (6.0 mL),
and the reaction mixture was stirred for 2 h at room temperature.
The reaction mixture was extracted with AcOEt, washed with
aqueous NaHCO3, and dried and concentrated. The resulting crude
product was purified by silica gel column chromatography (tolu-
ene/acetone = 30:1 to 10:1) to give the hemiacetal as a yellow
chromatography (toluene/AcOEt = 30:1 to 8:1) to give 17 (1.54 g,
25
83%) as a colorless oil. [
a]
+10.2 (c 1.0, CHCl3); Rf 0.48 (toluene/
D
AcOEt = 4:1); 1H NMR (400 MHz, CDCl3): d 8.72–6.72 (m, 49H, Ph
ꢁ10), 5.98 (d, 1H, H-4), 5.97 (t, 1H, H-2), 5.88 (d, 1H, H-4), 5.87
(d, 1H, H-400), 5.76 (t, 1H, H-20), 5.58 (dd, 1H, H-3), 5.57 (dd, 1H,
H-30), 5.42 (t, 1H, H-200), 5.36 (dd, 1H, H-300), 5.15 (d, 1H,
J1,2 = 7.8 Hz, H-1), 5.00 (d, 1H, J1,2 = 7.8 Hz, H-10), 4.53 (d, 1H,
J1,2 = 7.8 Hz, H-100), 4.32–4.09 (m, 6H, H-5, H-50, H-500, H-6a0, H-
6a00, H-6b00), 4.03 (dd, 1H, H-6b0), 3.94 (dd, 1H, H-6a), 3.69 (s, 3H,
–OMe), 3.66 (dd, 1H, H-6b), 2.64–2.51 (m, 4H, –CH2CH2–), 2.01
(s, 3H, –COCH3); 13C NMR (100 MHz, CDCl3): d 206.2, 171.6,
165.8, 165.5, 165.4, 165.3, 165.2, 155.8, 151.4, 133.4, 133.2,
133.1, 130.1, 129.9, 129.8, 129.7, 129.6, 129.4, 129.3, 129.2,
129.1, 129.0, 128.9, 128.6, 128.5, 128.4, 128.3, 128.2, 118.8,
114.6, 101.5 (C-1), 100.7 (C-10), 100.6 (C-100), 73.4 (C-5), 72.5 (C-
50), 71.9 (C-300), 71.6 (C-30), 71.5 (C-500), 71.1 (C-3), 69.9 (C-20),
69.7 (C-200), 69.3 (C-2), 68.4 (C-4), 68.1 (C-40), 67.8 (C-400), 67.1(C-
6), 66.6 (C-60), 61.5 (C-600), 55.8 (–OMe), 37.8, 29.6, 27.8; LC–MS
(ESI) calcd for C93H81O2+8 (M+H)+ m/z 1645.48; measure m/z
1645.49; HR-MS C93H80NaO+28 Calcd 1667.4734, Found 1667.4932
[M+Na]+.
oil. Cl3CCN (321 lL, 3.21 mmol) and DBU (72 lL, 0.48 mmol) were
added to a solution of the hemiacetal in CH2Cl2 (3.0 mL) at 0 °C and
stirred for 2 h at 0 °C under a nitrogen atmosphere. After comple-
tion of the reaction, the mixture was purified by silica gel column
chromatography (toluene/AcOEt = 50:1 to 10:1) to give 15 (1.18 g,
25
58%—two steps) as a yellow oil. [
a
]
D
ꢀ7.2 (c 1.0, CHCl3); Rf 0.45
(toluene/AcOEt = 8:1); 1H NMR (400 MHz, CDCl3): d 8.60, 8.58
(each s, 1H, NHCCl3 ꢁ2), 6.91 (d, 1H, J1,2 = 7.6 Hz, H-10 of b), 6.81
(d, 1H, J1,2 = 3.5 Hz, H-1 of a
); LC–MS (ESI) calcd for C68H59Cl3NO1+4
(M+H)+ m/z 1158.29; measured m/z 1158.29.
3.9. 4-Methoxyphenyl 2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl-
(1?2)-3,4,6-tri-O-benzoyl-
O-benzoyl-b- -galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-
galactopyranoside (16)
a-D-galactopyranosyl-(1?6)-2,3,4-tri-
D
D-
3.11. 4-Methoxyphenyl 3,4,6-tri-O-benzoyl-b-
(1?6)-2,3,4-tri-O-benzoyl-b- -galactopyranosyl-(1?6)-2,3,4-tri-
O-benzoyl-b- -galactopyranoside (18)
D-galactopyranosyl-
A
solution of 12 (700 mg, 0.65 mmol) and 15 (1.18 g,
D
1.10 mmol) containing activated type AW-300 molecular sieves
(7.5 g) in dry EtCN (15 mL) was stirred under a nitrogen atmo-
sphere for 2 h at room temperature. After cooling to ꢀ50 °C,
D
NH2NH2–AcOH (172 mg, 1.87 mmol) was added to a solution of
17 (1.54 g, 0.94 mmol) in THF (15 mL) and MeOH (1.5 mL), and the
reaction mixture was stirred for 3 h at room temperature. The reac-
tion mixture was diluted with AcOEt, washed with 5% HCl and
aqueous NaHCO3, dried, and concentrated. The crude product
TMSOTf (40 lL, 220 lmol) was added, and the mixture was stirred
for 1.5 h at ꢀ78 °C. The reaction was neutralized with Et3N, filtered,
and extracted with CHCl3. The organic solvent was washed with 5%
HCl and aqueous NaHCO3, dried, and concentrated to give a clear

I. Ohtsuka et al. / Carbohydrate Research 404 (2015) 9–16
15
was purified by silica gel column chromatography (toluene/ace-
The reaction mixture was extracted with AcOEt, washed with
aqueous NaHCO3, dried, and concentrated. The resulting crude
product was purified by silica gel column chromatography (tolu-
ene/acetone = 30:1 to 10:1) to give the hemiacetal as a yellow
tone = 40:1 to 20:1) to give 18 (1.23 g, 85%) as a colorless oil.
25
[
a]
+4.8 (c 1.0, CHCl3); Rf 0.45 (toluene/acetone = 4:1); 1H NMR
D
(400 MHz, CDCl3): d 8.09–6.75 (m, 49H, Ph ꢁ10), 5.95 (t, 1H, H-
2), 5.93–5.92 (m, 2H, H-4, H-40), 5.85 (d, 1H, H-400), 5.82 (t, 1H,
H-20), 5.59 (dd, 1H, H-3), 5.55 (dd, 1H, H-30), 5.34 (dd, 1H, H-300),
5.23 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.95 (d, 1H, J1,2 = 7.8 Hz, H-10), 4.53
(d, 1H, J1,2 = 7.8 Hz, H-100), 4.48 (q, 1H, H-5), 4.40 (dd, 1H, H-6a00),
4.28 (t, 1H, H-50), 4.17–4.09 (m, 5H, H-200, H-6a0, H-6b0, H-6b00, H-
6a), 3.78 (dd, 1H, H-6b), 3.69 (s, 3H, –OMe); 13C NMR (100 MHz,
CDCl3): d 165.9, 165.7, 165.5, 165.3, 165.2, 155.7, 151.4, 133.6,
133.4, 133.2, 133.0, 130.0, 129.7, 129.3, 129.2, 129.1, 129.0,
128.9, 128.8, 128.6, 128.5, 128.5, 128.5, 128.4, 128.3, 128.2,
125.3, 118.8, 114.6, 103.0 (C-100), 101.9 (C-10), 101.2 (C-1), 78.8
(C-5), 73.7 (C-300), 72.6 (C-50), 71.9 (C-3), 71.8 (C-30), 71.1 (C-500),
67.7 (C-20), 67.7 (C-6), 69.4 (C-2), 69.0 (C-200), 68.9 (C-60), 68.3 (C-
4), 67.9 (C-40), 67.4 (C-400), 61.7 (C-600), 55.7 (–OMe); LC–MS (ESI)
calcd for C88H75O+26 (M+H)+ m/z 1547.44; measured m/z 1547.43;
HR-MS C88H74NaO+26 Calcd 1569.4336, Found 1569.4635 [M+Na]+.
oil. Cl3CCN (321 lL, 3.21 mmol) and DBU (72 lL, 0.48 mmol) were
added to a solution of the hemiacetal in CH2Cl2 (3.0 mL) at 0 °C and
stirred for 2 h at 0 °C under a nitrogen atmosphere. After comple-
tion of the reaction, the mixture was purified by silica gel column
chromatography (toluene/AcOEt = 50:1 to 10:1) to give 20
25
(412 mg, 61%—two steps) as a colorless oil. [
a]
ꢀ3.3 (c 1.0,
D
CHCl3); Rf 0.55 (toluene/AcOEt = 8:1); 1H NMR (400 MHz, CDCl3):
d 8.52, 8.33 (each s, 2H, NHCCl3 ꢁ2), 6.79 (d, 1H, J1,2 = 7.8 Hz, H-1
of b), 6.77 (d, 1H, J1,2 = 3.9 Hz, H-1 of
a); LC–MS (ESI) calcd for
C117H103Cl3NO+30 (M+H)+ m/z 2106.56; measured m/z 2106.56.
3.14. 2,3,4,6-Tetra-O-benzyl-
O-benzoyl-b- -galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b- -galactopyranosyl-
a-D-glucopyranosyl-(1?2)-3,4,6-tri-
D
D-
D
(1?1)-(2S,3S,4R)-3,4-di-O-benzyl-2-hexadecanamido-octadecane
(22)
3.12. 4-Methoxyphenyl 2,3,4,6-tetra-O-benzyl-
anosyl-(1?2)-3,4,6-tri-O-benzoyl-b- -galactopyranosyl-
(1?6)-2,3,4-tri-O-benzoyl-b- -galactopyranosyl-(1?6)-
2,3,4-tri-O-benzoyl-b- -galactopyranoside (19)
a-D-glucopyr
D
A solution of 20 (412 mg, 196
benzyl-2-hexadecanamido-octadecane-3,4-diol
391 mol) containing activated type AW-300 molecular sieves
l
mol) and (2S,3S,4R)-3,4-di-O-
D
21 (288 mg,
D
l
(2.0 g) in dry CH2Cl2 (4.0 mL) was stirred under a nitrogen atmo-
A solution of 18 (1.23 g, 0.80 mmol) and 13 (1.51 g, 2.40 mmol)
containing activated type AW-300 molecular sieves (12 g) in dry
CH2Cl2 (15 mL) and dry Et2O (10 mL) was stirred under a nitrogen
atmosphere for 2 h at room temperature. After cooling to 0 °C, NIS
sphere for 5 h at room temperature. After cooling to 0 °C, TMSOTf
(26 lL, 117 lmol) was added, and the solution was stirred for
1.5 h at 0 °C. The mixture was neutralized with Et3N, filtered, and
extracted with CHCl3. The organic solvent was washed with 5%
HCl and aqueous NaHCO3, dried, and concentrated to give a clear
oil. The crude product was purified by silica gel column chroma-
(1.06 g 4.8 mmol) and TfOH (44 lL, 0.47 mmol) were added
sequentially, and the mixture was stirred for 1.5 h at 0 °C. The reac-
tion was neutralized with Et3N, filtered, and extracted with CHCl3.
The organic solvent was washed with aqueous sodium thiosulfate,
5% HCl, and aqueous NaHCO3, and then dried and concentrated to
give a clear oil. The crude product was purified by silica gel column
chromatography (toluene/acetone = 40:1 to 15:1) to give 19
tography (toluene/AcOEt = 40:1 to 10:1) to give 22 (210 mg, 40%)
25
as a colorless oil. [
a]
+6.8 (c 1.0, CHCl3); Rf 0.50 (toluene/
D
AcOEt = 8:1); 1H NMR (400 MHz, CDCl3): d 8.10–6.78 (m, 75H, Ph
ꢁ15), 5.89 (d, 1H), 5.72–5.61 (m, 2H), 5.59 (dd, 1H), 5.50 (dd,
1H), 5.41–5.33 (m, 2H), 5.40 (d, 1H, J1,2 = 3.6 Hz, H-1000), 4.83–4.56
(m, 7H), 4.62 (d, 1H, J1,2 = 7.9 Hz, H-100), 4.49 (d, 1H, J1,2 = 7.8 Hz,
H-1), 4.48 (d, 1H, J1,2 = 7.8 Hz, H-10), 4.41 (d, 1H), 4.35 (d, 1H),
4.23 (d, 1H), 4.12–3.99 (m, 4H), 3.95–3.74 (m, 6H), 3.67–3.49 (m,
5H), 3.31 (q, 1H), 3.15 (d, 1H), 3.07 (d, 1H), 2.93 (d, 1H), 1.57,
1.25, 0.87; 13C NMR (100 MHz, CDCl3): d 171.9, 165.6, 165.5,
165.4, 165.1, 164.9, 139.1, 138.8, 138.6, 138.5, 138.5, 137.8,
133.7, 133.4, 133.2, 133.0, 129.9, 129.8, 129.7, 129.4, 129.3,
129.2, 129.1, 129.0, 128.9, 128.7, 128.6, 128.4, 128.4, 128.1,
127.9, 127.8, 127.6, 127.4, 127.2, 127.1, 126.9, 102.1 (C-100), 101.3
(C-1), 100.7 (C-10), 96.6 (C-1000), 81.4, 79.7, 79.6, 75.5, 74.2, 73.9,
73.2, 72.8, 72.6, 72.5, 72.0, 71.7, 71.5, 71.2, 70.8, 70.4, 70.3, 69.7,
68.3, 67.7, 67.3, 67.1, 64.4, 60.9, 48.9, 36.2, 31.9, 29.8, 29.7, 29.6,
29.4, 29.2, 26.2, 25.4, 22.6, 14.1; LC–MS (ESI) calcd for
(1.33 g, 82%) as a yellow oil. [a]
25 +4.5 (c 1.0, CHCl3); Rf 0.53 (tolu-
D
ene/AcOEt = 8:1); 1H NMR (400 MHz, CDCl3): d 8.09–6.76 (m, 69H,
Ph ꢁ14), 5.59 (t, 1H, H-2), 5.93 (d, 1H, H-4), 5.89 (d, 1H, H-40), 5.71
(d, 1H, H-400), 5.70 (t, 1H, H-20), 5.52 (dd, 1H, H-3), 5.51 (dd, 1H, H-
30), 5.44 (dd, 1H, H-300), 5.41 (d, 1H, J1,2 = 3.8 Hz, H-1000), 5.11 (d, 1H,
J1,2 = 7.8 Hz, H-1). 4.76 (d, 1H, J1,2 = 7.8 Hz, H-10), 4.55 (d, 1H,
J1,2 = 7.8 Hz, H-100), 4.86–4.54 (m, 5H, PhCH2O ꢁ5), 4.37 (d, 1H,
PhCH2O), 4.22–4.08 (m, 4H, PhCH2O ꢁ2, H-200, H-6a00), 3.95 (dd,
1H, H-6b00), 3.92–3.69 (m, 9H, H-3000, H-5, H-50, H-500, H-6a, H-6a0,
–OMe), 3.65 (dd, 1H, H-6b0), 3.54–3.50 (m, H-2000, H-4000, H-5000),
3.43 (dd, 1H, H-6b), 3.08 (dd, 1H, H-6000), 2.97 (dd, 1H, H-6000); 13C
NMR (100 MHz, CDCl3): d 165.6, 165.4, 165.3, 165.2, 164.9, 155.9,
151.3, 138.7, 138.6, 138.5, 137.8, 133.5, 133.4, 133.3, 133.2,
133.1, 133.1, 133.0, 132.9, 130.2, 130.1, 129.9, 129.8, 129.7,
129.4, 129.3, 129.2, 129.1, 128.9, 128.5, 128.5, 128.4, 128.3,
128.2, 128.1, 128.0, 118.8, 114.7, 102.3 (C-100), 101.6 (C-1), 100.6
(C-10), 96.6 (C-1000), 81.5, 79.3, 75.4, 74.2, 73.6, 73.2, 72.6, 72.5,
72.0, 71.8, 71.7, 71.6, 70.3, 69.8, 69.6, 68.3, 67.7, 67.6, 67.3, 66.9,
64.5, 61.0, 55.6; LC–MS (ESI) calcd for C122H109O+31 (M+H)+ m/z
2069.69; measured m/z 2069.69; HR-MS C122H108NaO+31 Calcd
2091.6772, Found 2091.6970 [M+Na]+.
C
163H182NO+33 (M+H)+ m/z 2681.25; measured m/z 2681.24; HR-
MS C163H181NNaO33 Calcd 2070.2414, Found 2070.2610 [M+Na]+.
3.15.
a
-D
-Glucopyranosyl-(1?2)-b-D-galactopyranosyl-(1?6)-b-
D
-galactopyranosyl-(1?6)-b-
D
-galactopyranosyl-(1?1)-
(2S,3S,4R)-2-hexadecanamido-octadecane-3,4-di-ol (2)
Pd/C (10%, 350 mg) was added to a solution of 22 (210 mg,
78.4 lmol) in CH2Cl2 (2.0 mL), MeOH (2.0 mL), and AcOH
3.13. 2,3,4,6-Tetra-O-benzyl-
a
-
D
-glucopyranosyl-(1?2)-3,4,6-tri-
(2.0 mL), and the reaction mixture was stirred under a hydrogen
atmosphere at room temperature for 16 h. The mixture was fil-
tered, concentrated, and redissolved in MeOH (7.0 mL) and 1,4-
O-benzoyl-b- -galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-
D
galactopyranosyl-(1?6)-2,3,4-tri-O-benzoyl-b-D-galactopyran-
osyl trichloroacetimidate (20)
dioxane (7.0 mL). Then, 30% NaOMe (100 lL) in MeOH was added
to the solution, and the mixture was stirred for 5 h at room tem-
perature. The reaction was neutralized with Amberlite IR-120 (H+
form), filtered, and concentrated. The product was purified by
Sephadex LH-20 column chromatography (CHCl3/MeOH = 1:1) to
CAN (878 mg, 1.60 mmol) was added to a solution of 19
(663 mg, 0.32 mmol) in CH3CN (40 mL) and H2O (5.0 mL), and
the reaction mixture was stirred for 3 h at room temperature.

16
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give 2 (94 mg, quant.—two steps) as a white solid crystals. Mp
131–133 °C; [
25 +10.2 (c 1.0, MeOH); Rf 0.28 (CHCl3/MeOH = 4:1);
a]
D
1H NMR (400 MHz, CD3OD/CDCl3 = 1:1): d 5.28 (d, 1H, J1,2 = 3.7 Hz,
H-1000), 4.53 (d, 1H, J1,2 = 7.9 Hz, H-100), 4.36 (d, 1H, J1,2 = 7.8 Hz, H-
1), 4.34 (d, 1H, J1,2 = 7.8 Hz, H-10); 13C NMR (100 MHz, CD3OD/
CDCl3 = 1:1): d 174.9, 103.3 (C-100), 102.8 (C-1), 102.7 (C-10), 98.3
(C-1000), 77.5, 73.8, 73.5, 73.1, 72.9, 72.6, 71.7, 71.6, 71.4, 70.7,
69.9, 68.9, 68.7, 68.6, 68.0, 65.1, 61.1, 60.7, 48.4, 35.7, 31.3, 29.1,
29.0, 28.9, 28.8, 28.7, 26.5, 25.3, 22.0, 13.1; LC–MS (ESI) calcd for
C
C
58H110NO+24 (M+H)+ m/z 1204.73; measured m/z 1204.73; HR-MS
58H109NNaO24 Calcd 1226.7237, Found 1226.7360 [M+Na]+.
Acknowledgment
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The authors are grateful to Ms. J. Hada (Faculty of Pharmacy,
Keio University) for providing HR-MS data.
21. Demchenko, A. V. In Handbook of Chemical Glycosylation; Demchenko, A. V., Ed.;
Elsevier: Amsterdam, The Netherlands, 2008; pp 1–28.
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2014.11.
018.
References
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Further reading
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Elsevier: Amsterdam, The Netherlands, 2007; Vol. 3, pp 253–284.
6. Ziche, M.; Alessandri, G.; Gullino, P. M. Lab. Invest. 1989, 61, 629–634.
7. Ziche, M.; Alessandri, G.; Gullino, P. M. Lab. Invest. 1992, 67, 711–715.
3. Angata, T.; Varki, A. Chem. Rev. 2002, 102, 439–469.
17. Mallet, J. M.; Meyer, G.; Yyelin, F.; Jutand, A.; Amatore, C.; Sinayi, P. Carbohydr.
Res. 1993, 244, 237–346.