Synthesis of a novel glycosphingolipid from the millipede, Parafontaria laminata armigera, and the assembly of its carbohydrate moiety into multivalent structures

Article abstract of DOI:10.1016/j.carres.2006.04.019

A novel glycosphingolipid, β-d-Manp-(1→4)-[α-l-Fucp-(1→3)]-β-d-Glcp-(1→1)-Cer, found in the millipede, Parafontaria laminata armigera, and multivalent derivatives of its carbohydrate moiety were synthesized. As the key step, the target glycolipid (1) was

Full text of DOI:10.1016/j.carres.2006.04.019

Carbohydrate Research 341 (2006) 1341–1352
Synthesis of a novel glycosphingolipid from the millipede,
Parafontaria laminata armigera, and the assembly of its
carbohydrate moiety into multivalent structures
Noriyasu Hada, Yoshiko Sonoda and Tadahiro Takeda*
Kyoritsu University of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
Received 31 January 2006; received in revised form 12 April 2006; accepted 16 April 2006
Available online 15 May 2006
Abstract—A novel glycosphingolipid, b-D-Manp-(1!4)-[a-L-Fucp-(1!3)]-b-D-Glcp-(1!1)-Cer, found in the millipede, Parafon-
taria laminata armigera, and multivalent derivatives of its carbohydrate moiety were synthesized. As the key step, the target glyco-
lipid (1) was obtained through an inversion reaction at the 2-position of a b-glucopyranoside residue yielding a b-mannopyranoside.
In addition, the synthesis of fluorescently labeled trimer and tetramer glycoconjugates (2, 3) was achieved by iterative amide bond
formation using a monomer unit (24).
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: Glycosphingolipids; Parafontaria laminata armigera; Intermolecular nucleophile approach; Glycocluster
1. Introduction
ation. This earlier paper was the first report on the total
synthesis of b-D-Manp-(1!4)-[a-Fucp-(1!3)]-b-D-
Glcp-(1!1)-Cer by this method. Among b-mannopyr-
anoside syntheses, one approach is the so called inter-
molecular nucleophiles approach,7–10 in which an
inversion reaction at the 2-position of b-glucopurano-
side yields a b-mannopyranoside. The strategy, which
provides good yields, relies upon creating a highly reac-
tive leaving group at the position that is to be inverted
and then treating it with a strong nucleophile. The
nucleophile causes SN2 displacement and epimerizes
the position on the carbohydrate ring. We report here
the application of this approach for the synthesis of gly-
cosphingolipid 1.
In our continuing studies to elucidate the biological func-
tion of glycoconjugates, we have synthesized novel glyco-
sphingolipids found in various invertebrates1–6 that do
not have gangliosides. A number of glycosphingolipids
from a variety of invertebrate origins and related ana-
logues have been synthesized and their influence on pro-
liferation has been examined using mouse melanoma B16
cells. In these studies, the glycolipid, b-D-Manp-(1!4)-
[a-Fucp-(1!3)]-b-D-Glcp-(1!1)-Cer, which has been
isolated from the millipede Parafontaria laminata armi-
gera, has been observed to inhibit significantly cell
proliferation (details will be reported elsewhere).
In a previous paper,2 we reported the synthesis of this
glycosphingolipid. The key reaction in that synthesis
was a two-step glycosylation. The first step was the for-
mation of an orthoester from two sugar moieties and the
second step was the reductive cleavage of one of the
orthoester C–O bonds leading to b-selective mannosyl-
It is known that oligosaccharide chains generally
interact with their protein receptors in a multivalent
fashion to overcome the inherently low affinity of mono-
valent carbohydrate–protein interactions. Therefore, the
construction of a clustered glycoconjugates is an impor-
tant subject in glycoscience.11,12 For this reason, we have
used a new method to synthesize new peptidic glycoclus-
ters and glycodendrons (2, 3), which consist of a b-ala-
nine derivative linked to the sugar moiety of 1
(Fig. 1).13,14
*
Corresponding author. Tel.: +81 3 5400 2696; fax: +81 3 5400
2556; e-mail: takeda-td@kyoritsu-ph.ac.jp
0008-6215/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.04.019

1342
N. Hada et al. / Carbohydrate Research 341 (2006) 1341–1352
O
C
H
HO
O
15 31
HN
O
HO
HO
HO
O
(CH ) CH
O
2 12
3
O
OH
OH
OH
O
Me
OH
1
OH
OH
HO
HO
HO
HO
O
O
O
O
O
OH
OH
O
N
O
HN
HO
Me
O
OH
HO
HO
O
O
O
OH
HO
O
HO
O
OH
OH
O
N
HN
HO
O
O
HO
HO
O
O
O
Me
O
OH
O
N
H
OH
OH
HO
OH
O
Me
OH
HO
OH
O
O
HO
HO
OH
O
O
O
HO
O
OH
O
O
O
N
H
O
O
HO
HO
HO
N
OH
OH
O
HO
O
O
O
N
O
O
Me
OH
OH
H
OH
O
Me
OH
OH
HO
O
HO
OH
OH
O
HO
HO
OH
N
O
O
O
O
O
HO
HO
HO
N
O
N
H
O
O
O
OH
N
OH
O
HO
O
OH
H
DanHN
OH
Me
OH
DanHN
O
Me
OH
2
OH
3
OH
OH
OH
Figure 1. Structure of target glycoconjugates.
2. Results and discussion
2.1. Syntheses of monosaccharide derivatives
Glucopyranosyl donor 8 was obtained from phenyl
4,6-O-benzylidene-1-thio-b-D-glucopyranoside (7),16 via
a two-step procedure. First, reaction of the stannylidene
derivative of 7 with benzyl bromide was found to be
highly regiospecific in this case, which may be associated
with the electron donating nature of the sulfur aglycon.
Second, subsequent chloroacetylation of 7 afforded 8
(81% over two steps).
Syntheses of the glucopyranose building blocks 6a and 8
were carried out as depicted in Scheme 1. Compound 6a
was prepared from known 2-(trimethylsilyl)ethyl 4,6-O-
benzylidene-b-D-glucopyranoside (4)15 via successive
mono-alkylation of the diol, acylation and reductive ring
opening of the 4,6-acetal. Unfortunately, alkylation of 4
via a stanylene intermediate did not proceed in a regio-
specific manner. Although the formation of dialkylated
product was avoided, the two regioisomeric monoalkyl-
ated products could not be separated, nor could their
benzoylated derivatives 5a and 5b be separated. The
structures of 5a and 5b were confirmed after reductive
ring opening of the benzylidene acetal in 5, which yielded
a 1.5:1 ratio of 6a and 6b in a combined yield of 72%.
2.2. Synthesis of the target glycosphingolipid
Glycosylation of acceptor 6a with 8 in the presence of N-
iodosuccinimide (NIS), trifluoromethanesulfonic acid
(TfOH),17 and 4 A molecular sieves in dichloromethane
˚
gave the desired disaccharide (9) in 57% yield after puri-
fication. The stereochemistry of the newly formed glyco-
sidic linkage could be determined by 1H NMR
spectroscopy (H-10, 4.61 ppm, J = 7.9 Hz). Selective
BnO
O
O
Ph
Ph
O
O
O
c
a, b
O
HO
O
R O
2
HO
R O
2
O
O
O
TMS
TMS
TMS
OH
OR
1
OR
1
4
5a
5b
R
= Bz,
R = pMBn
2
= pMBn, R = Bz
2
6a
6b
R
= Bz,
R
= pMBn
2
1
1
1
R
R
= pMBn, R = Bz
2
1
Ph
O
Ph
O
O
O
d, e
O
O
SPh
SPh
BnO
HO
OClAc
OH
8
7
Scheme 1. Reagents: (a) (i) n-Bu2SnO, benzene; (ii) n-Bu4NBr, p-MBnCl, toluene; (b) BzCl, pyridine, 71% two steps; (c) NaBH3CN, HCl/Et2O,
THF, 72%; (d) (i) n-Bu2SnO, benzene; (ii) n-Bu4NBr, BnBr, toluene; (e) ClAcCl, CH2Cl2/pyridine, 81% two steps.

N. Hada et al. / Carbohydrate Research 341 (2006) 1341–1352
1343
removal of the chloroacetyl group in 9 with NaHCO3
gave 10, a disaccharide containing a b-D-glucopyranosyl
residue with the C20 hydroxyl group unsubstituted.
Compound 10 was converted into the triflate by treat-
ment with trifluoromethanesulfonic anhydride in the
presence of pyridine at 0 °C. The triflate was then sub-
jected to a nucleophilic substitution reaction with tetra-
butylammonium benzoate, which inverted the C20
stereocenter converting the b-D-glucopyranosyl moiety
to a b-D-mannopyranosyl residue (11) in good overall
yield from 10 (80%). The anomeric hydrogen atom of
the mannopyranosyl unit appeared in the NMR spec-
trum as a signal at d 4.79 (s, H-10). The b-D configura-
tion of the glycosidic bond was indicated by the
1JC1,H1 value of 157.3 Hz (C-10, 99.4 ppm) in the 13C
NMR spectrum.18 Removal of the 30-O-pMBn group
from 11 by DDQ gave the disaccharide acceptor 12.
Glycosylation of 12 with known fucopyranosyl donor
with trichloroacetonitrile in the presence of DBU gave
the corresponding a-trichloroacetimidate 16. Glycosyl-
ation of (2S,3R,4E)-3-O-benzoyl-2-hexadecanamido-4-
octadecene-1,3-diol 176 with 16 was carried out in the
presence of trimethylsilyl trifluoromethanesulfonate
˚
(TMSOTf) and 4 A molecular sieves to afford the de-
sired b-glycoside 18 in 35% yield. Finally, removal of
acyl groups in 18 under Zemplen conditions and column
chromatography on Sephadex LH-20 furnished a target
glycolipid 1 (Scheme 2). The structure and purity of 1
1
was demonstrated by H NMR spectroscopy and high-
resolution fast-atom-bombardment mass spectrometry.
2.3. Syntheses of multivalent glycoconjugates
We planned to use glycosyl donor 16 in the syntheses of
the multivalent glycoconjugates. Elongation of the gly-
cocluster was done using three iterative reactions: amide
bond formation, deprotection of the tert-butoxycar-
bonyl (Boc) group, and deprotection of the trichloro-
ethyl ester (Tce) group. The required building block 24
was prepared as follows. Coupling of trisaccharide imi-
date 16 with the spacer 19, which was obtained from
hexanolamine, in the presence of TMSOTf gave com-
pound 20 in quantitative yield. Selective removal of
the Troc group from 20 by zinc metal and acetic acid
gave the primary amine 21 also in quantitative yield.
Compound 21 was condensed in the presence of diethyl
1319 in the presence of NIS, TfOH and 4 A molecular
˚
sieves in dichloromethane at ꢀ60 °C gave the desired tri-
saccharide 14 (99%). The stereochemistry of the intro-
1
duced fucopyranosyl residue was evident by H NMR
spectroscopy (H-100, 5.37 ppm, J = 3.7 Hz). Removal of
the benzylidene and benzyl group from 14 by catalytic
hydrogenolysis over 10% Pd/C in THF/CH3OH/AcOH
(3:3:1) and subsequent acetylation gave 15. Selective re-
moval of the 2-(trimethylsilyl)ethyl (SE) group with tri-
fluoroacetic acid in dichloromethane, and treatment20
Ph
O
O
O
SPh
OAcCl
BnO
BnO
BnO
OR
c
O
BnO
O
O
a
8
O
BnO
O
O
O
RO
O
O
MBnO
O
TMS
O
OBz
Ph
p
TMS
+
OBz
O
Ph
OBz
BnO
HO
MBnO
O
11 R =
pMBn
9 R = ClAc
10 R = H
O
p
d
TMS
b
OBz
12 R = H
6
R O
3
O
R O
5
R O
5
3
O
R
1
R
R
H
R
R
R
5
R O
O
1
2
3
4
SPh
OBn
O
a
O
R O
4
Me
14 OSE
Bn Bz PhCH
12
+
OR
R
4
2
e
OBn
13
O
OBn
Me
OR
15 OSE
H
Ac
Ac
Bz
Bz
Ac
Ac
3
OR
3
f
3
OR
16
O
H OC(NH)CCl
O
3
C15H31
(CH2)12CH3
RO
O
HN
O
RO
RO
RO
O
O
C15H31
O
HN
OR'
g
OR'
OR'
16
+
(CH2)12CH3
HO
O
Me
OR
OBz
17
OR
OR
18 R = Ac, R' = Bz
1 R = H
h
˚
Scheme 2. Reagents: (a) NIS, TfOH, 4 A molecular sieves, CH2Cl2 9: 57%, 14: 99%; (b) NaHCO3, THF, 76%; (c) (i) Tf2O, CH2Cl2/pyridine; (ii) n-
Bu4NBz, DMF, 80%; (d) DDQ, CHCl3/H2O (19:1) 78%; (ii) n-Bu4NBr, BnBr, toluene; (e) (i) H2, Pd/C, CH3OH/AcOH; (ii) Ac2O/pyridine, 60%; (f)
˚
(i) TFA, CH2Cl2; (ii) CCl3CN, DBU, CH2Cl2, 98%; (g) TMSOTf, 4 A molecular sieves, CH2Cl2 35%; (h) NaOCH3, 1,4-dioxane/CH3OH, 94%.

1344
N. Hada et al. / Carbohydrate Research 341 (2006) 1341–1352
phosphorocyanidate (DEPC) in dry DMF with b-ala-
nine derivative 22, which was prepared according to a
previous paper,13 to give 23 in 66% yield. Subsequent re-
moval of the Tce group with zinc metal and acetic acid
afforded an 87% yield of 24, which has a free carboxylic
acid moiety. Coupling of 24 with 21 gave the dimer 25 in
71% yield. The Boc group of 25 was removed with 50%
TFA affording 26 (86% yield), which was subsequently
subjected to the next cycle for the elongation to give
the desired trimer glycocluster derivative 27 (79%). Dan-
syl glycine was introduced into the trimer 28 in the pres-
ence of DEPC to give compound 29 in 85% yield.
Finally, complete removal of the acyl groups provided
the fluorescently labeled glycocluster 2 in 91% yield
(Scheme 3). Furthermore, coupling of 28 with 24 in a
subsequent cycle gave the desired tetramer derivative
30 in 83% yield, and following the cleavage of the Boc
group (56% yield), the resulting amine was coupled with
dansyl glycine as described for 29 to give compound 32
in 94% yield. Complete removal of the acyl groups and
esters afforded 3 in 91% yield (Scheme 4). The structure
conjugates. This method should be of use in the prepa-
ration of a number of other such species. The results
of the biological testing of these compounds will be
reported in detail elsewhere.
3. Experimental
3.1. General methods
Optical rotations were measured with a Jasco digital
polarimeter. 1H NMR and 13C NMR spectra were
recorded with a JMN A500 FT NMR spectrometer with
(CH3)4Si as the internal standard for solutions in CDCl3.
MALDI-TOF mass spectra (MALDIMS) were recorded
on a Perseptive Voyager RP mass spectrometer. High-res-
olution mass spectra were recorded on a JEOL JMS-700
under FAB conditions (FABMS). TLC was performed
on Silica Gel 60 F254 (E. Merck) with detection by quench-
ing of UV fluorescence and by charring with 10% H2SO4.
Column chromatography was carried out on Silica Gel 60
(E. Merck). 2-(Trimethylsilyl)ethyl 4,6-O-benzylidene-b-
D-glucopyranoside (4),15 phenyl 4,6-O-benzylidene-1-
thio-b-D-glucopyranoside (3),16 phenyl 2,3,4-tri-O-benzyl-
1-thio-b-L-fucopyranoside (13)19 and (2S,3R,4E)-3-O-
benzoyl-2-hexadecanamido-4-octadecene-1,3-diol (17)6
were prepared as reported in the literature.
1
and purity of 2 and 3 were supported by H and 13C
NMR as well as mass spectrometry data.
In conclusion, the synthesis of oligosaccharides con-
taining the b-D-Manp-(1!4)-Glcp structural motifs
has been carried out. In addition, an efficient synthetic
strategy was developed to obtain new multivalent glyco-
HO
AcO
AcO
NHTroc
O
O
AcO
AcO
AcO
19
a
AcO
AcO
O
O
O
O
O
O
OBz
NHR
O
BzO
OBz
OBz
AcO
O
CCl
3
O
Me
O
OAc
Me
OAc
16
20 R=Troc
NH
b
OAc
OAc
OAc
OAc
R=H
21
AcO
AcO
AcO
O
COOTce
O
COOR
HOOC
O
N
O
O
AcO
Boc
OBz
22
OBz
O
AcO
HN
O
Me
O
O
AcO
AcO
OAc
c
NBoc
O
O
O
OAc
N
H
O
OBz
21
OAc
d
OBz
b
AcO
O
AcO
O
O
Me
OAc
AcO
AcO
NR
O
O
O
OAc
OAc
23 R=Tce
24 R=H
N
H
O
OBz
OBz
AcO
O
Me
OAc
25 R=Boc
26 R=H
OAc
OAc
e
R O
2
R O
2
2
O
R O
O
O
O
O
SO
2
OR
R O
2
1
OR
1
O
HN
O
Me
R O
Dan =
OR
2
OR
2
N
2
H C
3
CH
3
R O
2
O
O
R O
2
N
O
O
O
O
R O
2
f
N
H
O
R
R
R
3
Boc
1
2
OR
OR
1
R O
2
1
26
27
Bz
Bz
Bz
H
Ac
Ac
Ac
H
O
Me
OR
e
2
OR
2
28
29
2
H
R O
2
O
OR
2
O
R O
2
R O
2
g
h
NR
3
O
O
O
COCH NHDan
2
N
H
O
OR
OR
1
R O
2
1
O
COCH NHDan
Me
2
OR
2
OR
2
OR
2
Scheme 3. Reagents: (a) 19, TMSOTf, AW300 molecular sieves, CH2Cl2, quant; (b) Zn/AcOH, 21: quant 24: 87%; (c) 22, DEPC, Et3N, DMF, 66%;
(d) 21, DEPC, Et3N, DMF, 71%; (e) 50% TFA, 26: 86%, 28: 73%; (f) 24, DEPC, Et3N, DMF, 79%; (g) dansyl glycine, DEPC, Et3N, DMF, 85%;
(h) NaOCH3, 1,4-dioxane/CH3OH, 91%.

N. Hada et al. / Carbohydrate Research 341 (2006) 1341–1352
1345
R O
2
R O
2
O
R O
2
O
O
O
O
R O
2
OR
1
OR
1
O
N
O
HN
Me
OR
2
OR
2
OR
2
R O
2
O
O
R O
2
O
O
O
R O
O
2
O
N
H
OR
1
OR
R O
1
2
O
Me
OR
2
a
OR
2
2
28
OR
R O
2
O
O
O
R O
2
R O
2
R O
2
N
O
O
O
O
O
N
H
R
R
2
R
3
1
OR
OR
1
1
30
31
32
3
Bz
Bz
Bz
H
Ac
Ac
Ac
H
Boc
O
Me
OR
b
2
OR
2
2
H
OR
R O
2
O
NR
R O
2
c
d
3
O
O
R O
2
O
COCH NHDan
2
O
N
OR
OR
R O
1
H
1
2
O
Me
OR
COCH NHDan
2
2
OR
2
OR
2
Scheme 4. Reagents: (a) 24, DEPC, Et3N, DMF, 83%; (b) 50% TFA, 56%; (c) dansyl glycine, DEPC, Et3N, DMF, 94%; (d) NaOCH3, 1,4-dioxane/
CH3OH, 91%.
3.2. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzyl-
idene-3-O-p-methoxybenzyl-1-thio-b-D-glucopyranoside
(5a) and 2-(trimethylsilyl)ethyl 3-O-benzoyl-4,6-O-benz-
ylidene-2-O-p-methoxybenzyl-b-D-glucopyranoside (5b)
for 2 h at rt, then cooled to 0 °C. Hydrogen chloride
in Et2O was added until the solution was acidic (pH
paper, gas evolution). After 1 h, the reaction mixture
was poured into ice H2O and extracted with CHCl3.
The extract was washed sequentially with aq NaHCO3
and H2O, dried (Na2SO4), and concentrated. The product
was purified by silica gel column chromatography using
3:1 hexane/EtOAc as eluent to give 6a (1.24 g, 45%) and
A mixture of 2-(trimethylsilyl)ethyl 4,6-O-benzylidene-
b-D-glucopyranoside (4) (2.9 g, 7.88 mmol), n-Bu2SnO
(2.2 g, 8.83 mmol) and 80 mL of dry benzene was stirred
at reflux for 5 h. The benzene was distilled off and the
stannylidene derivative was redissolved in toluene
(20 mL), and n-Bu4NBr (2.5 g, 7.76 mmol) and p-meth-
oxybenzyl chloride (1.6 mL, 11.8 mmol) were added at
60 °C. After stirring the reaction mixture for 5 h, the
solution was concentrated. Purification of the residue
by column chromatography (10:1, toluene/EtOAc) on
silica gel gave a mixture of 2-(trimethylsilyl)ethyl 4,6-
O-benzylidene-3-O-p-methoxybenzyl-b-D-glucopyrano-
side and 2-(trimethylsilyl)ethyl 4,6-O-benzylidene-2-O-
p-methoxybenzyl-b-D-glucopyranoside (3.1 g, 81%);
MALDIMS m/z calcd for [C26H36O7Si+Na]+: 511.2.
Found: 511.3. To a solution of these compounds
(3.1 g, 6.34 mmol) in pyridine (20 mL) was added ben-
zoyl chloride (1.5 mL, 12.9 mmol), and the reaction mix-
ture was stirred for 4 h at rt. Toluene was added and the
solution was evaporated, the residue was extracted with
CHCl3, washed with 5% HCl, aq NaHCO3, and H2O,
dried (MgSO4), and concentrated. The product was
purified by silica gel column chromatography using 6:1
hexane/EtOAc as eluent to give the mixture of 5a and
5b (3.2 g, 85%): MALDIMS m/z calcd for [C33H40O8-
Si+Na]+: 615.3. Found: 615.5.
1
6b (851 mg, 29%): compound 6a: H NMR (CDCl3): d
8.00–6.64 (m, 14H, 3Ph), 5.18 (dd, 1H, J1,2 = 7.9 Hz,
J2,3 = 9.2 Hz, H-2), 4.62–4.50 (m, 4H, 2PhCH2), 4.57
(d, 1H, H-1), 3.98 (dt, 1H, CH2CH2Si), 3.79–3.71 (m,
3H, H-4, 6a, 6b), 3.67 (s, 3H, OCH3), 3.61 (dd, 1H, H-
3), 3.53–3.46 (m, 2H, H-5, CH2CH2Si), 2.81 (d, 1H,
OH), 0.87–0.74 (m, 2H, CH2CH2Si), ꢀ0.14 (s, 9H,
Si(CH3)3); 13C NMR (CDCl3): d 165.1, 159.2, 137.8,
132.9, 130.1, 129.9, 129.7, 129.6, 128.4, 127.8, 127.7,
113.7, 100.6 (C-1), 81.9 (C-3), 74.2 (C-5), 73.9 (PhCH2),
73.7 (PhCH2), 73.5 (C-2), 72.2 (C-4), 70.4 (C-6), 67.2
(CH2), 55.1 (OCH3), 17.9 (CH2CH2Si), ꢀ1.6 (Si(CH3)3);
MALDIMS m/z calcd for [C33H42O8Si+Na]+: 617.3.
Found: 617.4. Compound 6b: 1H NMR (CDCl3): d
7.96–6.58 (m, 14H, 3Ph), 5.18 (t, 1H, J2,3 3,4 = 9.2 Hz,
H-3), 4.72–4.53 (m, 4H, 2PhCH2), 4.48 (d, 1H,
J1,2 = 7.3 Hz, H-1), 4.00 (dt, 1H, CH2CH2Si), 3.78–3.59
(m, 4H, H-4, 6a, 6b, CH2), 3.68 (s, 3H, OCH3), 3.49 (dt,
1H, H-5), 3.44 (dd, 1H, H-2), 3.06 (d, 1H, OH), 0.71–
0.60 (m, 2H, CH2CH2Si), ꢀ0.17 (s, 9H, Si(CH3)3); 13C
NMR (CDCl3): d 167.1, 159.0, 137.8, 133.1, 130.1,
129.9, 129.8, 129.6, 128.4, 128.2, 127.8, 113.5, 103.1
(C-1), 78.0 (C-3), 77.8 (C-2), 74.3 (C-5), 73.63 (PhCH2),
73.60 (PhCH2), 71.0 (C-4), 70.1 (C-6), 67.6 (CH2), 55.1
(OCH3), 18.5 (CH2CH2Si), ꢀ1.4 (Si(CH3)3).
3.3. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-6-O-benzyl-3-O-
p-methoxybenzyl-b-D-glucopyranoside (6a)
3.4. Phenyl 4,6-O-benzylidene-3-O-benzyl-2-O-chloro-
acetyl-1-thio-b-D-glucopyranoside (8)
To a solution of compounds 5a and 5b (2.9 g,
4.89 mmol) and sodium cyanoborohydride (2.4 g,
38.2 mmol) in dry THF (50 mL) was added powdered
A mixture of phenyl 4,6-O-benzylidene-1-thio-b-D-
glucopyranoside (7) (1.0 g, 2.67 mmol), n-Bu2SnO
˚
3 A molecular sieves (3 g), and the mixture was stirred

1346
N. Hada et al. / Carbohydrate Research 341 (2006) 1341–1352
(731 mg, 2.94 mmol), and 20 mL of dry benzene was
stirred at reflux for 5 h. The benzene was distilled off
and the stannylidene derivative was redissolved in tolu-
ene (10 mL) and then n-Bu4NBr (1.29 g, 4.01 mmol) and
benzyl bromide (0.48 mL, 4.04 mmol) were added at
60 °C. After the reaction mixture was stirred for 5 h,
the solution was concentrated. Purification of the resi-
due by column chromatography (20:1, toluene/EtOAc)
on silica gel gave phenyl 4,6-O-benzylidene-3-O-benz-
yl-b-D-glucopyranoside (1.08 g, 84%); MALDIMS m/z
calcd for [C26H26O5S+Na]+: 473.2. Found: 473.3. To
a solution of this compound (1.08 g, 2.25 mmol) in
CH2Cl2/pyridine (4:1, 10 mL) was added chloroacetyl
chloride (0.35 mL) and the mixture was stirred for 2 h
at 0 °C. Toluene was added and evaporated, then ex-
tracted with CHCl3, washed with 5% HCl, aq NaHCO3,
and H2O, dried (MgSO4), and concentrated. The prod-
uct was purified by silica gel column chromatography
using 20:1 benzene/EtOAc as the eluent to give 8
d, 2H, CH2Cl), 3.75–3.39 (m, 11H, H-3, H-6a, H-6b, H-
60b, 30, 40, 50, OCH3, CH2CH2Si), 3.18 (dt, 1H, H-5),
0.90–0.80 (m, 2H, CH2CH2Si), ꢀ0.09 (s, 9H, Si(CH3)3);
13C NMR (CDCl3): d 165.8, 165.0, 138.1, 137.9, 137.1,
132.8, 130.4, 130.2, 129.8, 129.4, 129.1, 128.5, 128.4,
128.3, 128.18, 128.15, 128.0, 127.9, 127.8, 126.0, 113.5,
101.3 (PhCH), 100.6 (C-10), 100.1 (C-1), 81.7, 80.0,
78.2, 76.6, 75.1, 75.1, 75.0, 74.2, 74.0. 73.7, 73.3, 68.6,
67.8, 67.1, 66.0, 55.1 (OCH3), 40.5 (CH2Cl), 17.9
(CH2CH2Si), ꢀ1.5 (Si(CH3)3); MALDIMS m/z calcd
for [C55H64ClO14Si+Na]+: 1034.1. Found: 1034.0.
3.6. 2-(Trimethylsilyl)ethyl 4,6-O-benzylidene-3-O-benz-
yl-b-D-glucopranosyl-(1!4)-2-O-benzoyl-6-O-benzyl-3-
O-p-methoxybenzoyl-b-D-glucopyranoside (10)
A solution of 9 (980 mg, 0.97 mmol), saturated NaHCO3
in CH3OH/H2O (5:1 v/v) solution (48 mL), and THF
(12 mL) was stirred for 1 h at 50 °C. The solution was
evaporated, then extracted with CHCl3, washed with
H2O, dried (MgSO4), and concentrated. The product
was purified by silica gel column chromatography using
60:1 toluene/EtOAc as the eluent to give 10 (700 mg,
24
(1.17 g, 96%): ½aꢁD +12.5 (c 1.0, CHCl3); 1H NMR
(CDCl3): d 7.49–7.24 (m, 15H, 3Ph), 5.57 (s, 1H, PhCH),
5.02 (dd, 1H, J1,2 = 8.5 Hz, J2,3 = 10.3 Hz, H-2), 4.86,
4.63 (each d, 2H, PhCH2), 4.68 (d, 1H, H-1), 4.39 (dd,
1H, J5,6a = 10.4 Hz, J6a,6b = 4.7 Hz, H-6a), 3.94, 3.83
(each d, 2H, CH2Cl), 3.81–3.71 (m, 3H, H-3, 4, 6b),
3.50 (dt, 1H, H-5); 13C NMR (CDCl3): d 165.7, 137.9,
137.0, 133.0, 131.5, 129.1, 129.0, 128.4, 128.3, 128.0,
127.8, 125.9, 101.2 (PhCH), 86.2 (C-1), 81.2 (C-3), 79.4
(C-4), 74.4 (PhCH2), 72.8 (C-2), 70.5 (C-5), 68.4 (C-6),
24
1
77%): ½aꢁD +29.1 (c 2.0, CHCl3); H NMR (CDCl3): d
7.98–6.63 (m, 24H, 5Ph), 5.57 (s, 1H, PhCH), 5.22 (t,
1H, J1,2
, J2,3 = 9.2 Hz, H-2), 4.94–4.50 (m, 6H,
3PhCH2), 4.65 (d, 1H, J1 ,2 = 7.3 Hz, H-10), 4.51 (d,
0
0
1H, H-1), 4.16–4.11 (m, 2H, H-4, 6a), 4.00 (dd, 1H,
J5 ,6 a = 10.6 Hz, J6 a,6 b = 3.7 Hz, H-60a), 3.94 (dt, 1H,
0
0
0
0
40.6
(CH2Cl);
MALDIMS
m/z
calcd
for
CH2CH2Si), 3.82 (dd, 1H, J5 ,6 b = 1.8 Hz, H-60b), 3.77
(dd, 1H, H-3), 3.66 (s, 3H, OCH3), 3.61–3.49 (m, 6H,
H-6b, 20, 30, 40, 50, CH2CH2Si), 3.19 (dt, 1H, H-5),
0.91–0.80 (m, 2H, CH2CH2Si), ꢀ0.09 (s, 9H, Si(CH3)3);
13C NMR (CDCl3): d 103.4 (C-10), 101.1 (PhCH) 100.1
(C-1), 81.2 (C-3), 81.1 (C-30), 80.3 (C-40), 77.4 (C-5),
75.1 (C-20), 74.7 (C-5 PhCH2), 74.5 (PhCH2), 73.5 (C-
2), 68.6 (C-6), 68.2 (C-60), 67.0 (CH2), 66.2 (C-5), 55.0
(OCH3), 17.7 (CH2CH2Si), ꢀ1.4 (Si(CH3)3); MALDIMS
m/z calcd for [C53H62O13Si+Na]+: 957.4. Found: 957.2.
0
0
[C29H30ClO6S+Na]+: 564.1. Found: 564.0.
3.5. 2-(Trimethylsilyl)ethyl 4,6-O-benzylidene-3-O-benz-
yl-2-O-chloroacetyl-b-D-glucopranosyl-(1!4)-2-O-ben-
zoyl-6-O-benzyl-3-O-p-methoxybenzyl-b-D-glucopyran-
oside (9)
A solution of compound 6a (840 mg, 1.41 mmol) and 8
(1.3 g, 2.47 mmol) containing activated 4 A molecular
˚
sieves (1.5 g) in dry CH2Cl2 (10 mL) was stirred under
an atmosphere of argon for 2 h at rt. After cooling to
ꢀ40 °C, NIS (828 mg, 3.70 mmol) and TfOH (22 lL,
250 lmol) were successively added and stirring was con-
tinued at ꢀ40 °C for 30 min before the solution was neu-
tralized with Et3N. The reaction mixture was filtered,
and the filtrate was washed with sodium thiosulfate
and H2O, dried (MgSO4), and concentrated. The prod-
uct was purified by silica gel column chromatography
using 20:1 toluene/EtOAc as the eluant to give 9
3.7. 2-(Trimethylsilyl)ethyl 4,6-O-benzylidene-2-O-ben-
zoyl-3-O-benzyl-b-D-mannopyranosyl-(1!4)-2-O-ben-
zoyl-6-O-benzyl-3-O-p-methoxybenzyl-b-D-glucopyran-
oside (11)
To a solution of 10 (563 mg, 0.60 mmol) in CH2Cl2
(6 mL) and pyridine (3 mL), cooled to ꢀ20 °C was
added triflic anhydride (610 lL, 3.60 mmol), and the
reaction mixture was stirred for 1 h at ꢀ20 °C and then
for 1 h at 0 °C. Toluene was added and the solution was
evaporated, then extracted with CHCl3, washed with 5%
HCl, aq NaHCO3, and H2O, dried (MgSO4), and con-
centrated. The resulting residue was dissolved in DMF
(6 mL) and n-Bu4NOBz (1.75 g, 4.81 mmol), was added
and the mixture was stirred for 1.5 h at rt, then for 1 h at
100 °C. Toluene was added and the solution was con-
24
(810 mg, 57%). ½aꢁD +7.2 (c 0.5, CHCl3); 1H NMR
(CDCl3): d 7.96–6.63 (m, 24H, 5Ph), 5.50 (s, 1H, PhCH),
0
0
5.16 (t, 1H, J1,2, J2,3 = 7.9 Hz, H-2), 4.98 (t, 1H, J1 ,2
,
J2 ,3 = 7.9 Hz, H-20), 4.88–4.47 (m, 6H, 3PhCH2), 4.61
0
0
(d, 1H, J1 ,2 = 7.9 Hz, H-10), 4.47 (d, 1H, H-1), 4.23
0
0
(dd, 1H, J5 ,6 a = 10.6 Hz, J6 a,6 b = 3.7 Hz, H-60a), 4.07
(t, 1H, H-4), 3.94 (dt, 1H, CH2CH2Si), 3.82, 3.77 (each,
0
0
0
0

N. Hada et al. / Carbohydrate Research 341 (2006) 1341–1352
1347
centrated. The residue was purified by silica gel column
chromatography using 10:1 toluene/EtOAc as the eluent
3.9. 2-(Trimethylsilyl)ethyl 4,6-O-benzylidene-2-O-ben-
zoyl-3-O-benzyl-b-D-mannopyranosyl-(1!4)-[2,3,4-tri-O-
benzyl-a-L-fucopyranosyl-(1!3)]-2-O-benzoyl-6-O-benz-
yl-b-D-glucopyranoside (14)
25
1
to give 11 (501 mg 80%): ½aꢁD ꢀ15.3 (c 1.0 CHCl3); H
NMR (CDCl3): d 8.11–6.44 (m, 29H, 6Ph), 5.70 (d,
1H, J2 ,3 = 3.7 Hz, H-20), 5.60 (s, 1H, PhCH), 5.12
(dd, 1H, J1,2 = 7.9 Hz, J2,3 = 9.2 Hz, H-2), 4.79 (s, 1H,
H-10), 4.75–4.38 (m, 6H, 3PhCH2), 4.42 (d, 1H, H-1),
0
0
To a solution of 12 (221 mg, 0.24 mmol) and 13 (381 mg,
0.72 mmol) in dry CH2Cl2 (3 mL) was added powdered
4.30 (dd, 1H, J5 ,6 a = 10.4 Hz, J6 a,6 b = 4.3 Hz, H-60a),
4.14 (t, 1H, J3,4 4,5 = 9.2 Hz, H-4), 4.02 (t, 1H,
4 A molecular sieves (1 g), and the mixture was stirred
˚
0
0
0
0
for 2 h at rt, then cooled to ꢀ60 °C. NIS (241 mg,
1.08 mmol) and TfOH (6.0 lL, 72 lmol) were added to
the mixture, which was stirred for 10 min at ꢀ60 °C,
then neutralized with Et3N. The solids were filtered
and washed with CHCl3. The combined filtrate and
washings were successively washed with aq Na2S2O3
and H2O, dried (MgSO4), and concentrated. The prod-
uct was purified by silica gel column chromatography
using 3:1 hexane/EtOAc as eluent to give 14 (317 mg,
J3 ,4 4 ,5 = 9.2Hz, H-40), 3.92 (dt, 1H, OCH2CH2Si),
3.82–3.75 (m, 3H, H-6a, H-6b, H-60b), 3.63 (s, 3H,
CH3O), 3.56 (t, 1H, H-3), 3.55 (t, 1H, H-30), 3.49–3.42
(m, 2H, H-5, OCH2CH2Si), 3.20 (dt, 1H, H-50), 0.87–
0.75 (m, 2H, OCH2CH2Si), ꢀ0.12 (s, 9H, Si(CH3)3);
13C NMR (CDCl3): d 165.8, 164.9, 158.7, 137.9, 137.8,
137.4, 133.3, 132.7, 130.5, 130.2, 129.9, 129.8, 129.7,
129.4, 129.0, 128.9, 128.5, 128.4, 128.2, 127.6, 127.4,
0
0
0
0
24
1
126.1, 125.4, 113.2, 101.6 (PhCH), 100.6 (C-1, JC1,H1
=
99%): ½aꢁD ꢀ67.2 (c 1.6, CHCl3); H NMR (CDCl3): d
159.3Hz), 99.4 (C-10, JC1,H1 = 157.3 Hz), 79.9 (C-3), 78.3
(C-4), 78.0 (C-40), 75.8 (C-30), 75.2 (PhCH2), 74.5 (C-5),
74.0 (PhCH2), 73.6 (PhCH2), 73.1 (C-2), 71.5 (PhCH2),
69.7 (C-20), 68.7 (C-6), 68.5 (C-60), 67.03 (C-50), 66.98
(OCH2CH2Si), 55.0, 17.8, ꢀ1.5; MALDIMS m/z calcd
for [C60H66O14Si+Na]+: 1061.4. Found: 1061.2.
8.06–7.09 (m, 40H, 8Ph), 5.70 (d, 1H, J2 ,3 = 3.1 Hz,
H-20), 5.52 (s, 1H, PhCH), 5.40 (t, 1H,
J1,2 2,3 = 7.9 Hz, H-2), 5.37 (d, J1 ,2 = 3.7 Hz, H-100),
0 0
00 00
4.89 (s, 1H, H-10), 4.88–4.17 (m, 14H, H-3, 4, H-60a,
H-500, 5PhCH2), 4.40 (d, 1H, H-1), 3.97–3.78 (m, 5H,
H-6a, H-6a0, H-200, H-300, H-400, OCH2CH2Si), 3.62 (t,
1H, H-40), 3.51–3.43 (m, 4H, H-5, H-60b, H-30,
OCH2CH2Si), 3.12 (dt, 1H, H-50), 0.80–0.73 (m, 2H,
OCH2CH2Si), 0.72 (d, 3H, H-600), ꢀ0.10 (s, 9H,
Si(CH3)3); 13C NMR (CDCl3): d 166.0, 164.8, 139.2,
138.7, 138.1, 137.8, 137.7, 137.2, 133.3, 133.03, 129.94,
129.8, 129.0, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0,
127.9, 127.7, 127.4, 127.2, 126.8, 126.1, 125.3, 101.4,
100.54 (C-10), 100.46 (C-1), 95.5 (C-100), 78.8, 78.6,
78.4, 78.3, 76.3, 75.7, 75.2, 74.9, 74.8, 73.7, 73.5, 73.0,
72.3, 71.6, 70.2, 68.9, 68.6, 67.1, 67.0, 66.1, 17.8, 16.5,
ꢀ1.6; MALDIMS m/z calcd for [C79H86O17Si+Na]+:
1358.4. Found: 1358.6.
3.8. 2-(Trimethylsilyl)ethyl 4,6-O-benzylidene-2-O-ben-
zoyl-3-O-benzyl-b-D-mannopyranosyl-(1!4)-2-O-ben-
zoyl-6-O-benzyl-b-D-glucopyranoside (12)
To a solution of 11 (177 mg, 0.45 mmol) in CH2Cl2
(19 mL) and H2O (1 mL) was added DDQ (153 mg,
0.67 mmol), and the mixture was stirred for 10 h at rt.
The reaction mixture was filtered, and the filtrate was
washed with H2O, dried (MgSO4), and concentrated.
The residue was purified by silica gel column chroma-
tography using 8:1 toluene/EtOAc as the eluent to give
23
12 (323 mg, 78%): ½aꢁD ꢀ55.7 (c 1.6, CHCl3); 1H
NMR (CDCl3): d 8.07–7.15 (m, 25H, 5Ph,), 5.60 (s,
3.10. 2-(Trimethylsilyl)ethyl 3,4,6-tri-O-acetyl-2-O-ben-
zoyl-b-D-mannopyranosyl-(1!4)-[2,3,4-tri-O-acetyl-a-L-
fucopyranosyl-(1!3)]-2-O-benzoyl-6-O-acetyl-b-D-gluco-
pyranoside (15)
1H, PhCH), 5.58 (d, 1H, J2 ,3 = 3.7 Hz, H-20), 5.14
(dd, 1H, J1,2 = 8.6 Hz, J2,3 = 9.8 Hz, H-2), 4.79–4.47
(m, 4H, 2PhCH2), 4.54 (s, 1H, H-10), 4.49 (d, 1H, H-
0
0
0
0
0
0
1), 4.35 (dd, 1H, J5 ,6 a = 10.3 Hz, J6 a,6 b = 4.6 Hz, H-
60a), 4.03 (t, 1H, J3 ,4 4 ,5 = 9.8 Hz, H-40), 3.96 (m, 1H,
OCH2CH2Si), 3.89–3.72 (m, 6H, H-3, 4, H-6a, H-6b,
H-60b, OH), 3.60 (dd, 1H, H-30), 3.49 (dt, 1H,
OCH2CH2Si), 3.43 (dt, 1H, H-5), 3.37 (dt, 1H, H-50),
0.91–0.78 (m, 2H, OCH2CH2Si), ꢀ0.10 (s, 9H,
Si(CH3)3); 13C NMR (CDCl3): d 165.8, 165.3, 138.1,
137.7, 137.1, 133.2, 132.9, 130.1, 129.91, 129.86, 129.0,
128.5, 128.44, 128.36, 128.2, 128.1, 128.03, 127.95,
127.7, 127.5, 126.1, 101.6 (PhCH), 100.5 (C-1), 100.3
(C-10), 80.8 (C-4), 78.0 (C-40), 75.5 (C-30), 73.7 (C-5),
73.50 (2PhCH2), 73.45 (C-2), 71.8 (PhCH2), 69.2 (C-
20), 68.2 (C-3), 68.1 (C-50), 67.1 (C-6, C-60), 67.0
(OCH2CH2Si), 17.9, ꢀ1.5; MALDIMS m/z calcd for
[C52H58O13Si+Na]+: 941.4. Found: 941.0.
A solution of 14 (386 mg, 0.29 mmol) in THF (3.0 mL),
CH3OH (3.0 mL), and AcOH (1.0 mL) was hydrogenol-
ysed in the presence of 10% Pd/C (400 mg) for 3 h at rt,
then filtered and concentrated. The residue was acety-
lated with acetic anhydride (4 mL) in pyridine (6 mL).
The reaction mixture was poured into ice H2O and ex-
tracted with CHCl3. The extract was washed sequen-
tially with 5% HCl, aq NaHCO3, and H2O, dried
(MgSO4), and concentrated. The residue was purified
by silica gel column chromatography using 6:1 tolu-
0
0
0
0
24
ene/acetone as eluent to give 15 (315 mg, quant): ½aꢁD
ꢀ91.9 (c 1.5, CHCl3); 1H NMR (CDCl3): d 5.28 (d,
13
J1 ,2 = 3.1 Hz, H-100), 4.90 (s, 1H, H-10), 4.45 (d, 1H,
00 00
J1,2 = 7.9 Hz, H-1); C NMR (CDCl3): d 100.7 (C-10),

1348
N. Hada et al. / Carbohydrate Research 341 (2006) 1341–1352
100.1 (C-1), 95.2 (C-100); MALDIMS m/z calcd for
was filtered and concentrated. The product was purified
by Sephadex LH-20 column chromatography (1:1,
[C51H66O24Si+Na]+: 1114.1. Found: 1114.4.
24
CHCl3/CH3OH) to give 1 (18 mg, 94%): ½aꢁD ꢀ8.5 (c
3.11. 3,4,6-Tri-O-acetyl-2-O-benzoyl-b-D-mannopyran-
osyl-(1!4)-[2,3,4-tri-O-acetyl-a-L-fucopyranosyl-(1!3)]-
2-O-benzoyl-6-O-acetyl-a-D-glucopyranosyl trichloroacet-
imidate (16)
0.1, 1:1 CHCl3/CH3OH); 1H NMR (DMSO-d6/D2O,
19:1): d 5.24 (d, 1H, J1 ,2 = 4.3 Hz, H-100), 4.65 (s,
0 0 0 0
1H, H-10), 4.18 (d, 1H, J1,2 = 7.3 Hz, H-1); MALDIMS
m/z calcd for [C52H97NO17+Na]+: 1030.7. Found:
1030.2. FABMS m/z calcd for [C52H97NO17+Na]+:
1030.6654. Found: 1030.6632.
To a solution of 15 (320 mg, 0.29 mmol) in CH2Cl2
(2.0 mL), cooled to 0 °C was added CF3CO2H (4 mL),
and the mixture was stirred for 2.5 h at rt and concen-
trated. EtOAc and toluene (1:2) were added and evapo-
rated to give the reducing sugar. To a solution of the
residue in CH2Cl2 (1.0 mL) cooled at 0 °C were added
Cl3CCN (290 lL, 2.9 mmol) and DBU (43 lL,
0.29 mmol). The reaction mixture was stirred for 2 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/EtOAc) gave 16 (285 mg,
3.14. N-(2,2,2-Trichloroethoxycarbonyl)hexanolamine
(19)
To
a solution of 2,2,2-trichloroethylchloroformate
(4.7 mL, 0.03 mol) in 5 mL of dioxane was added at
0 °C a mixture of hexanolamine (5 g, 0.04 mol) and
MgO (3 g) in dioxane/H2O (1:1, 50 mL). The mixture
was stirred for 16 h at rt and then EtOAc was added,
the solids were filtered and washed with 5% HCl,
aq NaHCO3, and H2O, dried (MgSO4), and concen-
trated. The product was purified by silica gel column
chromatography (200:1, CHCl3/CH3OH) to give 19
25
1
86%): ½aꢁD ꢀ39.6 (c 1.6, CHCl3); H NMR (CDCl3): d
00 00
6.50 (d, 1H, J1,2 = 3.7 Hz, H-1), 5.43 (d, J1 ,2 = 3.1 Hz,
H-100), 4.92 (s, 1H, H-10); 13C NMR (CDCl3): d 100.6 (C-
1
10), 95.9 (C-100), 92.9 (C-1).
(8 g, 64%): H NMR (500 MHz, CDCl3): d 5.03 (1H,
s, NH), 4.72 (2H, s, CH2CCl3), 3.65 (2H, t, CH2OH),
3.24 (2H, dd, NHCH2), 1.61–1.36 (8H, m, 4CH2);
MALDIMS: m/z calcd for [C9H16Cl3NO3+Na]+:
314.0. Found: 314.3.
3.12. 3,4,6-Tri-O-acetyl-2-O-benzoyl-b-D-mannopyran-
osyl-(1!4)-[2,3,4-tri-O-acetyl-a-L-fucopyranosyl-(1!3)]-
2-O-benzoyl-6-O-acetyl-b-D-glucopyranosyl-(1!1)-
(2S,3R,4E)-3-O-benzoyl-2-hexadecanamido-4-octade-
cene-1,3-diol (18)
3.15. 6-N-(2,2,2-Trichloroethoxycarbonyl)aminohexyl
3,4,6-tri-O-acetyl-2-O-benzoyl-b-D-mannopyranosyl-
(1!4)-[2,3,4-tri-O-acetyl-a-L-fucopyranosyl-(1!3)-]2-
O-benzoyl-6-O-acetyl-b-D-glucopyranoside (20)
To a solution of 16 (63 mg, 55 lmol) and (2S,3R,4E)-3-
O-benzoyl-2-hexadecanamido-4-octadecene-1,3-diol 17
(53 mg, 83 lmol) in dry CH2Cl2 (1.0 mL) was added
˚
4 A molecular sieves (350 mg), and the mixture was stir-
To a solution of 16 (285 mg, 0.25 mmol) and 19 (95 mg,
0.33 mmol) in dry CH2Cl2 (2.0 mL) was added AW300
molecular sieves (500 mg), and the mixture was stirred
for 2 h at rt, then cooled to 0 °C. TMSOTf (5.8 lL,
25.0 lmol) was added and the mixture was stirred for
4 h at 0 °C, then neutralized with Et3N. The solids were
filtrated and washed with CHCl3. The combined filtrate
and washings were successively washed with H2O, dried
(MgSO4), and concentrated. The product was purified
by silica gel column chromatography using 5:1 tolu-
red for 2 h at rt, then cooled to 0 °C. TMSOTf (8 lL,
44 lmol) was added, and the mixture was stirred for
24 h at 0 °C, and then neutralized with Et3N. The solids
were filtrated and washed with CHCl3. The combined
filtrate and washings were successively washed with
H2O, dried (MgSO4), and concentrated. The product
was purified by silica gel column chromatography using
3:1 toluene/EtOAc as eluent to give 18 (31 mg, 35%):
25
1
½aꢁD ꢀ64.4 (c 1.0, CHCl3); H NMR (CDCl3): d 5.28
25
(d, J1 ,2 = 3.1 Hz, H-100), 4.83 (s, 1H, H-10), 4.40 (d,
ene/acetone as eluent to give 20 (316 mg, quant): ½aꢁD
00 00
1
1H, J1,2 = 7.9 Hz, H-1); 13C NMR (CDCl3): d 100.7
(C-10), 100.4 (C-1), 95.3 (C-100); MALDIMS m/z calcd
for [C87H123NO27+Na]+: 1636.8. Found: 1636.5.
ꢀ86.7 (c 1.4, CHCl3); H NMR (CDCl3): d 8.09–7.14
(m, 10H, 2Ph), 5.78 (d, 1H, J2 ,3 = 3.1 Hz, H-20), 5.51
0
0
0
0
0
0
0
(t, 1H, J3 ,4 , J4 ,5 = 10.4 Hz, H-4 ), 5.37 (dd, 1H,
J2 ,3 = 3.1 Hz, J3 ,4 = 11.0 Hz, H-300), 5.30 (d, 1H,
00 00
00 00
00 00
3.13. b-D-Mannopyranosyl-(1!4)-[a-L-fucopyranosyl-
(1!3)]-b-D-glucopyranosyl-(1!1)-(2S,3R,4E)-2-hexa-
decanamido-4-octadecene-1,3-diol (1)
J1 ,2 = 4.3 Hz, H-100), 5.26 (t, 1H, J1,2 2,3 = 8.6 Hz, H-
2), 5.23 (d, 1H, H-400), 5.15 (dd, 1H, H-30), 4.94 (dd,
1H, H-400), 4.90 (s, 1H, H-10), 4.86 (dd, 1H, H-500),
4.78 (dd, 1H, J5 ,6 a = 3.1 Hz, J6 a,6 b = 13.5 Hz, H-60a),
4.62 (s, 2H, CH2CCl3), 4.59 (dd, 1H, J5,6a = 2.5 Hz,
J6a,6b = 12.2 Hz, H-6a), 4.41 (dd, 1H, H-60b), 4.40 (d,
1H, H-1), 4.28 (dd, 1H, H-6b), 4.11 (t, 1H,
J3,4 4,5 = 9.1 Hz, H-4), 4.01 (t, 1H, H-3), 3.80 (dt, 1H,
0
0
0
0
To a solution of 18 (31 mg, 19.2 lmol) in CH3OH
(1.0 mL) and 1,4-dioxane (1.0 mL) was added NaOCH3
(20 mg) at rt and the mixture was stirred for 24 h, then
neutralized with Amberlite IR 120[H+]. The mixture

N. Hada et al. / Carbohydrate Research 341 (2006) 1341–1352
1349
OCH2CH2), 3.73 (dt, 1H, H-50), 3.60 (dt, 1H, H-5), 3.33
(dt, 1H, OCH2CH2), 3.00 (dd, 2H, CH2NH), 2.22, 2.18,
2.04, 2.03, 2.02, 1.92, 1.91 (each s, 21H, 7Ac), 1.45–1.06
(m, 8H, 4CH2), 0.61 (d, 3H, H-600); 13C NMR (CDCl3): d
170.4, 170.2, 170.1, 169.8, 169.7, 169.4, 165.8, 154.4,
137.8, 133.5, 129.9, 129.6, 129.4, 129.3 129.0, 128.5,
128.4, 128.2, 125.2, 100.8 (C-1), 100.7 (C-10), 95.7
(CCl3), 95.2 (C-100), 79.2 (C-3), 74.6 (C-2), 74.3
(CH2CCl3), 73.9 (C-4), 73.2 (C-50), 73.1 (C-5), 71.5 (C-
400), 71.2 (C-30), 69.9 (C-20), 69.8 (CH2), 67.8 (C-300),
67.4 (C-200), 64.7 (C-40), 64.5 (C-500), 62.1 (C-60), 60.8
(C-6), 40.9, 29.2, 29.0, 26.0, 25.3, 21.4, 21.0, 20.6, 20.5,
20.4, 15.7; MALDIMS m/z calcd for [C55H68ClNO26+
Na]+: 1286.3. Found: 1286.7.
(81 mg, 0.22 mmol) in DMF (3 mL) were added Et3N
(60 lL, 0.43 mmol) and DEPC (36 lL, 0.24 mmol).
The mixture was stirred for 16 h at rt. After completion
of the reaction, the mixture was extracted with CHCl3,
washed with H2O, dried (Na2SO4), and concentrated.
The product was purified by silica gel column chroma-
tography (3:1, toluene/acetone) to give 23 (186 mg,
23
1
66%): ½aꢁD ꢀ72.9 (c 1.0, CHCl3); H NMR (500 MHz,
CDCl3): d 8.09–7.14 (m, 10H, 2Ph), 6.11 (t 1H, NH),
5.77 (d, 1H, J2 ,3 = 3.1 Hz, H-20), 5.51 (t, 1H, J3 ,4
,
0
0
0
0
J4 ,5 = 9.8 Hz, H-40), 5.37 (dd, 1H, J2 ,3 = 3.0 Hz,
0
0
00 00
J3 ,4 = 11.0 Hz, H-300), 5.29 (d, 1H, J1 ,2 = 4.3 Hz,
H-100), 5.26 (t, 1H, J1,2 2,3 = 7.9 Hz, H-2), 5.23 (d, 1H,
H-400), 5.15 (dd, 1H, H-30), 4.94 (dd, 1H, H-400), 4.90
(s, 1H, H-10), 4.87 (dd, 1H, H-500), 4.77 (dd, 1H,
00 00
00 00
J5 ,6 a = 3.0 Hz, J6 a,6 b = 12.8 Hz, H-60a), 4.75 (s,
2H, CH2CCl3), 4.60 (dd, 1H, J5,6a = 2.4 Hz,
J6a,6b = 12.2 Hz, H-6a), 4.40 (dd, 1H, H-60b), 4.28 (d,
1H, H-1), 4.28 (dd, 1H, H-6b), 4.11 (t, 1H,
J3,4 4,5 = 9.1 Hz, H-4), 4.00 (t, 1H, H-3), 3.83 (s, 2H,
NCH2CO of b-alanine), 3.79 (dt, 1H, OCH2CH2 of
sugar unit), 3.72 (dt, 1H, H-50), 3.61 (m, 3H, H-5,
NCH2 of b-alanine), 3.33 (dt, 1H, OCH2CH2 of sugar
unit), 3.00 (dd, 2H, CH2NH of sugar unit), 2.77 (t,
2H, COCH2 of b-alanine), 2.23, 2.18, 2.04, 2.03, 2.02,
1.92, 1.91 (each s, 21H, 7Ac), 1.47–1.06 (17H, m, t-Bu,
4(CH2)), 0.62 (d, 3H, H-600); 13C NMR (CDCl3): d
170.4, 170.2, 170.1, 169.8, 169.7, 169.4, 169.0, 165.8,
164.8, 137.8, 133.5, 133.3, 129.9, 129.6, 129.33, 129.25
128.9, 128.5, 128.4, 128.1, 125.2, 100.8 (C-1), 100.7 (C-
10), 95.2 (C-100), 94.7, 81.1, 79.2, 74.5, 74.0, 73.9, 73.13,
73.08, 71.5, 71.1, 69.9, 69.8, 67.5, 67.4, 64.7, 64.5, 62.1,
60.8, 52.4, 44.9, 39.1, 33.1, 29.1, 29.0, 28.2, 26.3, 25.3,
21.4, 21.0, 20.6, 20.4, 15.7; MALDIMS m/z calcd for
[C64H83Cl3N2O29+Na]+: 1471.4. Found: 1471.5.
0
0
0
0
3.16. 6-Aminohexyl 3,4,6-tri-O-acetyl-2-O-benzoyl-b-D-
mannopyranosyl-(1!4)-[2,3,4-tri-O-acetyl-a-L-fucopyr-
anosyl-(1!3)]-2-O-benzoyl-6-O-acetyl-b-D-glucopyran-
oside (21)
To a solution of 20 (317 mg, 0.25 mmol) in HOAc (5 mL)
was added zinc powder (480 mg). The reaction mixture
was stirred for 16 h at rt. After completion of the reaction,
the mixture was filtered and the solids washed with
CHCl3. The filtrate was concentrated and purified by sil-
ica gel column chromatography (10:1, CHCl3/CH3OH)
23
to give 21 (274 mg, quant): ½aꢁD ꢀ92.5 (c 1.0, CHCl3);
1H NMR (CDCl3): d 8.08–7.16 (m, 10H, 2Ph), 5.77
(d, 1H, J2 ,3 = 3.1 Hz, H-20), 5.51 (t, 1H, J3 ,4
,
0
0
0
0
J4 ,5 = 10.4 Hz, H-40), 5.37 (dd, 1H, J2 ,3 = 3.1 Hz,
0
0
00 00
J3 ,4 = 11.0 Hz, H-300), 5.29 (d, 1H, J1 ,2 = 4.3 Hz,
H-100), 5.26 (t, 1H, J1,2 2,3 = 8.6 Hz, H-2), 5.23 (d, 1H,
H-400), 5.15 (dd, 1H, H-30), 4.93 (dd, 1H, H-400), 4.91
(s, 1H, H-10), 4.85 (dd, 1H, H-500), 4.78 (dd, 1H,
00 00
00 00
J5 ,6 a = 3.1 Hz, J6 a,6 b = 13.5 Hz, H-60a), 4.59 (dd, 1H,
J5,6a = 2.5 Hz, J6a,6b = 12.2 Hz, H-6a), 4.41 (dd, 1H, H-
60b), 4.40 (d, 1H, H-1), 4.28 (dd, 1H, H-6b), 4.11 (t, 1H,
J3,4 4,5 = 9.1 Hz, H-4), 4.01 (t, 1H, H-3), 3.80 (dt, 1H,
OCH2CH2), 3.73 (dt, 1H, H-50), 3.61 (dt, 1H, H-5), 3.33
(dt, 1H, OCH2CH2), 2.62 (dd, 2H, CH2NH), 2.23, 2.18,
2.043, 2.035, 2.02, 1.92, 1.91 (each s, 21H, 7Ac), 1.45–
1.06 (m, 8H, 4CH2), 0.61 (d, 3H, H-600); 13C NMR
(CDCl3): d 170.5, 170.4, 170.2, 169.9, 169.8, 169.5,
165.9, 164.9, 133.6, 133.5, 129.9, 129.7, 129.41, 129.36
129.0, 128.6, 128.2, 100.9 (C-1), 100.8 (C-10), 95.3 (C-
100), 79.3, 74.7, 74.0, 73.2, 71.6, 71.3, 70.0, 69.8, 67.53,
67.47, 64.8, 64.6, 62.2, 60.9, 39.8, 29.0, 28.0, 25.9, 25.1,
21.1, 20.71, 20.65, 20.61, 20.5, 15.8; MALDIMS m/z
calcd for [C52H68NO24+H]+: 1090.4. Found: 1090.8.
0
0
0
0
3.17.2. Compound 24. To a solution of 23 (186 mg,
0.13 mmol) in HOAc (2 mL) was added zinc powder
(240 mg). The mixture was stirred for 1 h at rt and after
completion of the reaction (TLC monitoring) the mix-
ture was filtered through Celite. The filtrate was concen-
trated and purified by silica gel column chromatography
23
(10:1, CHCl3/CH3OH) to give 24 (146 mg, 87%): ½aꢁD
1
ꢀ76.0 (c 1.9, CHCl3); H NMR (500 MHz, CDCl3): d
8.08–7.17 (m, 10H, 2Ph), 6.45 (t 1H, NH), 5.77 (d,
1H, J2 ,3 = 3.1 Hz, H-20), 5.51 (t, 1H, J3 ,4 , J4 ,5
=
0
0
0
0
0
0
9.8 Hz, H-40), 5.37 (dd, 1H, J2 ,3 = 3.0 Hz,
00 00
J3 ,4 = 11.0 Hz, H-300), 5.30 (d, 1H, J1 ,2 = 3.7 Hz,
H-100), 5.26 (t, 1H, J1,2 2,3 = 7.9 Hz, H-2), 5.23 (d, 1H,
H-400), 5.16 (dd, 1H, H-30), 4.94 (dd, 1H, H-400), 4.91
(s, 1H, H-10), 4.86 (dd, 1H, H-500), 4.77 (dd, 1H,
00 00
00 00
3.17. Syntheses of multivalent glycoconjugate building
blocks
J5 ,6 a = 3.0 Hz, J6 a,6 b = 12.8 Hz, H-60a), 4.60 (dd,
1H, J5,6a = 2.4 Hz, J6a,6b = 12.2 Hz, H-6a), 4.40 (dd,
1H, H-60b), 4.28 (d, 1H, H-1), 4.28 (dd, 1H, H-6b),
4.11 (t, 1H, J3,4 4,5 = 9.1 Hz, H-4) 4.01 (t, 1H, H-3),
3.88 (s, 2H, NCH2CO of b-alanine), 3.79 (dt, 1H,
0
0
0
0
3.17.1. Compound 23. To a solution of 21 (213 mg,
0.20 mmol) and 3-(tert-butoxycarbonyl-carboxymeth-
ylamino)-propionic acid 2,2,2-trichloroethylester 22

1350
N. Hada et al. / Carbohydrate Research 341 (2006) 1341–1352
OCH2CH2 of sugar unit), 3.72 (dt, 1H, H-50), 3.61 (m,
3H, H-5, NCH2 of b-alanine), 3.33 (dt, 1H, OCH2CH2
of sugar unit), 3.06 (dd, 2H, CH2NH of sugar unit),
2.57 (t, 2H, COCH2 of b-alanine), 2.23, 2.18, 2.04,
2.03, 2.02, 1.92, 1.91 (each s, 21H, 7Ac), 1.47–1.06
(17H, m, t-Bu, 4(CH2)), 0.62 (d, 3H, H-600); 13C NMR
(CDCl3): d 170.44, 170.36, 170.1, 169.9, 169.7, 169.4,
165.8, 165.0, 133.5, 133.4, 129.9, 129.6, 129.33, 129.2,
128.5, 128.4, 100.8 (C-1), 100.7 (C-10), 95.2 (C-100),
79.1, 77.2, 74.6, 73.9, 73.13, 73.08, 71.4, 71.2, 69.9,
69.8, 67.44, 67.38, 64.7, 64.5, 62.1, 60.8, 39.3, 29.0,
28.1, 26.2, 25.3, 21.0, 20.6, 20.54, 20.51, 20.41, 15.7;
MALDIMS m/z calcd for [C62H82N2O29+Na]+:
1341.5. Found: 1341.9.
completion of the reaction, the mixture was concen-
trated and purified by silica gel column chromatography
(10:1, CHCl3/CH3OH) to give 26 (74 mg, 86%). MALD-
IMS m/z calcd for [C109H139N3O50+Na]+: 2312.8.
Found: 2313.4.
3.19. Syntheses of trimeric glycoconjugate
3.19.1. Compound 27. To a solution of 26 (70 mg,
30.6 lmol) and 24 (44 mg, 33.7 lmol) in DMF (1 mL)
were added Et3N (9.1 lL, 66.0 lmol) and DEPC
(5.5 lL, 36.0 lmol). The mixture was stirred for 16 h
at rt. After completion of the reaction, the mixture
was extracted with CHCl3, washed with H2O, dried
(Na2SO4), and concentrated. The product was purified
by silica gel column chromatography (30:1, CHCl3/
3.18. Syntheses of dimeric glycoconjugate
23
CH3OH) to give 27 (85 mg, 79%): ½aꢁD ꢀ98.0 (c 2.6,
1
3.18.1. Compound 25. To a solution of 24 (71 mg,
54 lmol) and 21 (65 mg, 59 lmol) in DMF (1.5 mL)
were added Et3N (15 lL, 108 lmol) and DEPC (9 lL,
59 lmol). The mixture was stirred for 16 h at rt. After
completion of the reaction, the mixture was extracted
with EtOAc, washed with H2O, dried (Na2SO4), and
concentrated. The product was purified by silica gel col-
umn chromatography (20:1, CHCl3/CH3OH) to give 25
CHCl3); H NMR (500 MHz, CDCl3): d 8.09–7.33 (m,
30H, 6Ph), 5.78 (d, 3H, J2 ,3 = 3.1 Hz, H-20), 5.51 (t,
0
0
3H, J3 ,4
00 00
,
J4 ,5 = 10.4 Hz, H-40), 5.37 (dd, 3H,
0
0
0
0
J2 ,3 = 3.7 Hz, J3 ,4 = 10.9 Hz, H-300), 5.29 (d, 3H,
00 00
J1 ,2 = 3.7 Hz, H-100), 5.25 (t, 3H, J1,2 2,3 = 7.9 Hz, H-
00 00
2), 5.23 (d, 3H, H-400), 5.15 (dd, 3H, H-30), 4.95 (dd,
3H, H-400), 4.93 (s, 3H, H-10), 4.87 (dd, 3H, H-500),
4.77 (dd, 3H, J5 ,6 a = 3.0 Hz, J6 a,6 b = 12.8 Hz, H-60a),
4.60 (br dd, 3H, J5,6a = 2.4 Hz, J6a,6b = 12.8 Hz, H-
6a), 4.43 (d, 3H, H-1), 4.42 (dd, 3H, H-60b), 4.28 (dd,
3H, H-6b), 4.12 (t, 3H, J3,4 4,5 = 9.1 Hz, H-4) 4.00 (t,
3H, H-3), 3.83–3.75 (m, 8H, 3H-50, 2NCH2CO of b-ala-
nine, 3OCH2CH2 of sugar unit), 3.67–3.57 (m, 5H, 3H-
5, 2NCH2 of b-alanine), 3.33 (dt, 3H, OCH2CH2 of su-
gar unit), 3.00 (dd, 6H, CH2NH of sugar unit), 2.37 (t,
2H, COCH2 of b-alanine), 2.23, 2.18, 2.05, 2.04, 2.02,
1.92, 1.91 (each s, 63H, 21Ac), 1.34–1.05 (33H, m, t-
Bu, 12(CH2)), 0.62 (d, 9H, H-600); 13C NMR (CDCl3):
d 170.3, 170.2, 169.9, 169.7, 169.6, 169.3, 165.7, 164.7,
162.4, 133.4, 133.2, 129.8, 129.5, 129.20, 129.17, 128.4,
128.3, 100.8 (C-1), 100.5 (C-10), 95.1 (C-100), 79.1, 77.2,
74.5, 73.8, 73.0, 71.3, 71.1, 69.8, 67.34, 67.26, 64.6,
64.4, 62.0, 60.7, 39.2, 36.3, 31.2, 28.9, 28.1, 26.3, 25.2,
20.8, 20.5, 20.44, 20.39, 20.3, 15.5; MALDIMS m/z
calcd for [C171H219N5O78+Na]+: 3613.3. Found: 3613.0.
0
0
0
0
23
(92 mg, 71%): ½aꢁD ꢀ98.0 (c 1.2, CHCl3); 1H NMR
(500 MHz, CDCl3): d 8.09–7.029 (m, 20H, 4Ph), 5.78
0
0
0
0
(d, 2H, J2 ,3 = 3.1 Hz, H-2 ), 5.51 (t, 2H, J3 ,4
,
J4 ,5 = 9.8 Hz, H-40), 5.37 (dd, 2H, J2 ,3 = 3.2 Hz,
0
0
00 00
J3 ,4 = 11.0 Hz, H-300), 5.29 (d, 2H, J1 ,2 = 3.7 Hz, H-
100), 5.26 (t, 2H, J1,2 2,3 = 7.9 Hz, H-2), 5.23 (d, 2H, H-
400), 5.15 (dd, 2H, H-30), 4.94 (dd, 2H, H-400), 4.91 (s,
2H, H-10), 4.87 (dd, 2H, H-500), 4.77 (dd, 2H,
00 00
00 00
J5 ,6 a = 3.0 Hz, J6 a,6 b = 12.8 Hz, H-60a), 4.60 (br dd,
2H, J5,6a = 2.4 Hz, J6a,6b = 12.8 Hz, H-6a), 4.40 (d,
2H, H-1), 4.39 (dd, 2H, H-60b), 4.28 (dd, 2H, H-6b),
4.11 (t, 2H, J3,4 4,5 = 9.1 Hz, H-4) 4.00 (t, 2H, H-3),
3.81–3.73 (m, 6H, 2H-50, 2NCH2CO of b-alanine,
2OCH2CH2 of sugar unit), 3.61 (br dd, 2H, H-5), 3.56
(br t, 2H NCH2 of b-alanine), 3.33 (dt, 2H, OCH2CH2
of sugar unit), 3.00 (dd, 4H, CH2NH of sugar unit),
2.37 (t, 2H, COCH2 of b-alanine), 2.22, 2.18, 2.05,
2.04, 2.02, 1.92, 1.91 (each s, 42H, 14Ac), 1.47–1.06
(25H, m, t-Bu, 8(CH2)), 0.62 (d, 6H, H-600); 13C NMR
(CDCl3): d 170.4, 170.2, 170.1, 169.8, 169.7, 169.4,
169.0, 165.8, 164.9, 133.5, 133.3, 129.9, 129.6, 129.3,
129.2, 128.5, 128.4, 100.8 (C-1), 100.7 (C-10), 95.2 (C-
100), 80.6, 79.2, 77.3, 74.5, 73.9, 73.12, 73.07, 71.4, 71.1,
69.9, 67.4, 67.3, 64.7, 64.5, 62.1, 60.8, 39.4, 39.1, 29.2,
26.3, 25.3, 21.0, 20.6, 20.54, 20.51, 15.7; MALDIMS
m/z calcd for [C114H147N3O52+Na]+: 2412.9. Found:
2413.3.
0
0
0
0
3.19.2. Compound 28. Compound 28 was derived from
27 according to the procedure described for the synthe-
sis of 26; yield: 60 mg (73%): MALDIMS m/z calcd for
[C166H211N5O76+Na]+: 3513.3. Found: 3512.8.
3.19.3. Compound 29. To a solution of 28 (24 mg,
6.88 lmol) and dansyl glycine (6.3 mg, 20.6 lmol) in
DMF (1 mL) were added Et3N (5.2 lL, 38.0 lmol)
and DEPC (3.1 lL, 20.0 lmol) and the mixture was stir-
red for 16 h at rt. After completion of the reaction, the
mixture was extracted with CHCl3, washed with H2O,
dried (Na2SO4), and concentrated. The product was
purified by silica gel column chromatography (30:1,
3.18.2. Compound 26. To a solution of 25 (90 mg,
37.6 lmol) in CH2Cl2 (1 mL) was added CF3CO2H
(1 mL) and the mixture was stirred for 3 h at rt. After

N. Hada et al. / Carbohydrate Research 341 (2006) 1341–1352
1351
CHCl3/CH3OH) to give dansyl derivative 29 (22 mg,
OCH2CH2 of sugar unit), 3.00 (dd, 8H, CH2NH of
sugar unit), 2.37 (t, 2H, COCH2 of b-alanine), 2.22,
2.18, 2.04, 2.03, 2.02, 1.92, 1.91 (each s, 84H, 28Ac),
1.35–1.04 (41H, m, t-Bu, 16(CH2)), 0.62 (d, 12H,
H-600); 13C NMR (CDCl3): d 170.5, 170.3, 170.1, 169.8,
169.7, 169.4, 165.8, 164.9, 133.6, 133.4, 129.9, 129.6,
129.4, 129.3, 128.6, 128.5, 100.9 (C-1), 100.7 (C-10),
95.3 (C-100), 79.2, 74.6, 74.0, 73.2, 73.1, 71.5, 71.2,
70.0, 67.5, 67.4, 64.7, 64.5, 62.2, 60.9, 39.4, 39.2, 28.22,
28.17, 26.5, 26.4, 25.4, 21.0, 20.7, 20.61, 20.57, 20.5,
15.7; MALDIMS m/z calcd for [C228H291N7O104+Na]+:
4813.8. Found: 4814.0.
23
1
85%): ½aꢁD ꢀ74.4 (c 0.7, CHCl3); H NMR (500 MHz,
CDCl3): d 8.09–7.27 (m, 36H, C10H6 of dansyl glycine,
6Ph), 5.29 (d, 3H, J1 ,2 = 3.7 Hz, H-100), 4.93 (s, 3H,
00 00
H-10), 4.40 (d, 3H, J1,2 = 7.9 Hz, H-1), 3.97–3.54 (m,
15H, 3H-5, 3H-50, 2NCH2CO of b-alanine, 3OCH2CH2
of sugar unit, 2NCH2 of b-alanine, NHCH2CO of dan-
syl glycine), 3.33 (dt, 3H, OCH2CH2 of sugar unit), 3.00
(dd, 6H, CH2NH of sugar unit), 2.89 (6H, s, N(CH3)2 of
dansyl glycine), 2.77 (t, 2H, COCH2 of b-alanine), 2.23,
2.18, 2.05, 2.04, 2.02, 1.92, 1.91 (each s, 63H, 21Ac),
1.34–1.05 (24H, m, 12(CH2)), 0.61 (d, 9H, H-600); 13C
NMR (CDCl3): d 170.5, 170.3, 170.1, 169.9, 169.7,
169.4, 165.8, 164.9, 133.6, 133.4, 129.9, 129.6, 129.3,
128.9, 128.5, 100.9 (C-1), 100.7 (C-10), 95.3 (C-100),
79.2, 74.6, 74.0, 73.1, 71.5, 71.2, 70.0, 67.5, 67.4, 64.7,
64.5, 62.2, 60.9, 45.4, 39.4, 28.9, 26.4, 25.3, 21.0, 20.7,
20.6, 20.5, 15.7; MALDIMS m/z calcd for
[C180H225N7O79S+Na]+: 3802.4. Found: 3802.7.
3.20.2. Compound 31. Compound 31 was synthesized
from 30 according to the procedure described for the
synthesis of 26; yield: 24 mg (56%); MALDIMS m/z
calcd for [C223H283N7O102+Na]+: 4713.7. Found:
4713.9.
3.20.3. Compound 32. Compound 32 was synthesized
from 31 according to the procedure described for the
3.19.4. Compound 2. Compound 2 was synthesized
from 29 according to the procedure described for the
23
synthesis of 29; yield: 24 mg (94%): ½aꢁD ꢀ85.0 (c 0.8,
23
1
synthesis of 1; yield: 11 mg (91%): ½aꢁD ꢀ61.3 (c 0.4,
CHCl3); H NMR (500 MHz, CDCl3): d 8.09–7.27 (m,
1
CH3OH/H2O 1:1); H NMR (500 MHz, 1:1 CD3OD/
46H, C10H6 of dansyl glycine, 8Ph), 5.28 (d, 4H,
00 00
D2O): d 8.54–7.35 (m, 6H, C10H6 of dansyl glycine),
J1 ,2 = 3.7 Hz, H-100), 4.92 (s, 4H, H-10), 4.42 (d, 4H,
5.46 (d, 3H, J1 ,2 = 3.7 Hz, H-100), 4.56 (br d, 3H, H-
J1,2 = 7.9 Hz, H-1), 3.97–3.54 (m, 20H, 4H-5, 4H-50,
2NCH2CO of b-alanine, 4OCH2CH2 of sugar unit,
4NCH2 of b-alanine, NHCH2CO of dansyl glycine),
3.33 (dt, 4H, OCH2CH2 of sugar unit), 3.00 (dd, 8H,
CH2NH of sugar unit), 2.89 (6H, s, N(CH3)2 of dansyl
glycine), 2.77 (t, 2H, COCH2 of b-alanine), 2.23, 2.18,
2.05, 2.04, 2.02, 1.92, 1.91 (each s, 84H, 28Ac), 1.34–
1.05 (32H, m, 16(CH2)), 0.61 (d, 12H, H-600); 13C
NMR (CDCl3): d 170.5, 170.3, 170.1, 169.9, 169.7,
169.5, 165.8, 164.9, 133.6, 133.4, 129.9, 129.6, 129.3,
128.6, 128.5, 100.9 (C-1), 100.7 (C-10), 95.3 (C-100),
79.2, 74.6, 74.0, 73.1, 71.5, 71.2, 70.0, 67.5, 67.4, 64.7,
64.5, 62.2, 60.9, 39.4, 29.7, 29.2, 29.1, 26.41,
26.35, 25.4, 21.0, 20.7, 20.6, 20.5, 15.7; MALDIMS
m/z calcd for [C237H297N9O105S+Na]+: 5003.4. Found:
5003.9.
00 00
10), 4.36 (br d, 3H, H-1), 2.88, 2.87 (6H, each s,
N(CH3)2 of dansyl glycine), 1.18 (d, 9H, H-600); 13C
NMR (125 MHz, 1:1 CD3OD/D2O): d 174.0, 171.1,
170.8, 170.1, 152.4, 135.5, 131.3, 130.5, 130.4, 129.6,
124.7, 120.2, 120.1, 116.8, 103.5, 100.3, 99.8, 79.5,
78.6, 78.1, 76.4, 75.7, 75.3, 74.1, 73.3, 72.9, 71.7, 71.3,
71.1, 70.6, 69.8, 68.3, 68.1, 62.7, 61.8, 61.5, 52.5, 49.4,
45.9, 45.4, 44.9, 40.4, 30.0, 29.6, 27.1, 26.1, 16.7;
MALDIMS m/z calcd for [C96H159N7O52S+Na]+:
2298.0. Found: 2297.6.
3.20. Syntheses of tetrameric glycoconjugate
3.20.1. Compound 30. Compound 30 was synthesized
from 28 according to the procedure described for the
23
synthesis of 25; yield: 42 mg (83%): ½aꢁD ꢀ99.0 (c 1.0,
1
CHCl3); H NMR (500 MHz, CDCl3): d 8.08–7.27 (m,
3.20.4. Compound 3. Compound 3 was synthesized
from 32 according to the procedure described for the
40H, 8Ph), 5.78 (d, 4H, J2 ,3 = 3.1 Hz, H-20), 5.51 (t,
0
0
23
4H, J3 ,4 , J4 ,5 = 10.4 Hz, H-40), 5.37 (dd, 4H, J2 ,3
=
synthesis of 2; yield: 13.6 mg (91%): ½aꢁD ꢀ55.8 (c 0.5,
0
0
0
0
00 00
3.7 Hz, J3 ,4 = 10.9 Hz, H-300), 5.29 (d, 4H,
CH3OH/H2O 1:1); 1H NMR (500 MHz, CDCl3): d
8.39–7.28 (m, 6H, C10H6 of dansyl glycine), 5.42 (br t,
4H, H-100), 4.53 (br d, 4H, H-10), 4.33 (d, 4H,
J1,2 = 7.9 Hz, H-1), 2.84, 2.83 (6H, each s, N(CH3)2 of
dansyl glycine), 1.18 (d, 9H, H-600); 13C NMR
(125MHz, 1:1 CD3OD/D2O): d 103.4, 100.4, 99.7,
79.3, 79.0, 78.0, 76.4, 75.7, 75.3, 74.4, 73.3, 72.9,
71.7, 71.3, 70.6, 69.7, 68.3, 68.1, 62.7, 61.7, 61.5, 46.0,
40.4, 30.0, 29.6, 27.2, 27.1, 26.1, 16.7; MALDIMS m/z
calcd for [C125H209N9O69S+Na]+: 2995.3. Found:
2995.0.
00 00
J1 ,2 = 3.7 Hz, H-100), 5.28 (t, 4H, J1,2 2,3 = 7.9 Hz, H-
00 00
2), 5.23 (d, 4H, H-400), 5.15 (dd, 4H, H-30), 4.95 (dd,
4H, H-400), 4.91 (s, 4H, H-10), 4.85 (dd, 4H, H-500),
4.77 (dd, 4H, J5 ,6 a = 3.0 Hz, J6 a,6 b = 12.8 Hz, H-60a),
4.60 (br dd, 4H, J5,6a = 2.4 Hz, J6a,6b = 12.8 Hz, H-
6a), 4.41 (d, 4H, H-1), 4.40 (br d, 4H, H-60b), 4.28 (br
d, 4H, H-6b), 4.11 (t, 4H, J3,4 4,5 = 9.1 Hz, H-4) 4.00
(t, 4H, H-3), 3.83–3.75 (m, 10H, 4H-50, 2NCH2CO of
b-alanine, 4OCH2CH2 of sugar unit), 3.67–3.57 (m,
6H, 4H-5, 2NCH2 of b-alanine), 3.33 (dt, 4H,
0
0
0
0

1352
N. Hada et al. / Carbohydrate Research 341 (2006) 1341–1352
9. Sato, K.-I.; Yoshitomo, A.; Nanaumi, H.; Takai, Y.;
Supplementary data
Ishido, Y. J. Carbohydr. Chem. 1998, 17, 703–727.
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D. C. Biochemistry 1989, 28, 8388–8396.
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Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2006.04.019.
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