Systematic synthesis and MAG-binding activity of novel sulfated GM1b analogues as mimics of Chol-1 (α-series) gangliosides: Highly active ligands for neural siglecs

Article abstract of DOI:10.1016/S0008-6215(03)00245-3

Systematic synthesis and myelin-associated glycoprotein (MAG)-binding activity of novel sulfated GM1b analogues structurally related to Chol-1 (α-series) gangliosides, high-affinity ligands for neural siglecs, are described. The suitably protected ganglio

Full text of DOI:10.1016/S0008-6215(03)00245-3

Carbohydrate Research 338 (2003) 1621ꢁ1639
/
www.elsevier.com/locate/carres
Systematic synthesis and MAG-binding activity of novel sulfated
GM1b analogues as mimics of Chol-1 (a-series) gangliosides: highly
active ligands for neural siglecsꢀ
Hiromi Ito,a Hideharu Ishida,a Brian E. Collins,b Susan E. Fromholt,b
Ronald L. Schnaar,b Makoto Kisoa,*
a Department of Applied Bio-organic Chemistry, Gifu University, Gifu 501-1193, Japan
b Departments of Pharmacology and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Received 6 March 2003; accepted 2 May 2003
Abstract
Systematic synthesis and myelin-associated glycoprotein (MAG)-binding activity of novel sulfated GM1b analogues structurally
related to Chol-1 (a-series) gangliosides, high-affinity ligands for neural siglecs, are described. The suitably protected gangliotriose
derivatives, 2-(trimethylsilyl)ethyl 2-acetamido-2-deoxy-6-O-levulinoyl-b-
topyranosyl-(104)-2,3,6-tri-O-benzyl-b- -glucopyranoside and 2-(trimethylsilyl)ethyl 2-acetamido-2-deoxy-6-O-levulinoyl-b-
galactopyranosyl-(104)-2,6-di-O-benzyl-3-O-levulinoyl-b- -galactopyranosyl-(104)-2,3,6-tri-O-benzyl-b- -glucopyranoside
were each glycosylated with a-NeuAc-(203)-galactose donor to give the corresponding pentasaccharides in 94% (b1,3 glycoside
only) and 90% (b1,3:b1,4ꢀ2:1), respectively. After proper manipulation of the protecting groups, the pentasaccharides were
D
-galactopyranosyl-(10
/
4)-2,3,6-tri-O-benzyl-b-
D-galac-
/
D
D-
/
D
/
D
/
/
converted into three novel sulfated GM1b gangliosides by the successive introduction of the ceramide and sulfo groups, followed by
complete deprotection. Among the synthetic gangliosides, GSC-338 (II3III6-disulfate of iso-GM1b) was surprisingly found to be the
most potent MAG binding structure tested to date.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Sulfated GM1b gangliosides; Neural siglecs; Sialic acid
1. Introduction
domain in common, which is required for sialic acid
binding, a variable number of C-type Ig domains, a
single transmembrane domain, and a short cytoplasmic
tail. A nervous system siglec, MAG is a quantitatively
minor component of central and peripheral nervous
system myelin, and has been hypothesized to be
implicated in myelin-axon interactions.4 In particular,
MAG has been shown to be a major neurite outgrowth
inhibitor.5ꢁ7
In the course of study on sialic acid specificity of
siglec binding with a series of gangliosides, we have
demonstrated,8ꢁ10 in the first time that a cholinergic
neuron-specific polysialoganglioside GQ1ba11,12 (Fig. 1)
is a remarkably high affinity ligand for the neural
siglecs, MAG and SMP. A family of a-series ganglio-
sides, GT1aa13,14 and GD1a15 (Fig. 1), have also been
found to be high-affinity ligands for the neural siglecs.
The hierarchy of binding strength of a series of gang-
Siglecs2,3 (sialic acid-binding Ig-like lectins), such as the
macrophage receptor sialoadhesin (Siglec-1), the B-cell
antigen (CD22, Siglec-2), the marker of myelomonocytic
cells (CD33, Siglec-3), the myelin-associated glycopro-
tein (MAG, Siglec-4a), and its avian homologue,
Schwann cell myelin protein (SMP, Siglec-4b), are a
family of sialic acid-dependent adhesion molecules
belong to the immunoglobulin superfamily, previously
termed sialoadhesins or I-type lectins. These molecules
contain an N-terminal V-type immunoglobulin (Ig)
ꢀ Synthetic studies on sialoglycoconjugates, Part 130. For
Part 129, see Ref. 1.
* Corresponding author. Tel.: ꢂ
58-2932840.
E-mail address: kiso@cc.gifu-u.ac.jp (M. Kiso).
/
81-58-2932916; fax: ꢂ81-
/
0008-6215/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00245-3

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H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ1639
/
Fig. 1. Chol-1 (a-series) gangliosides.
liosides with MAG were GQ1baꢀ
GT1b, GD1aꢀGM3, GM4 (GQ1b, GD1b, GM1 and
GD3 failed to support binding). These results indicate
that not only the terminal NeuAca(203)Gal structure,
/
GT1aa, GD1aꢀ
/
mine donor 2 with the benyl protected lactose acceptor
320 gave 4 in 98% yield, which was then converted to 5.
Treatment of 5 or 8 with levulinic anhydride in pyridine
in the presence of 4-dimethylaminopyridine (DMAP)
gave the III6-levulinoyl (6) and II3III6-dilevulinoyl (9)
derivatives in 92 and 90% yields, respectively. The 3,4-
O-isopropylidene group in 6 or 9 was then cleaved by
treatment with 80% acetic acid at 40 8C to afford the
desired glycosyl acceptors 7 and 10 in high yields.
Dimethyl(methylthio)sulfonium triflate (DMTST)-pro-
/
/
which is essential for MAG binding,9 but also the
internal sialic acids a2,6-linked to GalNAc (III6) and
a2,3-linked to another Gal (II3) play important roles for
displaying enhanced avidity for neural siglec-binding.
Our recent study testing the role of the exocyclic glycerol
chain hydroxyls on the a2,6-NeuAc residue of GD1a
using its deoxy NeuAc analogues15 demonstrated that
each hydroxyl on the exocyclic glycerol chain is not
strictly required for enhanced siglec recognition. In fact,
all of the GD1a analogues tested could support the
adhesion as strong as the parent GD1a, raising the
possibility that the internal sialic acid may be replaced
by other anionic substituents as demonstrated in the
study on selectin ligands.16ꢁ18 To corroborate this
hypothesis we undertook, selectively, for the first time,
the total synthesis of the novel III6-sulfate [B; GSC-335]
of ganglioside GM1b [A] (a GD1a analogue), the
corresponding II3III6-disulfate [C; GSC-454] (a GT1aa
analogue), and a novel series (we call tentatively iso-
ganglio-series) ganglioside [D; GSC-338] (Fig. 2), and
examined their bindings by MAG.
moted glycosylation21 of 7 or 10 with a sialyl-a(20
/3)-
galactose donor 1122 was achieved (Scheme 1) b-stereo-
selectively to afford the protected pentasaccharide 12
(94%), and 13 (61%) accompanied by 14 (29%), respec-
tively. Although it was expected that the b(10
product (13) could be formed with high regioselectivity
4)-linked isomer 14 was
/3)-linked
at O-3 of Gal, the b(10
/
obtained in about 30% yield. It may result from the
steric hindrance and electronic effect of the levulinoyl
group at II3. The structures of 12ꢁ
14, were determined
/
2. Results and discussion
The most important problems in the synthesis of the
target compounds [BꢁC] are: (a) selective protection of
/
the II3 and III6 hydroxyl groups that undergo regiose-
lective sulfations; (b) efficient construction of the
terminal NeuAca(20/3)Gal structure linked to O-3 or
O-4 of the GalNAc residue; and (c) facile introduction
of the ceramide moiety. For the selective protection of
the II3 and III6 hydroxyl groups, we selected the suitably
protected ganglio-triose derivatives 5 and 819 as the key
intermediates (Scheme 1). Coupling of the galactosa-
Fig. 2. Structure of ganglioside GM1b (A) and novel sulfated
GM1b analogues (BꢁD) as the mimics of Chol-1 (a-series)
gangliosides.
/

H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ
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1639
1623
Scheme 2.
Selective cleavage of the 2-(trimethylsilyl)ethyl group
in 15, 16, 23, and treatment23 of the resulting 27, 28, and
38 with trichloroacetonitrile and DBU, afforded a series
of imidate derivatives 29, 30, and 39 (Scheme 3). The
direct glycosylation of the ceramide derivative 3124 with
29, 30, and 39 was carried out in the presence of
trimethylsilyl trifluoromethanesulfonate (TMSOTf)
˚
and 4A molecular sieves (AW-300) in dichloromethane
to yield the protected gangliosides 32, 33, and 40 in
about 40% yields. Most of the unreacted glycosyl donor
(1-OH form) and acceptor 31 were recovered. This result
is comparable to that obtained by an efficient b-
glycosidation procedure using the 2-O-pivaloyl group
as an auxiliary.25 Therefore, the yields of ꢁ
40% is
/
satisfactory considering the additional steps in the
azidosphingosine procedure, which has been employed
for various ganglioside synthesis.26,27 The levulinoyl
groups in 32, 33, and 40 were selectively removed by
treatment with hydrazine monoacetate in ethanol to give
34, 35, and 41, respectively, which were then sulfated
Scheme 1.
1
by the H NMR analysis of the peracylated derivatives
15, 16, and 23, respectively.
Hydrogenolytic removal of the benzyl groups in 12,
13 and 14, followed by acetylation, gave 15, 16, and 23,
respectively. The levulinoyl groups in 15, 16, and 23
were selectively removed by treatment with hydrazine
monoacetate in ethanol to give 17, 18, and 24, which
with a sulfur trioxideꢁ
Finally, removal of all protective groups under basic
conditions furnished the target compounds BꢁD in high
yields (Scheme 3 and Fig. 2).
/
pyridine complex in DMF.
/
were then sulfated with a sulfur trioxideꢁpyridine
/
In binding experiments with MAG-transfected COS
cells28 for the immobilized synthetic gangliosides, it was
demonstrated that the compound D (GSC-338; II3III6-
disulfo of iso-GM1b) was surprisingly found to be the
most potent siglec-binding structure tested to date (Fig.
3). It was 10-fold more potent than GT1aa in support-
ing MAG and SMP binding, and fourfold more potent
complex in DMF, yielding 19, 20, and 25. Removal of
all protective groups under basic conditions furnished
the corresponding sulfated GM1b pentasaccharides 21
(GSC-334), 22 (GSC-453) and 26 in which the reducing
end is protected by the 2-(trimethylsilyl)ethyl (SE) group
(Scheme 2).

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H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ
/
1639
Scheme 3.
than GQ1ba, which was the highest affinity ligand for
MAG so far.8ꢁ10 The III6-monosulfate B (GSC-335)
showed almost the same potency as GD1a or GT1aa,
and it was fourfold more potent than GM1b. Other
sulfated glycolipids, including sulfatide and sulfoglucur-
onyl glycolipids, do not support adhesion of MAG-
transfected COS cells.28
The validity of the MAG-Fc binding assay29 was
demonstrated using 10 gangliosides that had been tested
previously for MAG binding using MAG-transfected
COS cells.28 The rank-order and relative affinity of the
different gangliosides were the same in the two assays
(Fig. 4). The absolute binding affinity of each ganglio-
side was modestly lower in the MAG-Fc assay com-
pared to the cell binding assay, most likely due to the
lower degree of multivalency. Nevertheless, enhanced
MAG recognition of the a-series gangliosides was clear
in both assays.
The data in Fig. 5 demonstrate very highly enhanced
MAG-Fc binding to GSC-338. Fitting the data to a
rectangular hyperbola reveals apparent KD values of 38
pmol/well for GQ1ba, 7.4 pmol/well for GSC-454
(compound C) and 3.1 pmol/well for GSC-338, suggest-
ing that GSC-338 is over 12-fold more potent than
GQ1ba, itself a high affinity MAG ligand. Whereas
GQ1ba and GSC-338 supported similar maximum
binding (AU maxꢀ/2.5 under these conditions), GSC-
454 supported only a quarter of that (AU maxꢀ
/
0.64).
The reason for the lowered maximum binding to GSC-
454 is not known, but may reflect a different physical

H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ
/
1639
1625
These results indicate that the internal sialic acids
a2,6-linked to GalNAc (III6) and a2,3-linked to Gal
(II3) could be substituted by the sulfate groups to retain
or greatly enhance the siglec-mediated cell adhesion.
Systematic investigation for the specific counter-recep-
tor of siglecs may open new perspectives on develop-
ment of novel therapeutic agents.
3. Experimental
3.1. Synthesis
3.1.1. General methods. Optical rotations were deter-
mined with a HORIBA SEPA-300 polarimeter at 25 8C,
and IR spectra were recorded with a JASCO IRA-100
1
spectrophotometer. H NMR spectra were recorded at
400 or 500 MHz with Varian UNITY Inova-400- and
500-MHz spectrometers, respectively. Preparative chro-
matography was performed on silica gel (Fuji Silysia
Co., 300 mesh) with the solvent systems specified.
Concentrations were conducted in vacuo.
Fig. 3. MAG-mediated cell adhesion to the newly synthesized
III6-sulfate of GM1b (GSC-335) and II3III6-disulfate of iso-
GM1b (GSC-338) analogues and genuine gangliosides. X:
ganglioside added (pmol per well), Y: adhesion of MAG-
transfected COS cells (% of cells added). ^: GSC-338, m:
GT1aa, k: GD1a, I: GSC-335, 2: GM1b. For analytical
procedure see ref. [8,28].
3.1.2. Methyl 6-O-acetyl-2-deoxy-3,4-O-isopropylidene-
2-phthalimido-1-thio-b-
stirred solution of methyl 2-deoxy-3,4-O-isopropyli-
dene-2-phthalimido-1-thio-b-
-galactopyranoside30 (1;
D-galactopyranoside (2). To a
D
310 mg, 0.82 mmol) in pyridine (3 mL) was added acetic
anhydride (1.5 mL) at 0 8C. The mixture was stirred for
3 h at room temperature (rt), and MeOH (1.5 mL) was
added. The solution was concentrated to a syrup which
was extracted with chloroform. The extract was succes-
distribution of the ganglioside on the microwell. How-
ever, the apparent KD of GSC-454 (7.4 pmol/well)
reveals high affinity, although still ꢀ/twofold less avid
than for GSC-338.
Fig. 4. MAG-Fc binding to various natural and synthetic gangliosides. Natural gangliosides GM3, GD3, GM1, GD1a, GD1b,
GT1b, and GQ1b and synthetic gangliosides GD1a, GT1aa and GQ1ba were adsorbed on microwells at the indicated input
concentrations (pmol/well) and MAG-Fc binding was determined as described in Section 3, and are plotted as the absorbance at 405
nm (y-axis) vs. the pmol ganglioside added per well. Data are presented as the mean9S.E.M. for quintuplicate determinations.
/

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H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ1639
/
Fig. 5. MAG-Fc binding to GSC-338 (compound D), GSC-454 (compound C), GQ1ba, and GM1 (background). Gangliosides were
adsorbed on microwell plates at the indicated input concentrations (pmol/well) and MAG-Fc binding was determined as described
in Section 3, and are plotted as the absorbance at 405 nm (y-axis) vs. the pmol ganglioside added per well. Data are presented as the
mean9
/
S.E.M. for quadruplicate determinations. Data are representative of three separate experiments. Data points for each
gangliosides were fitted to a rectangular hyperbola (solid lines) to calculate apparent KD and maximum binding (see text).
sively washed with 2 M HCl, water, M Na2CO3, and
water, dried (Na2SO4) and concentrated. Column chro-
graphy (1:2 ethyl acetateꢁ
/
hexane) of the residue on
silica gel gave 4 (795 mg, 98%) as an amorphous mass.
1
matography (1:2 ethyl acetateꢁ
/
hexane) of the residue on
[a]D
ꢂ24.88 (c 0.5, CHCl3); H NMR (CDCl3): d 1.02
/
silica gel gave 2 (344 mg, quant) as an amorphous mass.
1
(m, 2 H, Me3SiCH2CH2), 1.35 and 1.52 (2 s, 6 H,
Me2C), 2.03 (s, 3 H, AcO), 3.00 (dd, 1 H, J1,2 7.6, J2,3 9.5
Hz, H-2II), 3.11 (dd, 1 H, J2,3 9.5, J3,4 2.7 Hz, H-3II),
3.27 (m, 1 H, H-5I), 3.31 (dd, 1 H, J1,2 7.8, J2,3 9.4 Hz,
[a]D
ꢂ64.18 (c 0.4, CHCl3); H NMR (CDCl3): d 1.33
/
and 1.64 (2 s, 6 H, Me2C), 2.12 and 2.15 (2 s, 6 H, AcO
and SMe), 4.20 (m, 1 H, H-5), 4.27 (dd, 1 H, J3,4 5.0, J4,5
2.2 Hz, H-4), 3.37 (dd, 1 H, J1,2 10.5, J2,3 8.9 Hz, H-2),
4.40 (dd, 1 H, Jgem 11.3, J5,6 4.5 Hz, H-6), 4.42 (dd, 1 H,
Jgem 11.3, J5,6 6.7 Hz, H-6?), 4.84 (dd, 1 H, J2,3 8.9, J3,4
H-2I), 3.53 (t, 1 H, J2,3
ꢀ
/
J3,4 9.4 Hz, H-3I), 3.68 (d, 1 H,
J4,5 9.4 Hz, H-4I),
J3,4 2.7 Hz, H-4II), 3.78 (t, 1 H, J3,4
ꢀ
/
4.14 (m, 1 H, H-5III), 4.18 (d, 1 H, J1,2 7.6 Hz, H-1II),
4.29 (m, 1 H, H-4III), 4.34 (d, 1 H, J1,2 7.8 Hz, H-1I),
4.39 (dd, 1 H, Jgem 11.4, J5,6 6.9 Hz, H-6III), 4.43 (t, 1 H,
5.0 Hz, H-3), 5.08 (d, 1 H, J1,2 10.5 Hz, H-1), and 7.72ꢁ
/
7.85 (m, 4 H, Ph). Anal. Calcd for C20H23NO7Si
(421.47): C, 57.00; H, 5.50; N, 3.32. Found: C, 56.99;
H, 5.41; N, 3.22.
J1,2
5.3 Hz, H-6?III), 5.10 (dd, 1 H, J2,3 8.9, J3,4 5.3 Hz, H-
3III), 5.18 (d, 1 H, J1,2 8.9 Hz, H-1III), and 6.95ꢁ
7.86 (m,
ꢀ
J2,3 8.9 Hz, H-2III), 4.51 (dd, 1 H, Jgem 11.4, J5,6
/
/
34 H, 7 Ph). Anal. Calcd for C78H89NO18Si (1356.65):
C, 69.06; H, 6.61; N, 1.03. Found: C, 68.77; H, 6.60; N,
0.92.
3.1.3. 2-(Trimethylsilyl)ethyl 6-O-acetyl-2-deoxy-3,4-O-
isopropylidene-2-phthalimido-b-
4)-2,3,6-tri-O-benzyl-b- -galactopyranosyl-(10
tri-O-benzyl-b- -glucopyranoside (4). To a solution of 2-
(trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-b- -galactopyra-
nosyl-(104)-2,3,6-tri-O-benzyl-b-
-glucopyranoside20
(3; 583 mg, 0.59 mmol) and 2 (300 mg, 0.71 mmol) in
D
-galactopyranosyl-(10
/
D
/
4)-2,3,6-
D
D
3.1.4. 2-(Trimethylsilyl)ethyl 2-acetamido-2-deoxy-3,4-
/
D
O-isopropylidene-b-
O-benzyl-b- -galactopyranosyl-(10
benzyl-b- -glucopyranoside (5). A solution of 4 (795 mg,
D
-galactopyranosyl-(10
/
4)-2,3,6-tri-
D
/
4)-2,3,6-tri-O-
˚
dichloromethane (7 mL) was added 4A molecular sieves
(1.0 g), and the mixture was stirred for 10 h at rt, then
D
0.59 mmol) in ethanol (20 mL) and hydrazine mono-
hydrate (0.70 mL) was heated for 5 h under reflux. After
cooling, insolubles were filtered off and washed with
EtOH. The filtrate and washings were combined and
evaporated to dryness, the residue was treated with
acetic anhydride (1.5 mL) and MeOH (20 mL) for 8 h at
rt, and the solution was concentrated. Column chroma-
cooled to ꢃ15 8C. To the mixture were added N-
/
iodosuccinimide (NIS; 240 mg, 1.07 mmol) and tri-
methylsilyl trifluoromethanesulfonate (TMSOTf; 14 mL,
0.11 mmol), and the stirring was continued for 2 h at ꢃ
/
15 8C. After completion of the reaction, the precipitate
was filtered off and washed with chloroform. The filtrate
and washings were combined, and the solution was
successively washed with M Na2CO3 and M Na2S2O3,
dried (Na2SO4) and concentrated. Column chromato-
tography (50:1 chloroformꢁ/methanol) of the residue on
silica gel gave 5 (710 mg, 98%) as an amorphous mass.
1
[a]D
ꢂ32.58 (c 0.3, CHCl3); H NMR (CDCl3): d 1.04
/

H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ
/
1639
1627
(m, 2 H, Me3SiCH2CH2), 1.31 and 1.50 (2 s, 6 H,
Me2C), 1.71 (s, 3 H, AcN), 3.28 (dd, 1 H, J2,3 9.5, J3,4 2.5
Hz, H-3II), 3.37 (m, 1 H, H-5I), 3.39 (dd, 1 H, J1,2 7.6,
J2,3 9.3 Hz, H-2I), 3.49 (dd, 1 H, J1,2 8.0, J2,3 9.5 Hz, H-
amorphous mass. [a]D
ꢂ
/
17.48 (c 0.5, CHCl3); 1H
NMR (CDCl3): d 1.03 (m, 2 H, Me3SiCH2CH2), 1.51
(s, 3 H, AcN), 2.17 (s, 3 H, CH3COCH2CH2), 2.40ꢁ2.80
/
(m, 4 H, CH3COCH2CH2), 3.37 (dd, 1 H, J1,2 7.8, J2,3
9.4 Hz, H-2I), 3.38 (m, 1 H, H-5I), 3.44 (dd, 1 H, J2,3 9.3,
J3,4 3.2 Hz, H-3II), 3.50 (m, 1 H, H-2III), 3.56 (t, 1 H,
2II), 3.52 (m, 1 H, H-2III), 3.55 (t, 1 H, J2,3
H-3I), 3.70 (m, 1 H, H-5II), 3.83 (m, 1 H, H-5III), 3.89 (t,
1 H, J3,4
J4,5 9.3 Hz, H-4I), 3.91 (d, 1 H, J3,4 2.5 Hz,
ꢀ/J3,4 9.3 Hz,
ꢀ
/
J2,3
H, H-2II), 3.83 (d, 1 H, J3,4 3.2 Hz, H-4II), 3.94 (t, 1 H,
J3,4ꢀ
J4,5 9.4 Hz, H-4I), 4.11 (m, 1 H, H-4III), 4.34 (dd, 1
ꢀ
J3,4 9.4 Hz, H-3I), 3.66 (m, 1 H, H-5III), 3.74 (m, 1
/
H-4II), 4.06 (dd, 1 H, J3,4 5.3, J4,5 2.1 Hz, H-4III), 4.32
(dd, 1 H, J2,3 8.5, J3,4 5.3 Hz, H-3III), 4.36 (d, 1 H, J1,2
8.0 Hz, H-1II), 4.38 (d, 1 H, J1,2 7.6 Hz, H-1I), 4.65 (d, 1
H, J1,2 8.7 Hz, H-1III), 5.53 (d, 1 H, J2,NH 7.3 Hz, NH),
/
H, Jgem 11.7, J5,6 4.8 Hz, H-6III), 4.38 (d, 1 H, J1,2 7.8
Hz, H-1I), 4.45 (d, 1 H, J1,2 8.0 Hz, H-1II), 4.46 (d, 1 H,
J1,2 7.5 Hz, H-1III), 4.52 (dd, 1 H, Jgem 11.7, J5,6 7.7 Hz,
and 7.20ꢁ7.43 (m, 30 H, 6 Ph). Anal. Calcd for
/
C70H87NO16Si (1226.54): C, 68.55; H, 7.15; N, 1.14.
Found: C, 68.39; H, 6.95; N, 1.06.
H-6?III), and 7.21ꢁ
7.47 (m, 30 H, 6 Ph). Anal. Calcd for
/
C72H89N3O18Si (1284.58): C, 67.32; H, 6.98; N, 1.09.
Found: C, 67.22; H, 6.96; N, 0.87.
3.1.5. 2-(Trimethylsilyl)ethyl 2-acetamido-2-deoxy-3,4-
O-isopropylidene-6-O-levulinoyl-b-
(104)-2,3,6-tri-O-benzyl-b- -galactopyranosyl-(10
2,3,6-tri-O-benzyl-b- -glucopyranoside (6). To a solution
D
-galactopyranosyl-
3.1.7. 2-(Trimethylsilyl)ethyl 2-acetamido-2-deoxy-3,4-
/
D
/4)-
O-isopropylidene-6-O-levulinoyl-b-
(104)-2,6-di-O-benzyl-3-O-levulinoyl-b-
pyranosyl-(104)-2,3,6-tri-O-benzyl-b- -glucopyranoside
(9). To a solution of 2-(trimethylsilyl)ethyl 2-acetamido-
2-deoxy-3,4-O-isopropylidene-b- -galactopyranosyl-
(104)-2,6-di-O-benzyl-b- -galactopyranosyl-(104)-
2,3,6-tri-O-benzyl-b-
-glucopyranoside14 (8; 1.0g, 0.88
D-galactopyranosyl-
D
/
D-galacto-
of 5 (1.5 g, 1.22 mmol) in pyridine (5 mL) and
dichloromethane (10 mL) were added levulinic anhy-
dride (0.81 g, 3.66 mmol) and 4-dimethylaminopyridine
(80 mg, 0.61 mmol). The mixture was stirred for 4 h at
rt, and MeOH (5 mL) was added. The solution was
concentrated, then extracted with chloroform. The
extract was succesively washed with 2 M HCl, water,
M Na2CO3, and water, dried (Na2SO4) and concen-
/
D
D
/
D
/
D
mmol) in pyridine (10 mL) was added levulinic anhy-
dride (0.75 g, 3.52 mmol) and 4-dimethylaminopyridine
(54 mg, 0.44 mmol), then workup as described for 6.
Column chromatography (ethyl acetate) of the residue
trated. Column chromatography (50:1 chloroformꢁ
/
methanol) of the residue on silica gel gave 6 (1.50 g,
92%) as an amorphous mass. [a]D
on silica gel gave 9 (1.05 g, 90%) as an amorphous mass.
1
ꢂ
/
33.58 (c 0.5,
[a]D
(m, 2 H, Me3SiCH2CH2), 1.36 and 1.56 (2 s, 6 H,
ꢂ
/
31.88 (c 0.4, CHCl3); H NMR (CDCl3): d 1.04
CHCl3); 1H NMR (CDCl3): d 1.03 (m, 2 H, Me3-
SiCH2CH2), 1.33 and 1.54 (2 s, 6 H, Me2C), 1.72 (s, 3 H,
Me2C), 1.95 (s, 3 H, AcN), 2.15 and 2.22 (2 s, 6 H, 2
AcN), 2.16 (s, 3 H, CH3COCH2CH2), 2.44ꢁ
/
2.77 (m, 4
C H 3 C O C H 2 C H 2 ) , 2 . 3 7 ꢁ 2 . 7 6 ( m , 8 H , 2
/
H, CH3COCH2CH2), 3.36 (dd, 1 H, J1,2 7.8, J2,3 9.2 Hz,
H-2I), 3.41 (dd, 1 H, J2,3 9.6, J3,4 3.4 Hz, H-3II), 3.51
(dd, 1 H, J1,2 7.6, J2,3 9.6 Hz, H-2II), 3.54 (t, 1 H, J1,2
CH3COCH2CH2), 3.02 (m, 1 H, H-2III), 3.34 (dd, 1 H,
J1,2 7.9, J2,3 9.2 Hz, H-2I), 3.40 (m, 1 H, H-5I), 3.59 (t, 1
ꢀ
/
H, J2,3
ꢀ
J3,4 9.2 Hz, H-3I), 3.64 (dd, 1 H, J1,2 7.8, J2,3
/
J2,3 8.7 Hz, H-2III), 3.56 (t, 1 H, J2,3
ꢀ
/
J3,4 9.2 Hz, H-3I),
10.1 Hz, H-2II), 3.91 (t, 1 H, J3,4
ꢀ
J4,5 9.2 Hz, H-4I),
/
3.92 (t, 1 H, J3,4
ꢀ
/
J4,5 9.2 Hz, H-4I), 3.95 (m, 1 H, H-
4.00 (m, 1 H, H-5III), 4.11 (d, 1 H, J3,4 3.0 Hz, H-4II),
4.16 (dd, 1 H, J3,4 5.0, J4,5 1.9 Hz, H-4III), 4.33 (dd, 1 H,
Jgem 11.4, J5,6 7.6 Hz, H-6III), 4.40 (d, 1 H, J1,2 7.9 Hz,
H-1I), 4.42 (dd, 1 H, Jgem 11.4, J5,6 6.6 Hz, H-6?III), 4.46
(d, 1 H, J1,2 7.8 Hz, H-1II), 4.57 (d, 1 H, J1,2 8.7 Hz, H-
1III), 4.59 (dd, 1 H, J2,3 8.5, J3,4 5.0 Hz, H-3III), 4.63 (dd,
5III), 4.08 (d, 1 H, J3,4 3.4 Hz, H-4II), 4.09 (dd, 1 H, J3,4
5.3, J4,5 2.3 Hz, H-4III), 4.18 (dd, 1 H, J2,3 8.2, J3,4 5.3
Hz, H-3III), 4.33 (dd, 1 H, Jgem 11.4, J5,6 7.1 Hz, H-6III),
4.38 (d, 1 H, J1,2 7.8 Hz, H-1I), 4.42 (d, 1 H, J1,2 7.6 Hz,
H-1II), 4.44 (dd, 1 H, Jgem 11.4, J5,6 5.2 Hz, H-6?III), 4.78
(d, 1 H, J1,2 8.7 Hz, H-1III), 5.53 (d, 1 H, J2,NH 7.3 Hz,
1 H, J2,3 10.1, J3,4 3.0 Hz, H-3II), and 7.12ꢁ
7.53 (m, 25
/
NH), and 7.19ꢁ
C75H93NO18Si (1324.64): C, 68.01; H, 7.08; N, 1.06.
Found: C, 67.72; H, 6.98; N, 1.04.
/
7.47 (m, 30 H, 6 Ph). Anal. Calcd for
H, 5 Ph). Anal. Calcd for C73H93NO20Si (1332.62): C,
65.80; H, 7.03; N, 1.05. Found: C, 65.63; H, 6.79; N,
0.85.
3.1.6. 2-(Trimethylsilyl)ethyl 2-acetamido-2-deoxy-6-O-
3.1.8. 2-(Trimethylsilyl)ethyl 2-acetamido-2-deoxy-6-O-
levulinoyl-b-
benzyl-b-
-galactopyranosyl-(10
b- -glucopyranoside (7). A solution of 6 (1.5 g, 1.13
D
-galactopyranosyl-(10
/
4)-2,3,6-tri-O-
levulinoyl-b-
3-O-levulinoyl-b-
benzyl-b- -glucopyranoside (10). A solution of 9 (1.0 g,
D
-galactopyranosyl-(10
/
4)-2,6-di-O-benzyl-
D
/
4)-2,3,6-tri-O-benzyl-
D
-galactopyranosyl-(10
/
4)-2,3,6-tri-O-
D
D
mmol) in 80% aq acetic acid (15 mL) was heated, with
stirring, for 48 h at 40 8C, then concentrated. Column
0.75 mmol) in 80% aq acetic acid (15 mL) was heated
with stirring for 24 h at 40 8C, then concentrated.
chromatography (50:1 chloroformꢁ/methanol) of the
Column chromatography (40:1 chloroformꢁ
/methanol)
residue on silica gel gave 7 (1.20 g, 83%) as an
of the residue on silica gel gave 10 (900 mg, 93%) as an

1628
H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ1639
/
amorphous mass. [a]D
ꢂ
/
21.28 (c 0.5, CHCl3); 1H NMR
(CDCl3): d 1.03 (m, 2 H, Me3SiCH2CH2), 1.93 (s, 3 H,
AcN), 2.14 and 2.19 (2 s, 6 H, 2 CH3COCH2CH2),
(d, 1 H, J5,NH 10.3 Hz, NH), 5.00 (d, 1 H, J1,2 8.0 Hz, H-
1IV), 5.04 (d, 1 H, J1,2 8.5 Hz, H-1III), 5.21 (d, 1 H, J2,NH
7.3 Hz, NH), 5.24 (dd, 1 H, J6,7 2.7, J7,8 9.6 Hz, H-7V),
5.35 (d, 1 H, J3,4 3.2 Hz, H-4IV), 5.47 (dd, 1 H, J1,2 8.0,
2.43ꢁ
2III), 3.37 (dd, 1 H, J1,2 8.0, J2,3 9.6 Hz, H-2I), 3.57 (dd, 1
H, J1,2 7.6, J2,3 10.3 Hz, H-2II), 3.59 (t, 1 H, J2,3
J3,4
9.6 Hz, H-3I), 3.73 (dd, 1 H, J3,4 3.2 Hz, H-4III), 3.74 (m,
1 H, H-5III), 3.87 (m, 1 H, H-3III), 3.96 (t, 1 H, J3,4
J4,5
/2.73 (m, 8 H, 2 CH3COCH2CH2), 3.04 (m, 1 H, H-
J2,3 10.1 Hz, H-2IV), 5.61 (m, 1 H, H-8V), and 7.16ꢁ
8.12
/
ꢀ
/
(m, 45 H, 9 Ph). Anal. Calcd for C119H138N2O38Si
(2232.48): C, 64.02; H, 6.23; N, 1.25. Found: C, 63.92;
H, 5.99; N, 1.16.
ꢀ
/
9.6 Hz, H-4I), 4.04 (d, 1 H, J3,4 3.2 Hz, H-4II), 4.27 (dd,
1 H, Jgem 11.2, J5,6 6.9 Hz, H-6III), 4.39 (d, 1 H, J1,2 8.0
Hz, H-1I), 4.45 (dd, 1 H, Jgem 11.2, J5,6 5.7 Hz, H-6?III),
4.48 (d, 1 H, J1,2 7.6 Hz, H-1II), 4.64 (d, 1 H, J1,2 8.0 Hz,
H-1III), 4.84 (dd, 1 H, J2,3 10.3, J3,4 3.2 Hz, H-3II), and
3.1.10. 2-(Trimethylsilyl)ethyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
galacto-2-nonulopyranosylonate-(20
benzoyl-b- 3)-2-acetamido-2-
-galactopyranosyl-(10
deoxy-6-O-levulinoyl-b- -galactopyranosyl-(104)-2,6-
di-O-benzyl-3-O-levulinoyl-b- -galactopyranosyl-(10
4)-2,3,6-tri-O-benzyl-b- -glucopyranoside (13). Glyco-
methyl
-glycero-a-
3)-2,4,6-tri-O-
5-acetamido-
D
D-
/
D
/
7.20ꢁ/7.47 (m, 25 H,
5
Ph). Anal. Calcd for
D
/
C70H89NO20Si (1292.56): C, 65.05; H, 6.94; N, 1.08.
Found: C, 64.77; H, 6.74; N, 1.00.
D
/
D
sylation of 10 (300 mg, 0.23 mmol) with 11 (393 mg,
0.39 mmol) in dichloromethane (7 mL) in the presence
˚
of DMTST (414 mg, 0.98 mmol) and 4A MS (1.0 g) for
24 h at 0 8C, then work up as described for 12, gave 13
(317 mg, 61%), accompanied by position-isomer 14 (150
3.1.9. 2-(Trimethylsilyl)ethyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
galacto-2-nonulopyranosylonate-(20
benzoyl-b- 4)-2-acetamido-2-
-galactopyranosyl-(10
deoxy-6-O-levulinoyl-b- -galactopyranosyl-(104)-2,3,6-
tri-O-benzyl-b- -galactopyranosyl-(104)-2,3,6-tri-O-
benzyl-b- -glucopyranoside (12). To a solution of 7 (938
mg, 0.73 mmol) and methyl methyl 5-acetamido-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy- -galacto-2-
-glycero-a-
methyl
-glycero-a-
3)-2,4,6-tri-O-
5-acetamido-
D
D-
/
D
/
D
/
mg, 29%) as an amorphous mass. Compound 13: [a]D
1
D
/
ꢂ
/
41.08 (c 0.4, CHCl3); H NMR (CDCl3): d 1.02 (m, 2
D
H, Me3SiCH2CH2), 1.11, 1.49, 1.76, 1.90, 2.07, 2.11,
2.19, and 2.20 (8 s, 24 H, 2 AcN, 4 AcO, and 2
D
D
CH3COCH2CH2), 1.61 (t, 1 H, Jgem
3Vax), 2.31ꢁ H, H-3Veq and
ꢀJ3ax,4 12.6 Hz, H-
/
nonulopyranosylonate-(20
/
3)-2,4,6-tri-O-benzoyl-b-
D
-
/
2.68 (m,
9
2
galactopyranoside (11; 1.27 g, 1.24 mmol) in dichlor-
˚
omethane (15 mL) were added 4A molecular sieves (3.0
CH3COCH2CH2), 3.32 (dd, 1 H, J1,2 8.0, J2,3 9.2 Hz,
H-2I), 3.53 (dd, 1 H, J1,2 7.8, J2,3 10.3 Hz, H-2II), 3.56 (t,
g), and the mixture was stirred for 6 h at rt, then cooled
to 0 8C. Dimethyl(methylthio)sulfonium triflate
(DMTST; 1.12 g, 2.48 mmol) was added, and the
mixture was stirred for 48 h at 7 8C, then filtered. The
precipitates were removed by filtration and washed
thoroughly with chloroform. The combined filtrate
and washings was successively washed with M NaHCO3
and water, dried (Na2SO4) and concentrated. Column
1 H, J2,3
2.7 Hz, H-6V), 3.70 (m, 1 H, H-2III), 3.75 (q, 1 H, J4,5
J5,6
J5,NH 10.3 Hz, H-5V), 3.85 (s, 3 H, MeO), 3.88 (t,
1 H, J3,4
J4,5 9.2 Hz, H-4I), 3.96 (dd, 1 H, Jgem 12.8,
ꢀ
J3,4 9.2 Hz, H-3I), 3.61 (dd, 1 H, J5,6 10.8, J6,7
/
ꢀ
/
ꢀ
/
ꢀ
/
J8,9 5.9 Hz, H-9V), 4.03 (d, 1 H, J3,4 2.7 Hz, H-4II), 4.08
(m, 1 H, H-4III), 4.25 (dd, 1 H, Jgem 12.8, J8,9 2.7 Hz, H-
9?V), 4.37 (d, 1 H, J1,2 8.0 Hz, H-1I), 4.42 (d, 1 H, J1,2 7.8
Hz, H-1II), 4.49 (m, 1 H, H-3III), 4.65 (dd, 1 H, J2,3 10.3,
J3,4 2.7 Hz, H-3II), 4.79 (m, 1 H, H-4V), 4.86 (dd, 1 H,
J2,3 10.1, J3,4 3.2 Hz, H-3IV), 4.97 (d, 1 H, J5,NH 10.3 Hz,
NH), 5.02 (d, 1 H, J1,2 7.8 Hz, H-1IV), 5.22 (dd, 1 H, J6,7
2.7, J7,8 9.8 Hz, H-7V), 5.34 (d, 1 H, J3,4 3.2 Hz, H-4IV),
5.48 (dd, 1 H, J1,2 7.8, J2,3 10.1 Hz, H-2IV), 5.52 (d, 1 H,
chromatography (40:1 chloroformꢁ
residue on silica gel gave 12 (1.55 g, 94%) as an
amorphous mass. [a]D
41.68 (c 0.5, CHCl3); 1H
/
methanol) of the
ꢂ
/
NMR (CDCl3): d 1.02 (m, 2 H, Me3SiCH2CH2), 0.93,
1.52, 1.77, 1.90, 2.08, 2.12, and 2.20 (7 s, 21 H, 2 AcN, 4
AcO, and CH3COCH2CH2), 1.60 (t, 1 H, Jgem
ꢀ
/
J3ax,4
J2,NH 7.8 Hz, NH), 5.59 (m, 1 H, H-8V), and 7.15ꢁ
8.13
/
11.2 Hz, H-3Vax), 2.39ꢁ2.67 (m, 5 H, H-3Veq and
/
(m, 45 H, 9 Ph). Anal. Calcd for C117H138N2O40Si
(2240.45): C, 62.72; H, 6.21; N, 1.25. Found: C, 62.72;
H, 5.93; N, 1.02.
CH3COCH2CH2), 3.30 (dd, 1 H, J2,3 9.6, J3,4 2.7 Hz, H-
3II), 3.34 (dd, 1 H, J1,2 8.0, J2,3 9.0 Hz, H-2I), 3.41 (dd, 1
H, J1,2 7.8, J2,3 9.6 Hz, H-2II), 3.46 (t, 1 H, H-2III), 3.53
(t, 1 H, J2,3
J6,7 2.7 Hz, H-6V), 3.76 (q, 1 H, J4,5 J5,6 J5,NH 10.3 Hz,
H-5V), 3.85 (t, 1 H, J3,4 J4,5 9.0 Hz, H-4I), 3.85 (s, 3 H,
ꢀ
/
J3,4 9.0 Hz, H-3I), 3.61 (dd, 1 H, J5,6 10.3,
3.1.11. 2-(Trimethylsilyl)ethyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
galacto-2-nonulopyranosylonate-(20
benzoyl-b- 4)-2-acetamido-2-
-galactopyranosyl-(10
deoxy-6-O-levulinoyl-b- -galactopyranosyl-(104)-2,6-
di-O-benzyl-3-O-levulinoyl-b- -galactopyranosyl-(10
4)-2,3,6-tri-O-benzyl-b- -glucopyranoside (14). [a]D
methyl
-glycero-a-
3)-2,4,6-tri-O-
5-acetamido-
D
D-
ꢀ
/
/
MeO), 3.98 (d, 1 H, J3,4 2.7 Hz, H-4II), 4.03 (dd, 1 H,
Jgem 12.6, J8,9 5.3 Hz, H-9V), 4.11 (d, 1 H, J3,4 2.5 Hz, H-
4III), 4.29 (dd, 1 H, Jgem 12.6, J8,9 2.5 Hz, H-9?V), 4.34 (d,
1 H, J1,2 7.8 Hz, H-1II), 4.37 (d, 1 H, J1,2 8.0 Hz, H-1I),
4.43 (dd, 1 H, J2,3 8.5, J3,4 2.5 Hz, H-3III), 4.79 (m, 1 H,
H-4V), 4.87 (dd, 1 H, J2,3 10.1, J3,4 3.2 Hz, H-3IV), 4.97
D
/
D
/
D
/
D
1
ꢂ
/
27.88 (c 0.8, CHCl3); H NMR (CDCl3): d 1.00 (m,
2 H, Me3SiCH2CH2), 1.66 (t, 1 H, Jgem J3ax,4 12.6 Hz,
ꢀ
/

H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ
/
1639
1629
H-3Vax), 1.90, 1.91, 2.06, 2.06, 2.07, 2.14, 2.16, and 2.19
(8 s, 24 H, 2 AcN, 4 AcO, and 2 CH3COCH2CH2),
2IV), 5.46 (d, 1 H, J3,4 3.7 Hz, H-4III), 5.61 (m, 1 H, H-
8V), 6.24 (d, 1 H, J2,NH 6.6 Hz, NH), and 7.27ꢁ
8.19 (m,
15 H, 3 Ph). Anal. Calcd for C91H116N2O45Si (1985.98):
C, 55.04; H, 5.89; N, 1.41. Found: C, 54.92; H, 5.62; N,
1.30.
/
2.31ꢁ
3.35 (m, 1 H, H-2I), 3.44 (m, 1 H, H-2II), 3.50 (m, 1 H,
H-2III), 3.55 (t, 1 H, J2,3 J3,4 9.2 Hz, H-3I), 3.81 (m, 1
H, H-5V), 3.82 (s, 3 H, MeO), 3.86 (t, 1 H, J3,4
J4,5 9.2
/
2.68 (m, 9 H, H-3Veq and 2 CH3COCH2CH2),
ꢀ
/
ꢀ
/
Hz, H-4I), 4.10 (d, 1 H, J3,4 3.0 Hz, H-4II), 4.36 (d, 1 H,
J1,2 7.8 Hz, H-1I), 4.45 (d, 1 H, J1,2 8.0 Hz, H-1II), 4.54
(dd, 1 H, J2,3 10.8, J3,4 3.0 Hz, H-3II), 4.80 (m, 1 H, H-
4V), 4.92 (d, 1 H, J1,2 7.8 Hz, H-1IV), 5.24 (dd, 1 H, J6,7
2.7, J7,8 9.3 Hz, H-7V), 5.40 (d, 1 H, J3,4 3.2 Hz, H-4IV),
5.51 (dd, 1 H, J1,2 7.8, J2,3 9.8 Hz, H-2IV), 5.60 (m, 1 H,
3.1.13. 2-(Trimethylsilyl)ethyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
galacto-2-nonulopyranosylonate-(20
benzoyl-b- 3)-2-acetamido-4-O-
-galactopyranosyl-(10
acetyl-2-deoxy-6-O-levulinoyl-b- -galactopyranosyl-
(104)-2,6-di-O-acetyl-3-O-levulinoyl-b- -galacto-
pyranosyl-(104)-2,3,6-tri-O-acetyl-b- -glucopyranoside
methyl
-glycero-a-
3)-2,4,6-tri-O-
5-acetamido-
D
D-
/
D
/
D
/
D
H-8V), 6.03 (d, 1 H, J2,NH 8.2 Hz, NH), and 7.15ꢁ
8.14
/
/
D
(m, 45 H, 9 Ph). Anal. Calcd for C117H138N2O40Si
(2240.45): C, 62.72; H, 6.21; N, 1.25. Found: C, 62.69;
H, 5.92; N, 1.18.
(16). Removal of the benzyl groups and subsequent O-
acetylation of 13 (500 mg, 0.22 mmol), as described for
15, gave 16 (445 mg, quant) as an amorphous mass. [a]D
1
ꢂ
/
10.68 (c 0.6, CHCl3); H NMR (CDCl3): d 0.92 (m, 2
H, Me3SiCH2CH2), 1.55, 1.57, 1.77, 1.91, 2.01, 2.05,
2.06, 2.06, 2.07, 2.09, 2.09, 2.13, 2.13, and 2.15 (14 s, 42
H, 2 AcN, 10 AcO, and 2 CH3COCH2CH2), 1.61 (t, 1
3.1.12. 2-(Trimethylsilyl)ethyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
galacto-2-nonulopyranosylonate-(20
benzoyl-b- 3)-2-acetamido-4-O-
-galactopyranosyl-(10
acetyl-2-deoxy-6-O-levulinoyl-b- -galactopyranosyl-
(104)-2,3,6-tri-O-acetyl-b- -galactopyranosyl-(10
2,3,6-tri-O-acetyl-b- -glucopyranoside (15). A solution
of 12 (1.14 g, 0.51 mmol) in ethanol (30 mL) was
hydrogenated in the presence of 20% Pd(OH)2 (1.1 g) for
48 h at 40 8C. The catalyst was removed by filtration,
and the solution was concentrated. The residue was
treated with acetic anhydride (5 mL) and pyridine (10
mL) for 24 h at rt The mixture was concentrated, and a
solution of the residue in chloroform was successively
washed with 2 M HCl and M Na2CO3, dried (Na2SO4)
and concentrated. Column chromatography (40:1
methyl
-glycero-a-
3)-2,4,6-tri-O-
5-acetamido-
D
D-
/
H, Jgem
H-3Veq and 2 CH3COCH2CH2), 3.03 (m, 1 H, H-2III),
3.60 (dd, 1 H, J5,6 10.5, J6,7 2.7 Hz H-6V), 3.74 (t, 1 H,
ꢀ
/
J3ax,4 12.4 Hz, H-3Vax), 2.42ꢁ
2.80 (m, 9 H,
/
D
/
D
/
D
/
4)-
J3,4
ꢀ
J4,5 9.4 Hz H-4I), 3.81 (s, 3 H, MeO), 4.03 (dd, 1
/
D
H, Jgem 12.3, J8,9 5.5 Hz, H-9V), 4.09 (d, 1 H, J3,4 3.2 Hz
H-4II), 4.27 (dd, 1 H, Jgem 12.3, J8,9 2.3 Hz, H-9?V), 4.37
(d, 1 H, J1,2 7.8 Hz, H-1II), 4.45 (d, 1 H, J1,2 8.0 Hz, H-
1I), 4.80 (m, 1 H, H-4V), 4.84 (dd, 1 H, J2,3 10.1, J3,4 3.2
Hz, H-3IV), 4.85 (dd, 1 H, J2,3 10.2, J3,4 3.2 Hz, H-3II),
4.88 (dd, 1 H, J1,2 8.0, J2,3 9.4 Hz, H-2I), 4.98 (d, 1 H,
J1,2 7.6 Hz, H-1IV), 5.00 (m, 1 H, H-3III), 5.06 (dd, 1 H,
J1,2 7.8, J2,3 10.2 Hz, H-2II), 5.10 (d, 1 H, J1,2 8.2 Hz, H-
1III), 5.14 (t, 1 H, J2,3
ꢀ
J3,4 9.4 Hz, H-3I), 5.23 (dd, 1 H,
/
J6,7 2.7, J7,8 9.8 Hz, H-7V), 5.35 (d, 1 H, J3,4 3.2 Hz, H-
4IV), 5.38 (dd, 1 H, J1,2 7.6, J2,3 10.1 Hz, H-2IV), 5.47 (d,
1 H, J3,4 3.4 Hz, H-4III), 5.60 (m, 1 H, H-8V), 6.03 (d, 1
chloroformꢁmethanol) of the residue on silica gel gave
/
15 (850 mg, 83%) as an amorphous mass. [a]D ꢂ30.08 (c
/
0.2, CHCl3); 1H NMR (CDCl3): d 0.92 (m, 2 H,
Me3SiCH2CH2), 1.54, 1.55, 1.66, 1.66, 1.78, 1.91, 2.00,
2.01, 2.05, 2.07, 2.07, 2.09, 2.13, and 2.14 (14 s, 42 H, 2
AcN, 11 AcO, and CH3COCH2CH2), 2.45 (dd, 1 H,
H, J2,NH 6.6 Hz, NH), and 7.39ꢁ8.19 (m, 15 H, 3 Ph).
/
Anal. Calcd for C94H120N2O46Si (2042.05): C, 55.29; H,
5.92; N, 1.37. Found: C, 55.25; H, 5.64; N, 1.28.
Jgem 12.7, J3eq,4 4.5 Hz, H-3Veq), 2.48ꢁ
CH3COCH2CH2), 2.98 (m, 1 H, H-2III), 3.59 (dd, 1 H,
J5,6 10.7, J6,7 2.9 Hz, H-6V), 3.73 (t, 1 H, J3,4
J4,5 9.4
/2.72 (m, 4 H,
3.1.14. 2-(Trimethylsilyl)ethyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
galacto-2-nonulopyranosylonate-(20
benzoyl-b- 3)-2-acetamido-4-O-
-galactopyranosyl-(10
acetyl-2-deoxy-b- 4)-2,3,6-tri-O-
-galactopyranosyl-(10
acetyl-b- 4)-2,3,6-tri-O-acetyl-b-
-galactopyranosyl-(10
-glucopyranoside (17). To a solution of 15 (60 mg, 0.030
methyl
-glycero-a-
3)-2,4,6-tri-O-
5-acetamido-
ꢀ
/
D
D-
Hz, H-4I), 3.77 (m, 1 H, H-5V), 3.82 (s, 3 H, MeO), 4.01
(dd, 1 H, Jgem 12.8, J8,9 5.7 Hz, H-9V), 4.10 (d, 1 H, J3,4
2.7 Hz, H-4II), 4.27 (dd, 1 H, Jgem 12.8, J8,9 2.4 Hz, H-
9?V), 4.36 (d, 1 H, J1,2 7.8 Hz, H-1II), 4.45 (d, 1 H, J1,2
8.0 Hz, H-1I), 4.80 (m, 1 H, H-4V), 4.83 (dd, 1 H, J2,3
10.3, J3,4 2.7 Hz, H-3II), 4.84 (dd, 1 H, J2,3 9.8, J3,4 3.2
Hz, H-3IV), 4.89 (dd, 1 H, J1,2 8.0, J2,3 9.4 Hz, H-2I),
4.96 (d, 1 H, J5,NH 10.1 Hz, NH), 4.99 (d, 1 H, J1,2 7.6
Hz, H-1IV), 5.02 (dd, 1 H, J2,3 9.0, J3,4 3.7 Hz, H-3III),
5.04 (dd, 1 H, J1,2 7.8, J2,3 10.3 Hz, H-2II), 5.04 (d, 1 H,
/
D
/
D
/
D
/
D
mmol) in ethanol (3 mL) was added hydrazine mono-
acetate (20 mg, 0.15 mmol), and the mixture was stirred
for 45 min at rt, then concentrated. Column chromato-
graphy (30:1 chloroformꢁ/methanol) of the residue on
silica gel gave 17 (47 mg, 82%) as an amorphous mass.
1
J1,2 8.2 Hz, H-1III), 5.12 (t, 1 H, J2,3
ꢀ
/
J3,4 9.4 Hz, H-3I),
[a]D
(m, 2 H, Me3SiCH2CH2), 1.33, 1.54, 1.76, 1.89, 1.98,
ꢂ15.38 (c 1.9, CHCl3); H NMR (CDCl3): d 0.90
/
5.22 (dd, 1 H, J6,7 2.9, J7,8 9.7 Hz, H-7V), 5.34 (d, 1 H,
J3,4 3.2 Hz, H-4IV), 5.38 (dd, 1 H, J1,2 7.6, J2,3 9.8 Hz, H-
2.01, 2.03, 2.03, 2.03, 2.05, 2.08, 2.08, and 2.14 (13 s, 39

1630
H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ1639
/
H, 2 AcN and 11 AcO), 1.59 (t, 1 H, Jgem
ꢀ
/
J3ax,4 12.7
3.1.16. 2-(Trimethylsilyl)ethyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
galacto-2-nonulopyranosylonate-(20
benzoyl-b- 3)-2-acetamido-4-O-
-galactopyranosyl-(10
acetyl-2-deoxy-6-O-sulfo-b- -galactopyranosyl-(104)-
2,3,6-tri-O-acetyl-b- -galactopyranosyl-(104)-2,3,6-tri-
O-acetyl-b- -glucopyranoside pyridinium salt (19). To a
solution of 17 (47 mg, 0.025 mmol) in N,N-dimethyl-
formamide (DMF; 1 mL) was added sulfur trioxideꢁ
methyl
-glycero-a-
3)-2,4,6-tri-O-
5-acetamido-
Hz, H-3Vax), 2.43 (dd, 1 H, Jgem 12.7, J3eq,4 4.7 Hz, H-
3Veq), 3.05 (m, 1 H, H-2III), 3.58 (dd, 1 H, J5,6 10.8, J6,7
D
D-
/
2.7 Hz, H-6V), 3.71 (t, 1 H, J3,4
ꢀ
/
J4,5 9.5 Hz, H-4I), 3.74
D
/
(m, 1 H, H-5V), 3.79 (s, 3 H, MeO), 4.01 (d, 1 H, J3,4 2.6
Hz, H-4II), 4.03 (dd, 1 H, Jgem 12.5, J8,9 4.9 Hz, H-9V),
4.25 (dd, 1 H, Jgem 12.5, J8,9 2.2 Hz, H-9?V), 4.37 (d, 1 H,
J1,2 7.8 Hz, H-1II), 4.44 (d, 1 H, J1,2 8.0 Hz, H-1I), 4.77
(dd, 1 H, J2,3 10.4, J3,4 2.6 Hz, H-3II), 4.78 (m, 1 H, H-
4V), 4.84 (dd, 1 H, J2,3 10.1, J3,4 3.2 Hz, H-3IV), 4.87 (dd,
1 H, J1,2 8.0, J2,3 9.5 Hz, H-2I), 4.98 (dd, 1 H, J2,3 10.2,
J3,4 3.4 Hz, H-3III), 4.99 (d, 1 H, J1,2 7.8 Hz, H-1IV), 5.04
D
/
D
/
D
/
pyridine complex (20 mg, 0.13 mmol), and the mixture
was stirred for 3 h at rt Methanol (1 mL) was added, and
the mixture was concentrated. Column chromatography
(dd, 1 H, J1,2 7.8, J2,3
ꢀ
/
10.4 Hz, H-2II), 5.05 (d, 1 H,
J3,4 9.5 Hz, H-3I),
(1:1 chloroformꢁ
LH-20 gave the crude product, and this was purified by
column chromatography (8:1 chloroformꢁmethanol) on
silica gel gave 19 (51 mg, quant) as an amorphous mass.
[a]D
15.98 (c 0.9, CHCl3); 1H NMR (CDCl3ꢁ
/methanol) of the residue on Sephadex
J1,2 8.0 Hz, H-1III), 5.13 (t, 1 H, J2,3
ꢀ
/
5.22 (dd, 1 H, J6,7 2.7, J7,8 9.6 Hz, H-7V), 5.30 (d, 1 H,
J3,4 3.2 Hz, H-4IV), 5.39 (d, 1 H, J3,4 3.4 Hz, H-4III), 5.39
(dd, 1 H, J1,2 7.8, J2,3 10.1 Hz, H-2IV), 5.59 (m, 1 H, H-
8V), 6.11 (d, 1 H, J2,NH 6.6 Hz, NH), and 7.26ꢁ
/
ꢂ
/
/
/8.18 (m,
CD3OD): d 0.88 (m, 2 H, Me3SiCH2CH2), 1.52, 1.75,
1.90, 1.98, 1.99, 2.00, 2.01, 2.03, 2.04, 2.05, 2.07, 2.07,
and 2.12 (13 s, 39 H, 2 AcN and 11 AcO), 1.60 (t, 1 H,
15 H, 3 Ph). Anal. Calcd for C86H110N2O43Si (1887.88):
C, 54.71; H, 5.87; N, 1.48. Found: C, 54.56; H, 5.68; N,
1.42.
Jgem
3.15 (m, 1 H, H-2III), 3.59 (dd, 1 H, J5,6 10.7, J6,7 2.5 Hz,
H-6V), 3.72 (t, 1 H, J3,4 J4,5 9.4 Hz, H-4I), 3.75 (m, 1
ꢀ
/
J3ax,4 12.4 Hz, H-3Vax), 2.42 (m, 1 H, H-3Veq),
ꢀ
/
3.1.15. 2-(Trimethylsilyl)ethyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
galacto-2-nonulopyranosylonate-(20
benzoyl-b- 3)-2-acetamido-4-O-
-galactopyranosyl-(10
acetyl-2-deoxy-b- 4)-2,6-di-O-
-galactopyranosyl-(10
acetyl-b- 4)-2,3,6-tri-O-acetyl-b-
-galactopyranosyl-(10
-glucopyranoside (18). Selective removal of the levuli-
methyl
-glycero-a-
3)-2,4,6-tri-O-
5-acetamido-
H, H-5V), 3.78 (s, 3 H, MeO), 3.98 (m, 1 H, H-9V), 4.12
(m, 2 H, H-5III and H-6III), 4.16 (m, 1 H, H-4II), 4.32 (m,
1 H, H-9?V), 4.40 (d, 1 H, J1,2 7.8 Hz, H-1II), 4.46 (d, 1
H, J1,2 8.0 Hz, H-1I), 4.58 (m, 1 H, H-6?III), 4.80 (m, 1 H,
H-4V), 4.83 (m, 2 H, H-3II and H-3IV), 4.85 (m, 2 H, H-
3II and H-3IV), 4.88 (dd, 1 H, J1,2 8.0, J2,3 9.4 Hz, H-2I),
4.89 (d, 1 H, J1,2 8.0 Hz, H-1III), 4.90 (m, 1 H, H-3III),
D
D-
/
D
/
D
/
D
/
D
noyl group in 16 (100 mg, 0.048 mmol) with hydrazine
monoacetate (46 mg, 0.48 mmol) in ethanol (3 mL) and
workup as described for 17, gave 18 (73 mg, 81%) as an
4.98 (dd, 1 H, J1,2 7.8, J2,3
H, J1,2 8.2 Hz, H-1IV), 5.15 (t, 1 H, J2,3
ꢀ
/
10.5 Hz, H-2II), 5.01 (d, 1
J3,4 9.4 Hz, H-
ꢀ
/
3I), 5.20 (dd, 1 H, J6,7 2.5, J7,8 9.6 Hz, H-7V), 5.36 (d, 1
H, J3,4 3.2 Hz, H-4IV), 5.37 (m, 1 H, H-2IV), 5.44 (d, 1 H,
amorphous mass. [a]D
ꢂ
29.98 (c 1.5, CHCl3); 1H NMR
/
(CDCl3): d 0.90 (m, 2 H, Me3SiCH2CH2), 1.39, 1.57,
1.77, 1.90, 2.02, 2.03, 2.06, 2.06, 2.08, 2.08, 2.09, and
2.16 (12 s, 36 H, 2 AcN and 10 AcO), 1.59 (t, 1 H,
J3,4 2.7 Hz, H-4III), 5.56 (m, 1 H, H-8V), and 7.36ꢁ
8.19
/
(m, 19 H, 4 Ph). Anal. Calcd for C91H114N3O46SSi
(2046.04): C, 53.42; H, 5.62; N, 2.05. Found: C, 53.40;
H, 5.45; N, 1.96.
Jgem
J3eq,4 4.6 Hz, H-3Veq), 3.22 (m, 1 H, H-2III), 3.46 (m, 1
H, H-3II), 3.60 (dd, 1 H, J5,6 12.0, J6,7 3.0 Hz, H-6V),
ꢀ
J3ax,4 12.6 Hz, H-3Vax), 2.44 (dd, 1 H, Jgem 12.6,
/
3.1.17. 2-(Trimethylsilyl)ethyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
galacto-2-nonulopyranosylonate-(20
benzoyl-b- 3)-2-acetamido-4-O-
-galactopyranosyl-(10
acetyl-2-deoxy-6-O-sulfo-b- -galactopyranosyl-(104)-
2,6-di-O-acetyl-3-O-sulfo-b- -galactopyranosyl-(104)-
2,3,6-tri-O-acetyl-b- -glucopyranoside dipyridinium salt
methyl
-glycero-a-
3)-2,4,6-tri-O-
5-acetamido-
3.72 (t, 1 H, J3,4
ꢀ
/
J4,5 9.4 Hz, H-4I), 3.77 (m, 1 H, H-
D
D-
5V), 3.81 (s, 3 H, MeO), 4.00 (m, 1 H, H-4II), 4.01 (m, 1
H, H-9V), 4.28 (m, 1 H, H-9?V), 4.29 (d, 1 H, J1,2 7.8 Hz,
H-1II), 4.38 (m, 1 H, H-3III), 4.44 (d, 1 H, J1,2 8.0 Hz, H-
/
D
/
D
/
1I), 4.70 (dd, 1 H, J1,2 7.8, J2,3
ꢀ
/
8.9 Hz, H-2II), 4.78 (m,
D
/
1 H, H-4V), 4.86 (dd, 1 H, J1,2 8.0, J2,3 9.4 Hz, H-2I),
4.87 (dd, 1 H, J2,3 9.8, J3,4 3.2 Hz, H-3IV), 4.98 (d, 1 H,
J1,2 7.6 Hz, H-1IV), 5.02 (d, 1 H, J5,NH 10.0 Hz, NH),
D
(20). To a solution of 18 (70 mg, 0.038 mmol) in DMF (2
mL) was added sulfur trioxide-pyridine complex (98 mg,
0.60 mmol), and the mixture was stirred for 48 h at
5.13 (t, 1 H, J2,3
ꢀ
J3,4 9.4 Hz, H-3I), 5.30 (dd, 1 H, J6,7
/
3.0, J7,8 9.8 Hz, H-7V), 5.35 (d, 1 H, J3,4 3.2 Hz, H-4IV),
5.41 (d, 1 H, J3,4 2.3 Hz, H-4III), 5.43 (dd, 1 H, J1,2 7.6,
J2,3 9.8 Hz, H-2IV), 5.54 (d, 1 H, J1,2 8.2 Hz, H-1III), 5.62
(m, 1 H, H-8V), 5.98 (d, 1 H, J2,NH 6.4 Hz, NH III), and
40 8C workup as described for 19 gave 20 (48 mg, 59%)
1
as an amorphous mass. [a]D
ꢂ
/
19.98 (c 1.0, CHCl3); H
NMR (CDCl3ꢁ
/
CD3OD): d 0.90 (m, 2 H, Me3-
SiCH2CH2), 1.38, 1.54, 1.72, 1.86, 1.99, 2.03, 2.04,
7.40ꢁ
/
8.19 (m, 15 H,
3
Ph). Anal. Calcd for
2.04, 2.08, 2.09, 2.10, and 2.14 (12 s, 36 H, 2 AcN and
ꢀ
J3ax,4 12.6 Hz, H-3Vax),
C84H108N2O42Si (1845.85): C, 54.66; H, 5.90; N, 1.52.
Found: C, 54.61; H, 5.84; N, 1.33.
10 AcO), 1.41 (t, 1 H, Jgem
2.41 (dd, 1 H, Jgem 12.6, J3ax,4 4.6 Hz, H-3Veq), 3.70 (dd,
/

H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ
/
1639
1631
1 H, J5,6 10.1, J6,7 2.7 Hz, H-6V), 3.78 (t, 1 H, J3,4
ꢀ
/
J4,5
C, 38.07; H, 5.40; N, 2.11. Found: C, 37.96; H, 5.30; N,
1.89.
9.4 Hz, H-4I), 3.80 (s, 3 H, MeO), 4.08 (m, 1 H, H-3III),
4.12 (m, 1 H, H-2III), 4.18 (dd, 1 H, Jgem 11.0, J5,6 8.0
Hz, H-6III), 4.25 (m, 1 H, H-9V), 4.28 (m, 1 H, H-4II),
4.31 (m, 1 H, H-5III), 4.38 (dd, 1 H, J2,3 10.1, J3,4 3.0 Hz,
H-3II), 4.49 (d, 1 H, J1,2 8.0 Hz, H-1II), 4.52 (dd, 1 H,
Jgem 11.0, J5,6 6.0 Hz, H-6?III), 4.56 (d, 1 H, J1,2 8.0 Hz,
H-1I), 4.66 (d, 1 H, J1,2 8.0 Hz, H-1III), 4.77 (dd, 1 H,
J1,2 8.0, J2,3 9.2 Hz, H-2I), 4.78 (m, 1 H, H-4V), 4.80 (dd,
3.1.20. 2-(Trimethylsilyl)ethyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
galacto-2-nonulopyranosylonate-(20
benzoyl-b- 4)-2-acetamido-3-O-
-galactopyranosyl-(10
acetyl-2-deoxy-6-O-levulinoyl-b- -galactopyranosyl-
(104)-2,6-di-O-acetyl-3-O-levulinoyl-b- -galacto-
pyranosyl-(104)-2,3,6-tri-O-acetyl-b- -glucopyranoside
methyl
-glycero-a-
3)-2,4,6-tri-O-
5-acetamido-
D
D-
/
D
/
D
/
D
1 H, J1,2 8.0, J2,3
ꢀ
/
10.1 Hz, H-2II), 4.94 (dd, 1 H, J2,3
J3,4 9.2 Hz,
/
D
9.9, J3,4 3.0 Hz, H-3IV), 5.07 (t, 1 H, J2,3
ꢀ
/
(23). Removal of the benzyl groups in 14 (350 mg, 0.16
mmol) and subsequent O-acetylation, as described for
15, gave 23 (290 mg, 91%) as an amorphous mass.
H-3I), 5.13 (d, 1 H, J1,2 7.8 Hz, H-1IV), 5.21 (dd, 1 H,
J6,7 2.7, J7,8 9.8 Hz, H-7V), 5.35 (dd, 1 H, J1,2 7.8, J2,3 9.0
Hz, H-2IV), 5.44 (d, 1 H, J3,4 3.0 Hz, H-4IV), 5.59 (d, 1
H, J3,4 2.7 Hz, H-4III), 5.64 (m, 1 H, H-8V), and 7.47ꢁ
[a]Dꢂ
7.68 (c 1.4, CHCl3); 1H NMR (CDCl3): d 0.91 (m,
/
/
2 H, Me3SiCH2CH2), 1.56, 1.78, 1.80, 1.94, 2.05, 2.05,
2.06, 2.09, 2.11, 2.12, 2.14, 2.14, 2.16, and 2.21 (14 s, 42
H, 2 AcN, 10 AcO, and 2 CH3COCH2CH2), 1.72 (t, 1
8.19 (m, 23 H, 5 Ph). Anal. Calcd for C94H116N4O48S2Si
(2162.16): C, 52.22; H, 5.41; N, 2.59. Found: C, 52.18;
H, 5.21; N, 2.39.
H, Jgem
ꢀ
/
J3ax,4 12.4 Hz, H-3Vax), 2.42ꢁ
2.70 (m, 9 H,
/
H-3Veq and 2 CH3COCH2CH2), 2.77 (m, 1 H, H-2III),
3.64 (m, 1 H, H-6V), 3.75 (m, 1 H, H-4I), 3.77 (m, 1 H,
H-5V), 3.86 (s, 3 H, MeO), 4.06 (m, 1 H, H-4II), 4.07 (dd,
1 H, Jgem 11.2, J8,9 5.5 Hz, H-9V), 4.25 (dd, 1 H, Jgem
11.2, J8,9 6.9 Hz, H-9?V), 4.36 (d, 1 H, J1,2 7.8 Hz, H-1II),
4.48 (d, 1 H, J1,2 8.0 Hz, H-1I), 4.83 (dd, 1 H, J2,3 9.8,
J3,4 2.8 Hz, H-3II), 4.86 (d, 1 H, J1,2 8.2 Hz, H-1IV), 4.89
(m, 1 H, H-3III), 4.91 (m, 1 H, H-4V), 4.95 (dd, 1 H, J1,2
8.0, J2,3 9.4 Hz, H-2I), 4.96 (dd, 1 H, J2,3 9.7, J3,4 1.8 Hz,
H-3IV), 4.98 (d, 1 H, J1,2 8.0 Hz, H-1III), 5.04 (m, 1 H,
H-4III), 5.08 (dd, 1 H, J1,2 7.8, J2,3 9.8 Hz, H-2II), 5.21 (t,
3.1.18. 2-(Trimethylsilyl)ethyl 5-acetamido-3,5-dideoxy-
D
-glycero-a-
3)-b-
-galactopyranosyl-(10
O-sulfo-b- -galactopyranosyl-(10
galactopyranosyl-(104)-b- -glucopyranoside disodium
D
-galacto-2-nonulopyranosylonic acid-(20
3)-2-acetamido-2-deoxy-6-
4)-b-
/
D
/
D
/
D-
/
D
salt (21). To a solution 19 (88 mg, 0.044 mmol) in
methanol was added sodium methoxide (10 mg). The
mixture was stirred for 48 h at rt, and water (0.5 mL)
was then added. After completion of the reaction (24 h),
the mixture was concentrated. Column chromatography
(methanol) of the residue on Sephadex LH-20 gave 21
1 H, J2,3
10.1 Hz, H-7V), 5.39 (d, 1 H, J3,4 1.6 Hz, H-4IV), 5.41
(dd, 1 H, J1,2 8.2, J2,3 9.7 Hz, H-2IV), 5.66 (m, 1 H, H-
ꢀ
J3,4 9.4 Hz, H-3I), 5.30 (dd, 1 H, J6,7 1.8, J7,8
/
(54 mg, quant) as an amorphous solid: [a]D
0.6 CH3OH); 1H NMR (CD3OD): d 1.00 (m, 2 H,
Me3SiCH2CH2), 1.76 (t, 1 H, Jgem J3ax,4 12.4 Hz, H-
ꢃ
/
13.68 (c
ꢀ
/
8V), and 7.40ꢁ
8.31 (m, 15 H, 3 Ph). Anal. Calcd for
C94H120N2O46Si (2042.05): C, 55.29; H, 5.92; N, 1.37.
Found: C, 55.04; H, 5.72; N, 1.34.
/
3Vax), 2.00 and 2.01 (2 s, 6 H, 2 AcN), 2.72 (dd, 1 H,
Jgem 12.4, J3eq,4 4.6 Hz, H-3Veq), 4.40 (d, 1 H, J1,2 7.8
Hz, H-1I), 4.43 (d, 1 H, J1,2 8.2 Hz, H-1II), 4.48 (d, 1 H,
J1,2 7.8 Hz, H-1IV), and 4.65 (d, 1 H, J1,2 8.5 Hz, H-1III).
Anal. Calcd for C42H72N2Na2O32SSi (1223.15): C,
41.24; H, 5.93; N, 2.29. Found: C, 41.11; H, 5.89; N,
2.01.
3.1.21. 2-(Trimethylsilyl)ethyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
galacto-2-nonulopyranosylonate-(20
benzoyl-b- 4)-2-acetamido-3-O-
-galactopyranosyl-(10
acetyl-2-deoxy-b- 4)-2,6-di-O-
-galactopyranosyl-(10
acetyl-b- 4)-2,3,6-tri-O-acetyl-b-
-galactopyranosyl-(10
-glucopyranoside (24). Selective removal of the levuli-
methyl
-glycero-a-
3)-2,4,6-tri-O-
5-acetamido-
D
D-
/
D
/
D
/
3.1.19. 2-(Trimethylsilyl)ethyl 5-acetamido-3,5-dideoxy-
D
/
D
-glycero-a-
3)-b-
-galactopyranosyl-(10
O-sulfo-b- -galactopyranosyl-(10
galactopyranosyl-(104)-b- -glucopyranoside trisodium
D
-galacto-2-nonulopyranosylonic acid-(20
3)-2-acetamido-2-deoxy-6-
4)-3-O-sulfo-b-
/
D
D
/
noyl group in 23 (70 mg, 0.034 mmol) with hydrazine
monoacetate (33 mg, 0.34 mmol) in ethanol (3 mL) and
D
/
D-
/
D
workup as described for 19 gave 24 (36 mg, 58%) as an
1
salt (22). To a solution 20 (47 mg, 0.022 mmol) in
amorphous mass. [a]Dꢂ
/
18.08 (c 0.7, CHCl3); H NMR
methanol was added sodium methoxide (10 mg).
Workup as described for 21 gave 22 (28 mg, quant) as
(CDCl3): d 0.90 (m, 2 H, Me3SiCH2CH2), 1.53, 1.78,
1.80, 1.93, 2.05, 2.06, 2.07, 2.11, 2.12, 2.18, 2.20, and
2.21 (12 s, 36 H, 2 AcN and 10 AcO), 1.72 (t, 1 H,
1
an amorphous solid: [a]D
NMR (CD3OD): d 1.02 (m, 2 H, Me3SiCH2CH2), 1.90
ꢃ
/
2.88 (c 0.6 CH3OH); H
Jgem
2.78 (m, 1 H, H-2III), 3.48 (m, 1 H, H-3II), 3.65 (dd, 1 H,
ꢀ
J3ax,4 12.4 Hz, H-3Vax), 2.46 (m, 1 H, H-3Veq),
/
(t, 1 H, Jgem
ꢀ
/
J3ax,4 12.8 Hz, H-3Vax), 2.01 and 2.06 (2
s, 6 H, 2 AcN), 2.78 (dd, 1 H, Jgem 12.8, J3eq,4 4.1 Hz, H-
3Veq), 4.30 (d, 1 H, J1,2 7.8 Hz, H-1I), 4.44 (d, 2 H, J1,2
7.8 Hz, H-1II and H-1IV), and 4.70 (d, 1 H, J1,2 8.7 Hz,
H-1III). Anal. Calcd for C42H71N2Na3O35S2Si (1325.20):
J5,6 10.5, J6,7 2.5 Hz, H-6V), 3.73 (t, 1 H, J3,4
ꢀJ4,5 9.4
/
Hz, H-4I), 3.84 (m, 1 H, H-5V), 3.88 (s, 3 H, MeO), 3.97
(m, 1 H, H-4II), 4.07 (dd, 1 H, Jgem 12.4, J8,9 3.0 Hz, H-
9V), 4.14 (dd, 1 H, Jgem 12.4, J8,9 3.3 Hz, H-9?V), 4.19 (d,

1632
H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ1639
/
1 H, J3,4 2.3 Hz, H-4III), 4.27 (d, 1 H, J1,2 7.8 Hz, H-1II),
4.47 (d, 1 H, J1,2 8.0 Hz, H-1I), 4.64 (d, 1 H, J2,NH 6.4
Hz, NH), 4.70 (dd, 1 H, J1,2 7.8, J2,3 8.3 Hz, H-2II), 4.87
(m, 1 H, H-4V), 4.88 (d, 1 H, J1,2 7.8 Hz, H-1IV), 4.90
(dd, 1 H, J2,3 10.1, J3,4 3.4 Hz, H-3IV), 4.93 (dd, 1 H, J1,2
8.0, J2,3 9.4 Hz, H-2I), 5.05 (d, 1 H, J5,NH 10.0 Hz, NH),
s, 6 H, 2 AcN), 2.78 (dd, 1 H, Jgem 12.8, J3eq,4 4.1 Hz, H-
3Veq), 4.35 (d, 1 H, J1,2 7.8 Hz, H-1I), 4.42 (m, 2 H, H-
1II and H-1IV), and 4.68 (d, 1 H, J1,2 8.7 Hz, H-1III).
Anal. Calcd for C42H71N2Na3O35S2Si (1325.20): C,
38.07; H, 5.40; N, 2.11. Found: C, 38.03; H, 5.38; N,
2.01.
5.20 (t, 1 H, J2,3
ꢀ
J3,4 9.4 Hz, H-3I), 5.30 (dd, 1 H, J6,7
/
2.5, J7,8 9.8 Hz, H-7V), 5.36 (d, 1 H, J3,4 3.4 Hz, H-4IV),
5.37 (dd, 1 H, J2,3 10.0, J3,4 2.3 Hz, H-3III), 5.42 (d, 1 H,
J1,2 7.8 Hz, H-1III), 5.48 (dd, 1 H, J1,2 7.8, J2,3 10.1 Hz,
3.1.24. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-4-O-acetyl-2-deoxy-6-O-levulinoyl-b-
galactopyranosyl-(104)-2,3,6-tri-O-acetyl-b-
galactopyranosyl-(104)-2,3,6-tri-O-acetyl-
D
-glycero-a-
D
-galacto-2-nonulopyranosylonate-
/
D
-galactopyranosyl-(103)-
/
H-2IV), 5.66 (m, 1 H, H-8V), and 7.29ꢁ
/8.35 (m, 15 H, 3
D-
Ph). Anal. Calcd for C84H108N2O42Si (1845.85): C,
54.66; H, 5.90; N, 1.52. Found: C, 54.39; H, 5.85; N,
1.24.
/
D-
/
D-
glucopyranose (27). To a solution of 15 (220 mg, 0.11
mmol) in dichloromethane (4 mL) was added trifluor-
oacetic acid (1.5 mL) at 0 8C. The mixture was stirred
for 3 h at rt and then concentrated. Column chromato-
3.1.22. 2-(Trimethylsilyl)ethyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
galacto-2-nonulopyranosylonate-(20
benzoyl-b- 4)-2-acetamido-3-O-
-galactopyranosyl-(10
acetyl-2-deoxy-6-O-sulfo-b- -galactopyranosyl-(104)-
2,6-di-O-acetyl-3-O-sulfo-b- -galactopyranosyl-(104)-
2,3,6-tri-O-acetyl-b- -glucopyranoside dipyridinium salt
(25). To a solution of 24 (36 mg, 0.019 mmol) in DMF (1
mL) was added sulfur trioxideꢁpyridine complex (25
methyl
-glycero-a-
3)-2,4,6-tri-O-
5-acetamido-
D
D-
/
graphy (20:1 chloroformꢁmethanol) of the residue on
/
D
/
silica gel gave 27 (208 mg, quant) as an amorphous
mass. Anal. Calcd for C86H104N2O45 (1885.75): C,
54.78; H, 5.56; N, 1.49. Found: C, 54.48; H, 5.34; N,
1.33.
D
/
D
/
D
/
3.1.25. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
mg, 0.15 mmol), and the mixture was stirred for 3 h at rt
Workup as described for 19 gave 25 (42 mg, quant) as an
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-4-O-acetyl-2-deoxy-6-O-levulinoyl-b-
galactopyranosyl-(104)-2,6-di-O-acetyl-3-O-levulinoyl-
b- 4)-2,3,6-tri-O-acetyl-
-galactopyranosyl-(10
D
-glycero-a-
D
-galacto-2-nonulopyranosylonate-
/
D
-galactopyranosyl-(103)-
/
1
amorphous mass. [a]Dꢂ
(CDCl3ꢁ
(t, 1 H, Jgem
/
13.98 (c 0.1, CHCl3); H NMR
D-
/
CD3OD): d 0.93 (m, 2 H, Me3SiCH2CH2), 1.60
/
ꢀ
/
J3ax,4 12.6 Hz, H-3Vax), 1.67, 1.77, 1.77,
D
/
D-
1.91, 1.03, 2.04, 2.05, 2.09, 2.11, 2.12, 2.12, and 2.17 (12
s, 36 H, 2 AcN and 10 AcO), 2.50 (dd, 1 H, Jgem 12.6,
J3ax,4 4.8 Hz, H-3Veq), 3.82 (m, 1 H, H-5V), 3.82 (s, 3 H,
MeO), 4.11 (dd, 1 H, Jgem 11.6, J5,6 5.5 Hz, H-9V), 4.19
(m, 1 H, H-9V), 4.21 (m, 1 H, H-4II), 4.30 (d, 1 H, J3,4 2.5
Hz, H-4III), 4.49 (d, 1 H, J1,2 7.8 Hz, H-1II), 4.56 (d, 1 H,
J1,2 7.8 Hz, H-1I), 4.86 (dd, 1 H, J1,2 7.8, J2,3 9.6 Hz, H-
glucopyranose (28). Selective removal of the 2-(tri-
methylsilyl)ethyl group of 16 (400 mg, 0.18 mmol)
with trifluoroacetic acid (1.5 mL) in dichloromethane
(3 mL), then workup as described for 27, gave 28 (360
mg, 95%) as an amorphous mass. Anal. Calcd for
C89H108N2O46 (1941.81): C, 55.05; H, 5.61; N, 1.44.
Found: C, 54.86; H, 5.59; N, 1.29
2I), 4.91 (dd, 1 H, J1,2 7.8, J2,3
ꢀ
/
10.1 Hz, H-2II), 4.96
(dd, 1 H, J2,3 10.1, J3,4 3.2 Hz, H-3IV), 4.99 (d, 1 H, J1,2
3.1.26. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
7.8 Hz, H-1IV), 5.13 (t, 1 H, J2,3
ꢀ
/
J3,4 9.6 Hz, H-3I), 5.30
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-3-O-acetyl-2-deoxy-6-O-levulinoyl-b-
galactopyranosyl-(104)-2,6-di-O-acetyl-3-O-levulinoyl-
b- 4)-2,3,6-tri-O-acetyl-
-galactopyranosyl-(10
D-glycero-a-
D-galacto-2-nonulopyranosylonate-
(dd, 1 H, J6,7 2.8, J7,8 9.8 Hz, H-7V), 5.43 (dd, 1 H, J1,2
7.8, J2,3 10.1 Hz, H-2IV), 5.46 (d, 1 H, J3,4 3.2 Hz, H-
/
D
-galactopyranosyl-(104)-
/
D-
4IV), 5.61 (m, 1 H, H-8V), and 7.43ꢁ
/
8.28 (m, 23 H, 5 Ph).
/
Anal. Calcd for C94H116N4O48S2Si (2162.16): C, 52.22;
H, 5.41; N, 2.59. Found: C, 51.99; H, 5.25; N, 2.59.
D
/
D-
glucopyranose (38). Selective removal of the 2-(tri-
methylsilyl)ethyl group of 23 (220 mg, 0.10 mmol)
with trifluoroacetic acid (1.5 mL) in dichloromethane
(3 mL), then workup as described for 27, gave 38 (206
mg, 99%) as an amorphous mass. Anal. Calcd for
C89H108N2O46 (1941.81): C, 55.05; H, 5.61; N, 1.44.
Found: C, 54.94; H, 5.58; N, 1.32
3.1.23. 2-(Trimethylsilyl)ethyl 5-acetamido-3,5-dideoxy-
D
-glycero-a-
3)-b-
-galactopyranosyl-(10
O-sulfo-b- -galactopyranosyl-(10
galactopyranosyl-(104)-b- -glucopyranoside trisodium
D
-galacto-2-nonulopyranosylonic acid-(20
4)-2-acetamido-2-deoxy-6-
4)-3-O-sulfo-b-
/
D
/
D
/
D-
/
D
salt (26). To a solution 25 (30 mg, 0.015 mmol) in
methanol was added sodium methoxide (10 mg).
Workup as described for 21 gave 26 (19 mg, quant) as
3.1.27. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-4-O-acetyl-2-deoxy-6-O-levulinoyl-b-
galactopyranosyl-(104)-2,3,6-tri-O-acetyl-b-
D-glycero-a-
D-galacto-2-nonulopyranosylonate-
1
an amorphous solid: [a]D
NMR (CD3OD): d 1.00 (m, 2 H, Me3SiCH2CH2), 1.85
ꢃ
/
14.88 (c 0.4 CH3OH); H
/
D-galactopyranosyl-(103)-
/
D-
(t, 1 H, Jgem
ꢀ
/
J3ax,4 12.8 Hz, H-3Vax), 2.01 and 2.07 (2
/
D-

H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ
/
1639
1633
galactopyranosyl-(10
/
4)-2,3,6-tri-O-acetyl-a-
D
-
7.27ꢁ
/
8.18 (m, 15 H, 3 Ph), and 8.66 (s, 1 H, Cꢀ
/
NH).
glucopyranosyl trichloroacetimidate (29). To a solution
of 27 (208 mg, 0.11 mmol) in dichloromethane (2 mL)
and trichloroacetonitrile (325 mL, 2.2 mmol) was added
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 20 mL, 0.13
mmol) at 0 8C, and the mixture was stirred for 30 min at
0 8C, then concentrated. Column chromatography (20:1
Anal. Calcd for C91H108Cl3N3O46 (2086.20): C, 52.39;
H, 5.22; N, 2.01. Found: C, 52.20; H, 5.15; N, 1.81.
3.1.29. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-4-O-acetyl-2-deoxy-6-O-levulinoyl-b-D-
D-glycero-a-
D-galacto-2-nonulopyranosylonate-
chloroformꢁ/methanol) of the residue on silica gel gave
/
D-galactopyranosyl-(103)-
/
29 (211 mg, 94%) as an amorphous mass. [a]D ꢂ
/
33.68 (c
0.3, CHCl3); 1H NMR (CDCl3): d 1.55, 1.56, 1.71, 1.77,
1.91, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.10, and
2.14 (14 s, 42 H, 2 AcN, 11 AcO, and CH3COCH2CH2),
galactopyranosyl-(10
galactopyranosyl-(10
glucopyranosyl-(10
octadecanamido-4-octadecene-1,3-diol (32). To a solution
of 29 (300 mg, 0.15 mmol) and (2S,3R,4E)-3-O-
benzoyl-2-octadecanamido-4-octadecene-1,3-diol (31;
198 mg, 0.30 mmol) in dichloromethane (4 mL) were
/
4)-2,3,6-tri-O-acetyl-b-
D
-
-
/
4)-2,3,6-tri-O-acetyl-b-
D
/
1)-(2S,3R,4E)-3-O-benzoyl-2-
1.61 (t, 1 H, Jgem
ꢀ
/
J3ax,4 12.8 Hz, H-3Vax), 2.44 (dd, 1
2.73 (m, 4 H,
H, Jgem 12.8, J3eq,4 4.8 Hz, H-3Veq), 2.48ꢁ
/
CH3COCH2CH2), 3.00 (m, 1 H, H-2III), 3.82 (s, 3 H,
MeO), 4.38 (d, 1 H, J1,2 7.7 Hz, H-1II), 4.78 (m, 1 H, H-
4V), 4.95 (d, 1 H, J5,NH 10.3 Hz, NH), 4.99 (d, 1 H, J1,2
7.7 Hz, H-1IV), 5.22 (dd, 1 H, J6,7 2.7, J7,8 10.6 Hz, H-
7V), 5.35 (d, 1 H, J3,4 2.9 Hz, H-4IV), 5.38 (dd, 1 H, J1,2
7.7, J2,3 9.9 Hz, H-2IV), 5.45 (d, 1 H, J3,4 2.2 Hz, H-4III),
5.61 (m, 1 H, H-8V), 6.22 (d, 1 H, J2,NH 6.6 Hz, Hz NH),
˚
added 4A molecular sieves (AW-300; 1.5 g), and the
mixture was stirred for 12 h at rt, then cooled to 0 8C.
TMSOTf (6 mL, 0.030 mmol) was added, and the
mixture was stirred for 30 h at 10 8C, then filtered.
The insoluble materials were washed with chloroform,
and the combined filtrate and washings was washed with
M NaHCO3 and water, dried (Na2SO4) and concen-
6.49 (d, 1 H, J1,2 3.7 Hz, H-1I), 7.27ꢁ
Ph), and 8.66 (s, 1 H, CꢀNH). Anal. Calcd for
C88H104Cl3N3O45 (2030.14): C, 52.06; H, 5.16; N, 2.07.
/8.19 (m, 15 H, 3
/
trated. Column chromatography (25:1 chloroformꢁ
methanol) of the residue on silica gel gave 32 (140 mg,
37%) as an amorphous mass. [a]D ꢂ18.98 (c 0.4,
/
Found: C, 52.04; H, 4.96; N, 2.05.
/
3.1.28. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
CHCl3); 1H NMR (CDCl3): d 0.88 (t, 6 H, JMe,CH2
6.7 Hz, 2 MeCH2), 1.26 (s, 52 H, 26 CH2), 1.53, 1.54,
1.77, 1.91, 1.92, 1.97, 2.01, 2.04, 2.05, 2.06, 2.08, 2.09,
2.13, and 2.14 (14 s, 42 H, 2 AcN, 11 AcO, and
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-4-O-acetyl-2-deoxy-6-O-levulinoyl-b-
galactopyranosyl-(104)-2,6-di-O-acetyl-3-O-levulinoyl-
b- 4)-2,3,6-tri-O-acetyl-a-
-galactopyranosyl-(10
D
-glycero-a-
D
-galacto-2-nonulopyranosylonate-
/
D-galactopyranosyl-(103)-
/
D-
/
CH3COCH2CH2), 1.61 (dd, 1 H, Jgem
ꢀ
/
J3ax,4 12.6 Hz,
D
/
D-
H-3Vax), 2.44 (dd, 1 H, Jgem 12.6, J3eq,4 4.6 Hz, H-
glucopyranosyl trichloroacetimidate (30). Treatment of
28 (120 mg, 0.064 mmol) with trichloroacetonitrile (273
mL, 1.92 mmol) and DBU (11 mL, 0.077 mmol) in
dichloromethane (2 mL), then workup as described for
29 gave 30 (130 mg, quant) as an amorphous mass. H
NMR (CDCl3): d 1.55, 1.56, 1.63, 1.78, 1.91, 2.03, 2.04,
2.06, 2.07, 2.09, 2.09, 2.13, 2.13, and 2.16 (14 s, 42 H, 2
3Veq), 2.48ꢁ
2.72 (m, 4 H, CH3COCH2CH2), 2.98 (m, 1
/
H, H-2III), 3.59 (dd, 1 H, J5,6 10.7, J6,7 2.7 Hz, H-6V),
3.61 (dd, 1 H, Jgem 10.3, J1,2 4.6 Hz, Cer-1), 3.77 (t, 1 H,
J3,4
ꢀ
J4,5 9.4 Hz, H-4I), 3.77 (m, 1 H, H-5V), 3.82 (s, 3
/
1
H, MeO), 3.99 (dd, 1 H, Jgem 10.3, J1,2 3.3 Hz, Cer-1?),
4.02 (dd, 1 H, Jgem 12.4, J8,9 5.8 Hz, H-9V), 4.10 (d, 1 H,
J3,4 2.5 Hz, H-4II), 4.12 (m, 1 H, H-5III), 4.20 (dd, 1 H,
Jgem 11.1, J5,6 4.0 Hz, H-6III), 4.27 (dd, 1 H, Jgem 12.4,
J8,9 2.3 Hz, H-9?V), 4.33 (d, 1 H, J1,2 7.8 Hz, H-1II), 4.41
(d, 1 H, J1,2 7.8 Hz, H-1I), 4.46 (m, 1 H, Cer-2), 4.48 (dd,
1 H, Jgem 11.1, J5,6 5.2 Hz, H-6?III), 4.80 (m, 1 H, H-4V),
4.81 (dd, 1 H, J2,3 10.3, J3,4 2.5 Hz, H-3II), 4.84 (dd, 1 H,
J2,3 10.1, J3,4 3.2 Hz, H-3IV),4.89 (dd, 1 H, J1,2 7.8, J2,3
9.4 Hz, H-2I), 4.93 (d, 1 H, J5,NH 8.5 Hz, NH), 4.98 (d, 1
H, J1,2 7.6 Hz, H-1IV), 5.04 (dd, 1 H, J2,3 10.5, J3,4 4.2
Hz, H-3III), 5.00 (dd, 1 H, J1,2 7.8, J2,3
5.04 (d, 1 H, J1,2 8.2 Hz, H-1III), 5.12 (t, 1 H, J2,3
9.4 Hz, H-3I), 5.21 (dd, 1 H, J6,7 2.7, J7,8 9.6 Hz, H-7V),
5.34 (d, 1 H, J3,4 3.2 Hz, H-4IV), 5.38 (dd, 1 H, J1,2 7.6,
J2,3 10.1 Hz, H-2IV), 5.45 (m, 1 H, Cer-4), 5.46 (d, 1 H,
J3,4 4.2 Hz, H-4III), 5.53 (m, 1 H, Cer-3), 5.60 (m, 1 H,
H-8V), 5.74 (m, 1 H, J2,NH 9.2 Hz, Cer-NH), 5.87 (m, 1
AcN, 10 AcO, and 2 CH3COCH2CH2), 2.42ꢁ2.79 (m, 9
/
H, H-3Veq and 2 CH3COCH2CH2), 3.00 (m, 1 H, H-
2III), 3.59 (dd, 1 H, J5,6 10.8, J6,7 2.7 Hz H-6V), 3.82 (s, 3
H, MeO), 4.03 (dd, 1 H, Jgem 12.4, J8,9 5.5 Hz, H-9V),
4.09 (d, 1 H, J3,4 2.7 Hz H-4II), 4.27 (dd, 1 H, Jgem 12.4,
J8,9 2.3 Hz, H-9?V), 4.37 (d, 1 H, J1,2 7.8 Hz, H-1II), 4.80
(m, 1 H, H-4V), 4.83 (dd, 1 H, J2,3 10.1, J3,4 3.2 Hz, H-
3IV), 4.84 (dd, 1 H, J2,3 10.3, J3,4 2.7 Hz, H-3II), 4.96 (d,
1 H, J5,NH 10.1 Hz, NH), 4.98 (d, 1 H, J1,2 7.6 Hz, H-
1IV), 5.01 (dd, 1 H, J2,3 10.8, J3,4 3.4 Hz, H-3IV), 5.05
(dd, 1 H, J1,2 3.7, J2,3 10.3 Hz, H-2I), 5.07 (dd, 1 H, J1,2
7.8, J2,3 10.3 Hz, H-2II), 5.09 (d, 1 H, J1,2 8.5 Hz, H-1III),
5.22 (dd, 1 H, J6,7 2.7, J7,8 9.8 Hz, H-7V), 5.35 (d, 1 H,
J3,4 3.4 Hz, H-4IV), 5.38 (dd, 1 H, J1,2 7.8, J2,3 10.1 Hz,
H-2IV), 5.47 (d, 1 H, J3,4 3.4 Hz, H-4III), 5.48 (t, 1 H,
ꢀ
/
10.3 Hz, H-2II),
J3,4
ꢀ
/
J2,3
ꢀ
/
J3,4 10.3 Hz, H-3I), 5.59 (m, 1 H, H-8V), 6.35 (d, 1
H, Cer-5), 5.97 (d, 1 H, J2,NH 6.6 Hz, NH), and 7.38ꢁ
8.19 (m, 20 H, 4 Ph). Anal. Calcd for C129H177N3O48
/
H, J2,NH 6.6 Hz, NH), 6.49 (d, 1 H, J1,2 3.7 Hz, H-1I),

1634
H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ1639
/
(2537.81): C, 61.05; H, 7.03; N, 1.16. Found: C, 60.76;
H, 6.94; N, 1.61.
workup as described for 17, gave 34 (116 mg, 87%) as an
amorphous mass. [a]D
15.78 (c 1.3, CHCl3); 1H NMR
ꢂ
/
(CDCl3): d 0.88 (t, 6 H, JMe,CH2 6.8 Hz, 2 MeCH2), 1.26
(s, 52 H, 26 CH2), 1.31, 1.55, 1.77, 1.91, 1.97, 2.02, 2.03,
2.04, 2.05, 2.06, 2.08, 2.09, and 2.16 (13 s, 39 H, 2 AcN
3.1.30. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-4-O-acetyl-2-deoxy-6-O-levulinoyl-b-
galactopyranosyl-(104)-2,6-di-O-acetyl-3-O-levulinoyl-
b- 4)-2,3,6-tri-O-acetyl-b-
-galactopyranosyl-(10
glucopyranosyl-(101)-(2S,3R,4E)-3-O-benzoyl-2-
D-glycero-a-D-galacto-2-nonulopyranosylonate-
/
D
-galactopyranosyl-(103)-
/
and 11 AcO), 1.60 (dd, 1 H, Jgem
ꢀJ3ax,4 12.6 Hz, H-
/
D-
3Vax), 2.44 (dd, 1 H, Jgem 12.6, J3eq,4 4.6 Hz, H-3Veq),
3.01 (m, 1 H, H-5III), 3.05 (m, 1 H, H-2III), 3.26 (m, 1 H,
H-6III), 3.43 (m, 1 H, H-6?III), 3.59 (dd, 1 H, J5,6 10.8,
J6,7 2.7 Hz, H-6V), 3.62 (dd, 1 H, Jgem 10.3, J1,2 4.6 Hz,
/
D
/
D-
/
octadecanamido-4-octadecene-1,3-diol (33). Glycosyla-
tion of 31 (85 mg, 0.13 mmol) with 30 (130 mg, 0.065
mmol) in dichloromethane (2 mL) in the presence of
TMSOTf (2.4 mL, 0.013 mmol) and AW-300 (1.5 g) for
24 h at 10 8C, then workup as described for 32, gave 33
Cer-1), 3.72 (t, 1 H, J3,4
ꢀ
J4,5 9.4 Hz, H-4I), 3.77 (m, 1
/
H, H-5V), 3.80 (s, 3 H, MeO), 3.99 (dd, 1 H, Jgem 10.3,
J1,2 3.7 Hz, Cer-1?), 4.02 (d, 1 H, J3,4 2.5 Hz, H-4II), 4.05
(dd, 1 H, Jgem 12.6, J8,9 5.3 Hz, H-9V), 4.26 (dd, 1 H,
Jgem 12.6, J8,9 2.3 Hz, H-9?V), 4.36 (d, 1 H, J1,2 7.8 Hz,
H-1II), 4.42 (d, 1 H, J1,2 7.8 Hz, H-1I), 4.47 (m, 1 H, Cer-
2), 4.77 (dd, 1 H, J2,3 10.0, J3,4 2.5 Hz, H-3II), 4.80 (m, 1
H, H-4V), 4.85 (dd, 1 H, J2,3 10.3, J3,4 3.2 Hz, H-3IV),
4.88 (dd, 1 H, J1,2 7.8, J2,3 9.4 Hz, H-2I), 4.98 (dd, 1 H,
J2,3 10.0, J3,4 3.7 Hz, H-3III), 5.00 (d, 1 H, J1,2 7.6 Hz, H-
1IV), 5.02 (dd, 1 H, J1,2 7.8, J2,3 10.0 Hz, H-2II), 5.06 (d,
(58 mg, 36%) as an amorphous mass. [a]D ꢂ14.68 (c 0.6,
/
1
CHCl3); H NMR (CDCl3): d 0.88 (t, 6 H, JMe,CH2 6.9
Hz, 2 MeCH2), 1.25 (s, 52 H, 26 CH2), 1.55, 1.77, 1.91,
1.92, 2.01, 2.02, 2.04, 2.05, 2.08, 2.09, 2.13, 2.13, 2.13,
and 2.15 (14 s, 42 H, 2 AcN, 10 AcO, and 2
CH3COCH2CH2), 1.61 (dd, 1 H, Jgem
H-3Vax), 2.41ꢁ
ꢀ
/
J3ax,4 12.4 Hz,
2
/
2.79 (m,
9
H, H-3Veq and
1 H, J1,2 8.2 Hz, H-1III), 5.14 (t, 1 H, J2,3
ꢀJ3,4 9.4 Hz,
/
CH3COCH2CH2), 3.01 (m, 1 H, H-2III), 3.60 (dd, 1 H,
J5,6 10.8, J6,7 2.7 Hz, H-6V), 3.62 (dd, 1 H, Jgem 10.3, J1,2
H-3I), 5.23 (dd, 1 H, J6,7 2.7, J7,8 9.6 Hz, H-7V), 5.33 (d,
1 H, J3,4 3.2 Hz, H-4IV), 5.40 (d, 1 H, J3,4 3.7 Hz, H-4III),
5.43 (dd, 1 H, J1,2 7.6, J2,3 10.3 Hz, H-2IV), 5.45 (m, 1 H,
Cer-4), 5.53 (m, 1 H, Cer-3), 5.60 (m, 1 H, H-8V), 5.75
(m, 1 H, J2,NH 9.2 Hz, Cer-NH), 5.86 (m, 1 H, Cer-5),
4.4 Hz, Cer-1), 3.70 (t, 1 H, J3,4
ꢀ
J4,5 9.4 Hz, H-4I), 3.75
/
(m, 1 H, H-5V), 3.81 (s, 3 H, MeO), 3.99 (dd, 1 H, Jgem
10.3, J1,2 3.7 Hz, Cer-1?), 4.03 (dd, 1 H, Jgem 12.6, J8,9
5.1 Hz, H-9V), 4.08 (d, 1 H, J3,4 2.1 Hz, H-4II), 4.26 (dd,
1 H, Jgem 12.6, J8,9 2.3 Hz, H-9?V), 4.32 (d, 1 H, J1,2 8.0
Hz, H-1II), 4.41 (d, 1 H, J1,2 7.8 Hz, H-1I), 4.47 (m, 1 H,
Cer-2), 4.78 (m, 1 H, H-4V), 4.82 (m, 1 H, H-3II), 4.83
(dd, 1 H, J2,3 10.1, J3,4 3.2 Hz, H-3IV), 4.89 (dd, 1 H, J1,2
7.8, J2,3 9.4 Hz, H-2I), 4.97 (d, 1 H, J5,NH 8.5 Hz, NH),
4.98 (d, 1 H, J1,2 7.8 Hz, H-1IV), 4.99 (m, 1 H, NH), 5.00
(dd, 1 H, J2,3 10.5, J3,4 3.2 Hz, H-3III), 5.03 (dd, 1 H, J1,2
8.0, J2,3 10.3 Hz, H-2II), 5.08 (d, 1 H, J1,2 8.2 Hz, H-1III),
5.96 (d, 1 H, J2,NH 6.6 Hz, NH), and 7.28ꢁ8.20 (m, 20
/
H, 4 Ph). Anal. Calcd for C124H171N3O46 (2439.71): C,
61.05; H, 7.07; N, 1.72. Found: C, 60.84; H, 6.97; N,
1.57.
3.1.32. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-4-O-acetyl-2-deoxy-b-
(104)-2,6-di-O-acetyl-b- -galactopyranosyl-(10
2,3,6-tri-O-acetyl-b- -glucopyranosyl-(101)-
(2S,3R,4E)-3-O-benzoyl-2-octadecanamido-4-
D
-glycero-a-
D
-galacto-2-nonulopyranosylonate-
-galactopyranosyl-(103)-
-galactopyranosyl-
4)-
/
D
/
D
5.12 (t, 1 H, J2,3
ꢀ
/
J3,4 9.4 Hz, H-3I), 5.22 (dd, 1 H, J6,7
/
D
/
2.7, J7,8 9.6 Hz, H-7V), 5.34 (d, 1 H, J3,4 3.2 Hz, H-4IV),
5.38 (dd, 1 H, J1,2 7.8, J2,3 10.1 Hz, H-2IV), 5.45 (m, 1 H,
Cer-4), 5.46 (d, 1 H, J3,4 3.2 Hz, H-4III), 5.53 (m, 1 H,
Cer-3), 5.58 (m, 1 H, H-8V), 5.75 (m, 1 H, J2,NH 9.2 Hz,
Cer-NH), 5.87 (m, 1 H, Cer-5), 6.21 (d, 1 H, J2,NH 6.6
D
/
octadecene-1,3-diol (35). Selective removal of the levuli-
noyl group in 33 (58 mg, 0.022 mmol) with hydrazine
monoacetate (21 mg, 0.22 mmol) in ethanol (2 mL), and
workup as described for 17, gave 35 (46 mg, 88%) as an
Hz, NH), and 7.39ꢁ8.20 (m, 20 H, 4 Ph). Anal. Calcd
/
for C132H181N3O49 (2593.87): C, 61.12; H, 7.03; N, 1.62.
Found: C, 61.09; H, 6.93; N, 1.41.
amorphous mass. [a]D
ꢂ
37.88 (c 0.2, CHCl3); 1H NMR
/
(CDCl3): d 0.88 (t, 6 H, JMe,CH2 6.9 Hz, 2 MeCH2), 1.26
(s, 52 H, 26 CH2), 1.38, 1.57, 1.78, 1.90, 1.93, 2.03, 2.03,
2.04, 2.07, 2.09, 2.10, and 2.16 (12 s, 36 H, 2 AcN and 10
AcO), 2.45 (dd, 1 H, Jgem 12.6, J3eq,4 4.6 Hz, H-3Veq),
3.22 (m, 1 H, H-2III), 3.42 (m, 1 H, H-3II), 3.59 (dd, 1 H,
3.1.31. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-4-O-acetyl-2-deoxy-b-
(104)-2,3,6-tri-O-acetyl-b- -galactopyranosyl-(10
2,3,6-tri-O-acetyl-b- -glucopyranosyl-(101)-
(2S,3R,4E)-3-O-benzoyl-2-octadecanamido-4-
D
-glycero-a-
D
-galacto-2-nonulopyranosylonate-
-galactopyranosyl-(103)-
-galactopyranosyl-
4)-
/
D
/
D
J5,6 10.8, J6,7 3.0 Hz, H-6V), 3.70 (t, 1 H, J3,4
ꢀJ4,5 9.4
/
/
D
/
Hz, H-4I), 3.80 (m, 1 H, H-5V), 3.81 (s, 3 H, MeO), 4.00
(s, 1 H, H-4II), 4.02 (dd, 1 H, Jgem 12.6, J8,9 6.2 Hz, H-
9V), 4.26 (d, 1 H, J1,2 7.8 Hz, H-1II), 4.32 (m, 1 H, H-
9?V), 4.37 (dd, 1 H, J2,3 10.5, J3,4 3.0 Hz, H-3III), 4.41 (d,
1 H, J1,2 8.0 Hz, H-1I), 4.67 (dd, 1 H, J1,2 7.8, J2,3 9.2
Hz, H-2II), 4.80 (m, 1 H, H-4V), 4.87 (dd, 1 H, J1,2 8.0,
D
/
octadecene-1,3-diol (34). Selective removal of the levuli-
noyl group in 32 (140 mg, 0.055 mmol) with hydrazine
monoacetate (52 mg, 0.55 mmol) in ethanol (2 mL), and

H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ
/
1639
1635
J2,3 9.4 Hz, H-2I), 4.88 (dd, 1 H, J2,3 9.8, J3,4 3.2 Hz, H-
3IV), 4.95 (d, 1 H, J5,NH 9.6 Hz, NH),4.97 (d, 1 H, J1,2
galactopyranosyl-(10
/
4)-2,3,6-tri-O-acetyl-b-
D-
glucopyranosyl-(101)-(2S,3R,4E)-3-O-benzoyl-2-
/
7.6 Hz, H-1IV), 5.12 (t, 1 H, J2,3
ꢀ
/
J3,4 9.4 Hz, H-3I), 5.23
octadecanamido-4-octadecene-1,3-diol dipyridine salt
(37). To a solution of 35 (46 mg, 0.019 mmol) in
DMF (2.0 mL) was added sulfur trioxideꢁpyridine
complex (31 mg, 0.19 mmol), and the mixture was
stirred for 3 h at rt. Workup as described for 19 gave 37
(dd, 1 H, J6,7 3.0, J7,8 9.8 Hz, H-7V), 5.35 (d, 1 H, J3,4 3.2
Hz, H-4IV), 5.42 (d, 1 H, J3,4 3.0 Hz, H-4III), 5.43 (dd, 1
H, J1,2 7.6, J2,3 9.8 Hz, H-2IV), 5.45 (m, 1 H, Cer-4), 5.54
(m, 1 H, Cer-3), 5.54 (d, 1 H, J1,2 8.0 Hz, H-1III), 5.62
(m, 1 H, H-8V), 5.75 (m, 1 H, J2,NH 9.4 Hz, Cer-NH),
5.86 (m, 1 H, Cer-5), 5.92 (d, 1 H, J2,NH 6.6 Hz, NH),
/
(33 mg, 64%) as an amorphous mass. [a]D ꢂ22.68 (c 0.7,
/
1
CHCl3); H NMR (CDCl3): d 0.88 (t, 6 H, JMe,CH2 6.9
Hz, 2 MeCH2), 1.26 (s, 52 H, 26 CH2), 1.40, 1.57, 1.72,
1.86, 1.97, 2.01, 2.01, 2.04, 2.05, 2.08, 2.11, and 2.13 (12
s, 36 H, 2 AcN and 10 AcO), 2.40 (dd, 1 H, Jgem 12.6,
J3eq,4 4.8 Hz, H-3Veq), 3.67 (dd, 1 H, J5,6 11.0, J6,7 2.5
and 7.27ꢁ
/
8.20 (m, 20 H, 4 Ph). Anal. Calcd for
122H169N3O45 (2397.67): C, 61.12; H, 7.10; N, 1.75.
C
Found: C, 60.82; H, 6.91; N, 1.70.
3.1.33. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
Hz, H-6V), 3.76 (t, 1 H, J3,4
ꢀ
J4,5 9.4 Hz, H-4I), 3.78 (s,
/
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-4-O-acetyl-2-deoxy-6-O-sulfo-b-
D
-glycero-a-
D
-galacto-2-nonulopyranosylonate-
3 H, MeO), 4.00 (s, 1 H, H-4II), 4.01 (dd, 1 H, Jgem 12.4,
J8,9 5.3 Hz, H-9V), 4.25 (m, 1 H, H-9?V), 4.31 (d, 1 H, J3,4
2.9 Hz, H-4II), 4.71 (m, 1 H, H-3III), 4.47 (d, 1 H, J1,2 8.0
Hz, H-1II), 4.50 (m, 1 H, H-3II),4.61 (d, 1 H, J1,2 8.0 Hz,
H-1I), 4.77 (m, 1 H, H-4V), 4.82 (dd, 1 H, J1,2 8.0, J2,3
9.4 Hz, H-2I), 4.86 (m, 1 H, H-2II), 4.92 (dd, 1 H, J2,3
10.1, J3,4 3.0 Hz, H-3IV), 5.08 (d, 1 H, J1,2 8.0 Hz, H-
/
D
-galactopyranosyl-(103)-
/
D-
D-
D
-
galactopyranosyl-(10
galactopyranosyl-(10
glucopyranosyl-(10
/
4)-2,3,6-tri-O-acetyl-b-
4)-2,3,6-tri-O-acetyl-b-
/
/
1)-(2S,3R,4E)-3-O-benzoyl-2-
octadecanamido-4-octadecene-1,3-diol pyridinium salt
(36). To a solution of 34 (110 mg, 0.045 mmol) in
DMF (2 mL) was added sulfur trioxideꢁpyridine
complex (37 mg, 0.22 mmol), and the mixture was
1IV), 5.09 (t, 1 H, J2,3
ꢀ
J3,4 9.4 Hz, H-3I), 5.23 (dd, 1 H,
/
/
J6,7 2.5, J7,8 9.8 Hz, H-7V), 5.35 (dd, 1 H, J1,2 8.0, J2,3
10.1 Hz, H-2IV), 5.45 (d, 1 H, J3,4 3.0 Hz, H-4IV), 5.49
(m, 1 H, Cer-3), 5.53 (s, 1 H, H-4III), 5.55 (m, 1 H, Cer-
stirred for 3 h at rt. Workup as described for 19 gave 36
(83 mg, 71%) as an amorphous mass. [a]D ꢂ
/
12.88 (c 1.8,
4), 5.60 (m, 1 H, H-8V), 5.88 (m, 1 H, Cer-5), and 7.35ꢁ
/
CHCl3); 1H NMR (CD3ODꢁ
/D2O): d 0.89 (t, 6 H,
8.08 (m, 28 H, 6 Ph). Anal. Calcd for C132H177N5O51S2
(2713.98): C, 58.42; H, 6.57; N, 2.58. Found: C, 58.18;
H, 6.55; N, 2.58.
JMe,CH2 6.9 Hz, 2 MeCH2), 1.26 (s, 52 H, 26 CH2), 1.50,
1.55, 1.75, 1.76, 1.91, 1.94, 1.98, 2.02, 2.04, 2.06, 2.09,
2.12, and 2.15 (13 s, 39 H, 2 AcN and 11 AcO), 1.55 (m,
1 H, H-3Vax), 2.45 (dd, 1 H, Jgem 12.5, J3eq,4 4.4 Hz, H-
3Veq), 3.35 (m, 1 H, H-2III), 3.63 (dd, 1 H, J5,6 11.2, J6,7
2.7 Hz, H-6V), 3.66 (dd, 1 H, Jgem 9.9, J1,2 4.0 Hz, Cer-
1), 3.70 (m, 1 H, H-5V), 3.72 (m, 1 H, H-4I), 3.81 (s, 3 H,
MeO), 3.98 (dd, 1 H, Jgem 9.9, J1,2 4.6 Hz, Cer-1?), 4.02
(dd, 1 H, Jgem 12.2, J8,9 6.6 Hz, H-9V), 4.08 (d, 1 H, J3,4
2.5 Hz, H-4II), 4.27 (dd, 1 H, Jgem 12.2, J8,9 2.6 Hz, H-
9?V), 4.33 (m, 1 H, H-1II), 4.43 (m, 1 H, Cer-2), 4.47 (d, 1
H, J1,2 8.0 Hz, H-1I), 4.76 (m, 1 H, H-4V), 4.87 (m, 2 H,
H-3II and H-3IV), 4.89 (dd, 1 H, J1,2 8.0, J2,3 9.6 Hz, H-
2I), 4.92 (d, 1 H, J1,2 8.2 Hz, H-1III), 4.95 (m, 1 H, H-
3III), 5.00 (dd, 1 H, J1,2 7.8, J2,3 10.0 Hz, H-2II), 5.02 (d,
3.1.35. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-3-O-acetyl-2-deoxy-6-O-levulinoyl-b-
galactopyranosyl-(104)-2,6-di-O-acetyl-3-O-levulinoyl-
b- 4)-2,3,6-tri-O-acetyl-
-galactopyranosyl-(10
D-glycero-a-
D-galacto-2-nonulopyranosylonate-
/
D
-galactopyranosyl-(104)-
/
D-
/
D
/
D-
glucopyranosyl trichloroacetimidate (39). Treatment of
38 (206 mg, 0.11 mmol) with trichloroacetonitrile (469
mL, 3.18 mmol) and DBU (19 mL, 0.13 mmol) in
dichloromethane (2 mL), then workup as described for
29, gave 39 (184 mg, 83%) as an amorphous mass. The
anomeric ratio (a:b) was estimated as 1:1 from the ratio
of the intensities of the anomeric proton signals of
galactose unit in the 1H NMR spectrum. Anal. Calcd for
C91H108Cl3N3O46 (2086.20): C, 52.39; H, 5.22; N, 2.01.
Found: C, 52.23; H, 4.99; N, 1.84.
1 H, J1,2 8.0 Hz, H-1IV), 5.11 (t, 1 H, J2,3
ꢀJ3,4 9.6 Hz,
/
H-3I), 5.20 (dd, 1 H, J6,7 2.7, J7,8 9.6 Hz, H-7V), 5.38 (dd,
1 H, J1,2 8.0, J2,3 10.1 Hz, H-2IV), 5.41 (d, 1 H, J3,4 3.2
Hz, H-4IV), 5.45 (m, 1 H, Cer-4), 5.48 (d, 1 H, J3,4 3.7
Hz, H-4III), 5.54 (m, 1 H, Cer-3), 5.60 (m, 1 H, H-8V),
5.89 (m, 1 H, Cer-5), and 7.25ꢁ/8.19 (m, 24 H, 5 Ph).
3.1.36. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
Anal. Calcd for C129H175N4O49S (2597.86): C, 59.64; H,
6.79; N, 2.16. Found: C, 59.41; H, 6.51; N, 1.87.
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-3-O-acetyl-2-deoxy-6-O-levulinoyl-b-
galactopyranosyl-(104)-2,6-di-O-acetyl-3-O-levulinoyl-
b- 4)-2,3,6-tri-O-acetyl-b-
-galactopyranosyl-(10
glucopyranosyl-(101)-(2S,3R,4E)-3-O-benzoyl-2-
D
-glycero-a-
D
-galacto-2-nonulopyranosylonate-
/
D
-galactopyranosyl-(104)-
/
D-
3.1.34. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
/
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-4-O-acetyl-2-deoxy-6-O-sulfo-b-
galactopyranosyl-(104)-2,6-di-O-acetyl-3-O-sulfo-b-
D
-glycero-a-
D
-galacto-2-nonulopyranosylonate-
D
/
D-
/
D
-galactopyranosyl-(103)-
/
/
D-
octadecanamido-4-octadecene-1,3-diol (40). Glycosyla-
/
D-
tion of 31 (118 mg, 0.18 mmol) with 39 (184 mg, 0.088

1636
H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ1639
/
mmol) in dichloromethane (3 mL) in the presence of
TMSOTf (3.4 mL, 0.017 mmol) and AW-300 (1.0 g) for
15 h at 10 8C, then workup as described for 32, gave 40
Hz, H-2I), 5.18 (t, 1 H, J2,3
ꢀ
J3,4 9.4 Hz, H-3I), 5.30 (dd,
/
1 H, J6,7 2.7, J7,8 10.0 Hz, H-7V), 5.32 (dd, 1 H, J2,3 10.4,
J3,4 2.5 Hz, H-3III), 5.36 (d, 1 H, J3,4 3.2 Hz, H-4IV), 5.41
(d, 1 H, J1,2 8.0 Hz, H-1III), 5.45 (m, 1 H, Cer-4), 5.47
(dd, 1 H, J1,2 7.8, J2,3 9.4 Hz, H-2IV), 5.54 (m, 1 H, Cer-
3), 5.66 (m, 1 H, H-8V), 5.77 (m, 1 H, J2,NH 9.2 Hz, Cer-
(75 mg, 33%) as an amorphous mass. [a]Dꢂ17.88 (c 1.1,
/
1
CHCl3); H NMR (CDCl3): d 0.88 (t, 6 H, JMe,CH2 6.9
Hz, 2 MeCH2), 1.26 (s, 52 H, 26 CH2), 1.55, 1.76, 1.80,
1.93, 1.94, 2.03, 2.05, 2.06, 2.12, 2.13, 2.14, 2.15, 2.16,
and 2.22 (14 s, 42 H, 2 AcN, 10 AcO, and 2
NH), 5.87 (m, 1 H, Cer-5), and 7.40ꢁ8.35 (m, 20 H, 4
Ph). Anal. Calcd for C122H169N3O45 (2397.67): C, 61.12;
H, 7.10; N, 1.75. Found: C, 61.08; H, 6.97; N, 1.59.
/
CH3COCH2CH2), 1.72 (dd, 1 H, Jgem
ꢀJ3ax,4 12.4 Hz,
/
H-3Vax), 2.41ꢁ H, H-3Veq and
/
2.70 (m,
9
2
CH3COCH2CH2), 2.75 (m, 1 H, H-2III), 3.64 (dd, 1 H,
J5,6 10.6, J6,7 2.7 Hz, H-6V), 3.72 (m, 1 H, H-4I), 3.82 (m,
1 H, H-5V), 3.86 (s, 3 H, MeO), 4.06 (d, 1 H, J3,4 2.7 Hz,
H-4II), 4.08 (dd, 1 H, Jgem 12.0, J8,9 4.6 Hz, H-9V), 4.16
(m, 1 H, H-9?V), 4.32 (d, 1 H, J1,2 7.8 Hz, H-1II), 4.44 (d,
1 H, J1,2 7.8 Hz, H-1I), 4.81 (dd, 1 H, J2,3 10.3, J3,4 2.7
Hz, H-3II), 4.83 (m, 1 H, H-3IV), 4.86 (d, 1 H, J1,2 7.8
Hz, H-1IV), 4.88 (m, 1 H, H-4V), 4.94 (d, 1 H, J1,2 8.2
Hz, H-1III),4.96 (dd, 1 H, J1,2 7.8, J2,3 9.4 Hz, H-2I), 5.05
3.1.38. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-3-O-acetyl-2-deoxy-6-O-sulfo-b-
pyranosyl-(104)-2,6-di-O-acetyl-3-O-sulfo-b-
pyranosyl-(104)-2,3,6-tri-O-acetyl-b- -glucopyranosyl-
(101)-(2S,3R,4E)-3-O-benzoyl-2-octadecanamido-4-
D
-glycero-a-
D
-galacto-2-nonulopyranosylonate-
-galactopyranosyl-(104)-
-galacto-
-galacto-
/
D
/
D
/
D
/
D
/
octadecene-1,3-diol dipyridinium salt (42). To a solution
of 41 (34 mg, 0.014 mmol) in DMF (1.5 mL) was added
(dd, 1 H, J1,2 7.8, J2,3 10.3 Hz, H-2II), 5.20 (t, 1 H, J2,3
ꢀ
/
sulfur trioxideꢁpyridine complex (18 mg, 0.11 mmol),
/
J3,4 9.4 Hz, H-3I), 5.30 (dd, 1 H, J6,7 2.7, J7,8 9.8 Hz, H-
7V), 5.38 (d, 1 H, J3,4 2.7 Hz, H-4IV), 5.40 (dd, 1 H, J1,2
7.8, J2,3 10.1 Hz, H-2IV), 5.45 (m, 1 H, Cer-4), 5.55 (m, 1
H, Cer-3), 5.66 (m, 1 H, H-8V), 5.77 (d, 1 H, J2,NH 9.2
Hz, Cer-NH), 5.83 (d, 1 H, J2,NH 8.9 Hz, NH), 5.87 (m,
and the mixture was stirred for 3 h at rt. Workup as
described for 36 gave 42 (33 mg, 87%) as an amorphous
1
mass. [a]Dꢂ
/
14.98 (c 0.7, CHCl3); H NMR (CDCl3): d
0.88 (t, 6 H, JMe,CH2 6.7 Hz, 2 MeCH2), 1.26 (s, 52 H, 26
CH2), 1.60, 1.76, 1.84, 1.92, 1.95, 2.04, 2.05, 2.07, 2.09,
2.13, 2.15, and 2.20 (12 s, 36 H, 2 AcN and 10 AcO),
2.47 (dd, 1 H, Jgem 12.4, J3eq,4 4.3 Hz, H-3Veq), 2.85 (m,
1 H, H-2III), 3.83 (s, 3 H, MeO), 4.36 (d, 1 H, J1,2 8.0 Hz,
H-1II), 4.47 (d, 1 H, J1,2 8.0 Hz, H-1I), 4.78 (m, 1 H, H-
1 H, Cer-5), and 7.40ꢁ8.34 (m, 20 H, 4 Ph). Anal. Calcd
/
for C132H181N3O49 (2593.87): C, 61.12; H, 7.03; N, 1.62.
Found: C, 61.00; H, 6.82; N, 1.36.
3.1.37. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
4V), 4.90 (d, 1 H, J1,2 8.2 Hz, H-1IV), 5.11 (t, 1 H, J2,3
ꢀ
/
dideoxy-
(203)-2,4,6-tri-O-benzoyl-b-
2-acetamido-3-O-acetyl-2-deoxy-b-
(104)-2,6-di-O-acetyl-b- -galactopyranosyl-(10
2,3,6-tri-O-acetyl-b- -glucopyranosyl-(101)-
(2S,3R,4E)-3-O-benzoyl-2-octadecanamido-4-
D
-glycero-a-
D
-galacto-2-nonulopyranosylonate-
-galactopyranosyl-(104)-
-galactopyranosyl-
4)-
J3,4 9.5 Hz, H-3I), 5.20 (d, 1 H, J1,2 8.0 Hz, H-1III), 5.25
(m, 1 H, H-7V), 5.36 (dd, 1 H, J1,2 8.2, J2,3 9.4 Hz, H-
2IV), 5.37 (m, 1 H, H-4IV), 5.46 (m, 1 H, Cer-4), 5.55 (m,
1 H, Cer-3), 5.57 (m, 1 H, H-8V), 5.89 (m, 1 H, Cer-5),
/
D
/
D
/
D
/
D
/
and 7.40ꢁ
132H177N5O51S2 (2713.98): C, 58.42; H, 6.57; N, 2.58.
Found: C, 58.23; H, 6.41; N, 2.53.
/
8.26 (m, 28 H, 6 Ph). Anal. Calcd for
C
octadecene-1,3-diol (41). Selective removal of the levuli-
noyl group in 40 (75 mg, 0.029 mmol) with hydrazine
monoacetate (27 mg, 0.29 mmol) in ethanol (2 mL), and
workup as described for 37, gave 39 (35 mg, 50%) as an
3.1.39. 5-Acetamido-3,5-dideoxy-
2-nonulopyranosylonic
acid-(20
osyl-(103)-2-acetamido-2-deoxy-6-O-sulfo-b-
pyranosyl-(104)-b- -galactopyranosyl-(104)-b-
glucopyranosyl-(101)-(2S,3R,4E)-2-octadecanamido-4-
D
-glycero-a-
3)-b- -galactopyran-
-galacto-
D
-galacto-
/
D
amorphous mass. [a]D
ꢂ
/
22.58 (c 0.6, CHCl3); 1H NMR
/
D
(CDCl3): d 0.88 (t, 6 H, JMe,CH2 6.9 Hz, 2 MeCH2), 1.25
(s, 52 H, 26 CH2), 1.52, 1.78, 1.80, 1.93, 1.94, 2.04, 2.05,
2.05, 2.13, 2.18, 2.20, and 2.21 (12 s, 36 H, 2 AcN and 10
/
D
/
D-
/
octadecene-1,3-diol disodium salt (B, GSC-335). To a
solution 36 (83 mg, 28 mmol) in methanol (2 mL) and
dioxane (1 mL) was added sodium methoxide (10 mg);
then water (0.5 mL) was added. After completion of the
reaction (24 h), the mixture was concentrated. Column
AcO), 1.72 (dd, 1 H, Jgem
2.47 (dd, 1 H, Jgem 12.4, J3eq,4 4.6 Hz, H-3Veq), 2.77 (m,
ꢀ
J3ax,4 12.4 Hz, H-3Vax),
/
1 H, H-2III), 3.41 (m, 1 H, H-3II), 3.64 (m, 1 H, H-6V),
3.70 (t, 1 H, J3,4
ꢀ
J4,5 9.4 Hz, H-4I), 3.84 (m, 1 H, H-
/
5V), 3.89 (s, 3 H, MeO), 3.96 (d, 1 H, J3,4 2.5 Hz, H-4II),
4.07 (dd, 1 H, Jgem 12.2, J8,9 4.7 Hz, H-9V), 4.13 (dd, 1
H, Jgem 12.2, J8,9 2.5 Hz, H-9?V), 4.18 (d, 1 H, J3,4 2.5
Hz, H-4III), 4.23 (d, 1 H, J1,2 7.8 Hz, H-1II), 4.44 (d, 1 H,
J1,2 7.8 Hz, H-1I), 4.56 (d, 1 H, J2,NH 6.4 Hz, NH), 4.67
(dd, 1 H, J1,2 7.8, J2,3 9.4 Hz, H-2II), 4.86 (m, 1 H, H-
4V), 4.88 (d, 1 H, J1,2 7.8 Hz, H-1IV), 4.91 (dd, 1 H, J2,3
9.4, J3,4 3.2 Hz, H-3IV), 4.94 (dd, 1 H, J1,2 7.8, J2,3 9.4
chromatography (5:4:0.7:0.07 CHCl3ꢁ
/
MeOHꢁ
/
H2Oꢁ
/
Et3N) of the residue on Sephadex LH-20 gave B
(GSC-335) (53 mg, quant) as an amorphous mass. [a]D
1
H2O); H NMR
D2O): d 0.85 (t, 6 H, JMe,CH2 7.0 Hz, 2
ꢂ
/
14.18 (c 0.2, 5:4:0.7 CHCl3ꢁ
/
MeOHꢁ
/
((CD3)2SOꢁ
/
MeCH2), 1.23 (s, 52 H, 26 CH2), 1.40 (m, 2 H,
COCH2CH2), 1.80 and 1.90 (2 s, 6 H, 2 AcN), 2.74
(m, 1 H, H-3Veq), 4.17 (d, 1 H, J1,2 7.3 Hz, H-1I), 4.19

H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ
/
1639
1637
(d, 1 H, J1,2 7.0 Hz, H-1II), 4.25 (d, 1 H, J1,2 7.3 Hz, H-
1IV), 4.52 (d, 1 H, J1,2 8.4 Hz, H-1III), 5.33 (m, 1 H, Cer-
4), and 5.52 (m, 1 H, Cer-5).
[3?-O-sulfo lactosyl ceramide]ꢃ, 726.5 [glucosyl
ceramide]ꢃ, 564.6 [ceramide]ꢃ; calcd for C73H129N3-
NaO37S2 [Mꢃ2Na]: 1726.7644; found: 1726.88.
/
FABMS (negative-ion mode, triethanolamine ma-
trix): m/z 1646.9 [Mꢃ
1333.8 [MꢃNaꢃ
NeuAc]ꢃ, 1171.8 [1333.8ꢃ
888.7 [lactosyl ceramide]ꢃ, 726.7 [glucosyl ceramide]ꢃ,
564.6 [ceramide]ꢃ; calcd for C73H129N3NaO34S [Mꢃ
/
Na]ꢃ, 1624.9 [Mꢃ
/
2Na]ꢃ,
/
/
/
Gal]ꢃ,
4. MAG-glycolipid binding assays
/
Na]: 1646.8076; found: 1646.86.
MAG binding was quantified by measuring the adhe-
sion of MAG-transfected cells or the binding or MAG-
Fc chimeric protein to glycolipids adsorbed on multiwell
plates. To simulate a membrane monolayer, glyco-
sphingolipids were stably co-adsorbed with phosphati-
dylcholine and cholesterol onto the bottom of 96-well
polystyrene multiwell plates as described in detail else-
where.28 Briefly, lipids were dissolved at the desired
concentrations in ethanol containing 1 mM phosphati-
dylcholine and 4 mM cholesterol. An equal volume of
water was added, and 50 mL of the solution was placed
in each well of a 96-well multiwell plate (polystyrene,
Corning-Costar #9017). Plates were left uncovered for
90 min to allow partial evaporation of the ethanol. This
results in the lipids adsorbing onto the plastic surface as
a monolayer, where they are stable in aqueous solutions.
Prior studies indicated that co-adsorption of glycolipids
with phosphatidylcholine and cholesterol resulted in
3.1.40. 5-Acetamido-3,5-dideoxy-
D
-glycero-a-
3)-b- -galactopyran-
3)-2-acetamido-2-deoxy-6-O-sulfo-b- -galacto-
pyranosyl-(104)-3-O-sulfo-b- -galactopyranosyl-(10
4)-b- 1)-(2S,3R,4E)-2-octadecana-
-glucopyranosyl-(10
D
-galacto-
2-nonulopyranosylonic
osyl-(10
acid-(20
/
D
/
D
/
D
/
D
/
mido-4-octadecene-1,3-diol trisodium salt (C, GSC-454).
To a solution of 37 (33 mg, 0.012 mmol) in methanol
(3.0 mL) was added sodium methoxide (10 mg), and
workup as described for B, gave C (GSC-454) (21 mg,
quant) as an amorphous mass. [a]D
1
ꢂ
/
0.38 (c 0.6, 5:4:0.7
CHCl3ꢁ
/
MeOHꢁ
/
H2O); H NMR ((CD3)2SOꢁD2O): d
/
0.83 (t, 6 H, JMe,CH2 7.0 Hz, 2 MeCH2), 1.22 (s, 52 H, 26
CH2), 1.41 (m, 2 H, COCH2CH2), 1.85 and 1.92 (2 s, 6
H, 2 AcN), 2.74 (m, 1 H, H-3Veq), 4.08 (m, 1 H, H-1I),
4.28 (d, 1 H, J1,2 7.1 Hz, H-1II), 4.31 (m, 1 H, H-1IV),
4.50 (d, 1 H, J1,2 7.6 Hz, H-1III), 5.27 (m, 1 H, Cer-4),
and 5.54 (m, 1 H, Cer-5).
equivalent adsorption efficiency (ꢁ50% of the added
/
FABMS (negative-ion mode, triethanolamine ma-
ganglioside), higher adsorption stability and enhanced
recognition of immobilized glycolipids.28 After ganglio-
side adsorption, the wells were washed with water and
trix): m/z 1726.8 [Mꢃ
SO3Naꢂ
H]ꢃ, 1435.8 [Mꢃ
[1435.8ꢃ
/
2Na]ꢃ, 1625.0 [Mꢃ
/
2Naꢃ
NeuAc]ꢃ, 1333.8
Gal]ꢃ, 968.7
/
/
/
Naꢃ
/
/
SO3Naꢂ
/
H]ꢃ, 1171.7 [1333.8ꢃ
/
blocked by addition of 200 mL of BSAꢁ/PBS (1 mg/mL
[3?-O-sulfo lactosyl ceramide]ꢃ, 726.6 [glucosyl
bovine serum albumin in Dulbecco’s phosphate buffered
saline) for 30 min at 37 8C.
ceramide]ꢃ, 564.6 [ceramide]ꢃ; calcd for C73H129N3-
NaO37S2 (Mꢃ
/
2Na): 1726.7644; found: 1726.80.
To ensure that the different gangliosides tested
adsorbed equivalently control experiments were per-
formed, followed by lipid recovery and quantification.
Synthetic and natural gangliosides were adsorbed at an
input concentration of 200 pmol/well, blocked with
BSA-containing buffer, then incubated with untrans-
fected COS-1 cells (50,000 cells/well) for 45 min at 37 8C.
The cells, which were non-adherent, were detached by
controlled centrifugation in a custom-designed Plexiglas
adhesion chamber.28 After cell detachment, gangliosides
were recovered from the microwell surfaces by washing
each well with water then incubating with 200 mL of
butanol. The butanol extract was evaporated, and the
recovered gangliosides dissolved in a small volume of
solvent and analyzed by quantitative thin-layer chro-
matography using resorcinol reagent for ganglioside
detection.31 Adsorption efficiency and stability among
the different gangliosides tested varied over a twofold
range. Most gangliosides (e.g., GM1a, GD1a) were
recovered at 50% of the amount added. GSC335 was
recovered at 35% and GSC338 at 26% of the amount
added. Results are reported as cell adhesion or chimeric
protein binding as a function of glycolipid added to the
3.1.41. 5-Acetamido-3,5-dideoxy-
2-nonulopyranosylonic
acid-(20
osyl-(104)-2-acetamido-2-deoxy-6-O-sulfo-b-
pyranosyl-(104)-3-O-sulfo-b- -galactopyranosyl-(10
4)-b- 1)-(2S,3R,4E)-2-octadecana-
-glucopyranosyl-(10
D
-glycero-a-
3)-b- -galactopyran-
-galacto-
D
-galacto-
/
D
/
D
/
D
/
D
/
mido-4-octadecene-1,3-diol trisodium salt (D, GSC-338).
To a solution of 42 (33 mg, 0.012 mmol) in methanol (4
mL) was added sodium methoxide (10 mg), and workup
as described for B, gave (D, GSC-338) (21 mg, quant) as
an amorphous mass. [a]D ꢃ
/
12.78 (c 0.2, 5:4:0.7 CHCl3ꢁ
/
1
MeOHꢁ
/
H2O); H NMR ((CD3)2SOꢁD2O): d 0.85 (t, 6
/
H, JMe,CH2 7.0 Hz, 2 MeCH2), 1.23 (s, 52 H, 26 CH2),
1.44 (m, 2 H, COCH2CH2), 1.87 and 1.90 (2 s, 6 H, 2
AcN), 2.71 (m, 1 H, H-3Veq), 4.17 (d, 1 H, J1,2 7.7 Hz,
H-1I), 4.23 (d, 1 H, J1,2 7.7 Hz, H-1II), 4.29 (d, 1 H, J1,2
8.1 Hz, H-1IV), 4.42 (d, 1 H, J1,2 7.7 Hz, H-1III), 5.32 (m,
1 H, Cer-4), and 5.52 (m, 1 H, Cer-5).
FABMS (negative-ion mode, triethanolamine ma-
trix): m/z 1726.9 [Mꢃ
SO3Naꢂ
H]ꢃ, 1435.8 [Mꢃ
[1435.8ꢃSO3Naꢂ
H]ꢃ, 1171.8 [1333.7ꢃ
/
2Na]ꢃ, 1625.0 [Mꢃ
/
2Naꢃ
NeuAc]ꢃ, 1333.7
Gal]ꢃ, 968.7
/
/
/
Naꢃ
/
/
/
/

1638
H. Ito et al. / Carbohydrate Research 338 (2003) 1621ꢁ1639
/
well without correcting for adsorption efficiency over
this range.
Hercules, CA). The data were fitted to rectangular
hyperbolae with the ganglioside added per well as the
‘x’ value and the absorbance at 405 as the ‘y’ value, to
provide an ‘apparent KD’ for comparison of ganglioside
potencies in supporting MAG-Fc binding.
Adhesion of MAG-transfected COS-1 cells to immo-
bilized glycolipids in microwell plates was determined as
described previously.28 Briefly, COS-1 cells were tran-
siently transfected with an expression vector bearing the
full length long form of rat MAG. After culturing to
allow expression (48ꢁ72 h) cells were collected in an
/
Acknowledgements
EDTA-containing buffer, washed by centrifugation,
resuspended in buffer, and added at 50,000 cells/200
mL buffer to each well of glycolipid-adsorbed 96-well
microplates. After 45 min to allow adhesion to proceed,
the plate was immersed, inverted while immersed, placed
in a custom-designed adhesion chamber, and subjected
to a centrifugal detachment force of 110g for 10 min to
remove non-adherent cells. The adhesion chamber was
immersed in buffer, the plate recovered and righted, and
adherent cells quantified by lysis (using Triton X-100)
and analysis of released lactate dehydrogenase activity
using a kinetic 96-well plate reader. Non-transfected
COS-1 cells, or cells transfected with control expression
vector, did not adhere to any glycolipid tested (Ref. 8
and data not shown).
This study was supported in part by a Grant-in-Aid
(No. 12306007 to M.K.) for Scientific Research from
Japan Society for the Promotion of Science, and
National Institutes of Health grant NS37096 (to
R.L.S). We are grateful to Dr Takao Ikami for the
mass spectrometric analyses.
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/
PBS containing 100 ng of MAG-Fc
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/
PBS containing 15 ng of biotinylated goat
/
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/
/
/
washed with PBS, and 50 mL of BSAꢁ
/
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/
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/
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/
/
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