Syntheses of model compounds related to an antigenic epitope in pectic polysaccharides from Bupleurum falcatum L.

Article abstract of DOI:10.1016/S0008-6215(01)00148-3

Stereocontrolled syntheses of model compounds related to a category of the major antigenic epitope against anti-bupleurum 2IIc/PG-1-IgG from an anti-ulcer pectic polysaccharide are described. Glycosylation of the glucuronic acid donors methyl(2,3-di-O-ben

Full text of DOI:10.1016/S0008-6215(01)00148-3

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Carbohydrate Research 334 (2001) 7–17
Syntheses of model compounds related to an antigenic
epitope in pectic polysaccharides from Bupleurum
falcatum L.
Noriyasu Hada,a Tomoko Ogino,a Haruki Yamada,b Tadahiro Takedaa,*
aKyoritsu College of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo 105-8512, Japan
bOriental Medicine Research Center, The Kitasato Institute, Shiroganedai 5-9-1, Minato-ku, Tokyo,
108-8642, Japan
Received 18 January 2001; accepted 19 May 2001
Abstract
Stereocontrolled syntheses of model compounds related to a category of the major antigenic epitope against
anti-bupleurum 2IIc/PG-1-IgG from an anti-ulcer pectic polysaccharide are described. Glycosylation of the glu-
curonic acid donors methyl(2,3-di-O-benzoyl-4-O-methyl-a-
methyl (2,3-di-O-benzoyl-4-O-methyl-b- -glucopyranosyl)uronate-(1“6)-2,3,4-tri-O-benzoyl-a-
trichloroacetimidate with the common acceptor 2-(trimethylsilyl)ethyl 2,3,4-tri-O-benzyl-b- -galactopyranoside in
the presence of trimethylsilyl triflate (TMSOTf) gave the desired di- and trisaccharide derivatives. Furthermore the
products were transformed into the oligo-valent clustering saccharides, N,N%,N%%-tri-{5-[4-O-methyl-b- -glucopyran-
osyluronic acid-(1“6)-b- -galactopyranosyloxy]pentylcarbonylaminoethyl}-1,3,5-benzenetriamide and N,N%,N%%-tri-
{5-[4-O-methyl-b- -glucopyranosyluronic acid -galactopyranosyloxy]-
D
-glucopyranosyl trichloroacetimidate)uronate and
D
D
-galactopyranosyl
D
D
D
D
(1“6)-b-
D
-galactopyranosyl-(1“6)-b-
D
pentylcarbonylaminoethyl}-1,3,5-benzenetriamide. © 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Antigenic epitope; Anti-ulcer pectic polysaccharide; Bupleurum falcatum; Oligovalent di- and trisaccharides; Chemical
synthesis
1. Introduction
reported that the potent anti-ulcer pectic
polysaccharide (Bupleuran 2IIc) was isolated
from the hot-water extract of the roots of
Bupleurum falcatum. Bupleuran 2IIc consists
of a galacturonan region, a ‘ramified’ region
(PG-1) composed of a rhamnogalacturonan
core having neutral sugar side chains, and a
rhamnogalacturonan II-like region;3 the
‘ramified’ region has been considered to be an
important part of the expression of the activ-
ity. The backbone of PG-1 is composed of an
In the last few years, it has become appar-
ent that pectic polysaccharides including
pectin from medicinal herbs display various in
vitro and in vivo pharmacological activities in
addition to the application for drug delivery.
Therefore, it is possible to consider pectic
polysaccharides, not only as one of the active
ingredients of medicinal herbs, but also as
medicinals.1 Recently, Yamada et al.2 have
a-
L
-Rha-(1“4)-a- -GalA-(1“2)-repeating
D
* Corresponding author. Tel.: +81-3-54002696; fax: +81-
3-54002556.
E-mail address: takeda-td@kyoritsu-ph.ac.jp (T. Takeda).
unit, which was reported in our previous
paper4 as the synthetic tetrasaccharide.
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00148-3

8
N. Hada et al. / Carbohydrate Research 334 (2001) 7–17
On the other hand, a polyclonal antibody
presentation of specific sugar epitope in one
molecule can result in remarkably increased
adhesion. It is expected that multivalent sac-
charides would bind to the cell-surface adhe-
sion molecule more tightly than monovalent
ones.
(anti-bupleuran 2IIc/PG-1-IgG) against the
PG-1 of the bupleuran 2IIc was prepared, and
the major antigenic epitope against this anti-
body was characterized as a 6-linked galacto-
syl chain containing terminal glucuronic acid
(GlcA)
(GlcA4Me), which were substituted to (1“3)-
b- -galactosyl chains in the ‘ramified’ region
or
4-O-methyl-glucuronic
acid
We therefore initiated the synthesis of sim-
ple readily accessible oligo-valent saccharides,
and we report herein the synthesis of mono-
D
of bupleuran 2IIc.5 Furthermore, among the
structural region of bupleuran 2IIc, the
‘ramified‘ region (PG-1), showed potent mito-
genic activity, suggesting it was the active
site.6 The proposed structure of the antigenic
epitope in PG-1 was the target for synthetic
studies as part of our investigations on the
synthesis of oligosaccharides of biological in-
terest. In this time, di-, and trisaccharide
derivatives including GlcA4Me were chosen as
target compounds. Concerning the synthesis
of oligosaccharides, suitable glycomimetics
that are able to compete or perform even
better than the naturally occurring carbohy-
drate ligands are needed for the development
of suitably bioactive compounds. For this pur-
pose clustering glycosides7 have proved to be
advantageous in many instances, as the multi-
and trivalent analogs (1–4) of b-
(1“6)-b- -Gal- and b- -GlcA4Me-(1“6)-b-
-Gal-(1“6)-b- -Gal.
D
-GlcA4Me-
D
D
D
D
2. Results and discussion
Syntheses of monosaccharide deri6ati6es.—
Syntheses of the additional glucuronic acid
methyl ester building blocks 9 were carried
out as depicted in Scheme 1. Compound 6 was
prepared from known 2-(trimethylsilyl)ethyl
2,3-di-O-benzyl-b-
-glucopyranoside (5)8 by
D
the following three-step procedure. Regiose-
lective tritylation of the starting material with
trityl chloride, followed by methylation and
subsequent acid-hydrolysis of the trityl group,
gave compound 6. Oxidation and esterifica-

N. Hada et al. / Carbohydrate Research 334 (2001) 7–17
9
tion of 6 was carried out with K2Cr2O7 and
HCl–MeOH. Removal of the benzyl groups
from 7 by catalytic hydrogenolysis over 10%
Pd–C and subsequent benzoylation gave
methyl[2 - (trimethylsilyl)ethyl(2,3 - di - O - ben-
undec-7-ene (DBU) in dichloromethane for 2
h at 0 °C, gave the corresponding a-trichloro-
acetimidate 9 (70%) as the sole product
(Scheme 1).
Syntheses of target oligosaccharide ana-
logues.—The glycosylation of 9 with 2-
zoyl-4-O-methyl-b- -glucopyranosid)uronate)]
D
(8). For selective removal of the 2-(trimethyl-
silyl)ethyl (SE) group, 8 was treated9 with tri-
fluoroacetic acid in dichloromethane for 1 h at
0 °C to give the 1-hydroxy compound, which
on further treatment10 with trichloroacetoni-
trile in the presence of 1,8-diazabicyclo[5.4.0]-
(trimethylsilyl)ethyl
2,3,4-tri-O-benzyl-b- -
D
galactopyranoside (10) in the presence of
,
trimethylsilyl triflate (TMSOTf) and 4 A MS
in dichloromethane for 1 h at 0 °C gave the
desired disaccharide 11 (81%), as evidenced by
1H NMR spectroscopy (H-1%, 4.81 ppm, J 7.9
Scheme 1.
Scheme 2.

10
N. Hada et al. / Carbohydrate Research 334 (2001) 7–17
Hz). Compound 11 was subjected to O-deben-
zylation, followed by benzoylation, to give
compound 12. Selective removal of the SE
group and treatment with trichloroacetonitrile
in the presence of DBU gave only the corre-
sponding a-trichloroacetimidate 13. Glycosy-
lation of 13 with 10 in the presence of
We
chose
5-[2-(2,2,2-trichloroethoxycar-
bonyl)aminoethyleneaminocarbonyl]pentanol
(20) as a spacer, which was prepared as de-
picted in Scheme 3. Compound 20 was pre-
pared from o-caprolactone by the following
procedure. o-Caprolactone was converted to
5-methoxycarbonylpentanol by NaOMe, and
subsequent benzylation gave benzyl 5-
methoxycarbonylpentyl ether (17). Compound
17 was converted to the primary amine 18 by
reaction with neat anhydrous ethylene-
diamine11 and was protected by 2,2,2-tri-
chloroethyl carbamate (19). Finally removal
of the benzyl groups by catalytic hydrogenoly-
sis over 10% Pd–C gave the target spacer,
5 - [2 - (2,2,2 - trichloroethoxycarbonyl)amino-
,
TMSOTf and 4 A MS in dichloromethane for
1 h at 0 °C gave the desired trisaccharide 14
1
(85%), as evidenced by H NMR spectroscopy
(H-1%, 4.80 ppm, J 7.9 Hz), showing the newly
formed glycosidic linkages to be b. Compound
14 was converted by O-debenzylation and
subsequent benzoylation to compound 15. Se-
lective removal of the SE group, and treat-
ment with trichloroacetonitrile in the presence
of DBU gave only the corresponding a-
trichloroacetimidate 16. In an alternate proce-
dure, a part of 12 and 15 was converted by
debenzoylation and hydrolysis of the ester to
give the target compounds 1 and 2 (Scheme
2).
ethyleneaminocarbonyl]pentanol
(TOF-MS
m/z: 371.3 [M+Na]+) (20) (Scheme 3). Cou-
pling of 13 and/or 16 with the spacer 20 in the
,
presence of TMSOTf and 4 A MS for 1 h at
0 °C afforded the desired di- and trisaccharide
derivatives 21 (65%) and 22 (60%), as evi-
1
Next, concerning the synthesis of sugar
clusters, spacer groups are needed as linkers
between the core and the carbohydrate chain.
denced by H NMR spectroscopy (21: H-1,
4.58 ppm, J 7.9 Hz, 22: H-1, 4.68 ppm, J 7.9
Hz). Removal of the Troc group from 21 and
Scheme 3.
Scheme 4.

N. Hada et al. / Carbohydrate Research 334 (2001) 7–17
11

12
N. Hada et al. / Carbohydrate Research 334 (2001) 7–17
22 by Zn–AcOH gave the primary amines 23
and 24 (Scheme 4). We chose 1,3,5-benzenetri-
carbonyl trichioride12 a core for the synthesis
of the sugar cluster. Coupling of 23 and/or 24
with the core, 1,3,5-benzenetricarbonyl
trichloride in the presence of triethylamine
afforded the desired trivalent oligosaccharide
cluster 25 (64%) and 26 (78%), as evidenced
by TOF-MS (25: [M+Na]+=3360, 26: [M+
Na]+=4782) (Scheme 5). Finally, removal of
all acyl groups and esters with sodium
methoxide in 5:1 methanol–water for 4 h at
room temperature afforded the desired
oligosaccharide clusters 3 and 4. Compound 3
revealed an [M+Na]+ ion peak at m/z 1757
and compound 4 revealed an [M+Na]+ ion
peak at m/z 2243 in the TOF-MS spectrum.
Mitogenic activity of these compounds on
spleen cells and Peyer’s patch cells were evalu-
ated, and the results will be reported in detail
elsewhere.
methylsilyl)ethyl 2,3-di-O-benzyl-6-O-trityl-b-
-glucopyranoside. To a solution of this com-
D
pound in DMF (15 mL) was added NaH in
oil (110 mg) and iodomethane (1 mL). The
mixture was stirred for 4 h at rt, and then
MeOH was added to destroy excess NaH. The
reaction mixture was poured into ice-water
and extracted with CHCl3. The extract was
successively washed with aq NaHCO3 and
water, dried (Na2SO4), and concentrated. This
compound was treated with 80% AcOH (10
mL) at 55 °C for 10 h, and following removal
of AcOH and water by coevaporation with
toluene, the crude product was purified by
column chromatography (50:1 benzene–ace-
tone) on silica gel to give compound 6 (1.31 g,
1
three steps 70%): H NMR (CDCl3): l 7.34–
7.25 (m, 10 H, 2×Ph), 4.95-4.69 (m, 4 H, 2
PhCH2), 4.42 (d, 1 H, J1,2 7.9 Hz, H-1), 4.00–
3.95 (m, 1 H, ꢀOCH2CH2ꢀ), 3.93–3.76 (br,d, 2
H, H-6a,6b), 3.63–3.26 (m, 5 H, H-2,3,4,5,
ꢀOCH2CH2ꢀ), 3.55 (s, 3 H, OMe), 0.97–0.86
(m, 2 H, ꢀOCH2CH2ꢀ), −0.06 (s, 9 H,
Si(CH3)3): MALDI-TOFMS: Calcd for
C26H38O6Si: m/z 474.2. Found: m/z 497.2
[M+Na]+.
3. Experimental
Optical rotations were determined with a
1
13
Methyl [2-(trimethylsilyl)ethyl 2,3-di-O-ben-
JASCO digital polarimeter. H and C NMR
spectra were recorded on a JNM A 500 FT
NMR spectrometer in CDCl3 with Me4Si as
the internal standard. MALDI-TOFMS was
recorded on a Perceptive Voyager RP mass
spectrometer. TLC was performed on Silica
Gel 60 F254 (E. Merck) with detection by
quenching of UV fluorescence and by spraying
with 10% H2SO4. Column chromatography
was carried out on Silica Gel 60 (E. Merck).
zyl-4-O-methyl-i- -glucopyranosid]uronate
D
(7).—To a solution of 6 (1.22 g, 5.06 mmol)
in acetone (30 mL) was added K2Cr2O7 (2.8
g), 3.5 M H2SO4(11 mL), and the mixture was
stirred for 1 h at 55 °C, at the end of which
time water was added. The residue was
washed with CHCl3, and the washings were
successively washed with water, dried
(Na2SO4), and concentrated. The resulting
compound was treated with 10% HCl–MeOH
(60 mL) at rt for 2 h and then concentrated.
The product was purified by silica-gel column
chromatography using 8:1 hexane–EtOAc as
2-(Trimethylsilyl)ethyl
2,3-di-O-benzyl-b- -
D
glucopyranoside (5) was prepared by a litera-
ture method.8
2-(Trimethylsilyl)ethyl 2,3-di-O-benzyl-4-O-
1
eluent to give 7 (1.88 g, 73.9%): H NMR
methyl-i- -glucopyranoside (6).—To a solu-
D
(CDCl3): l 7.36–7.26 (m, 10 H, 2 Ph), 4.94–
4.68 (m, 4 H, 2 PhCH2), 4.42 (d, 1 H, J1,2 7.5
Hz, H-1), 4.00–3.94 (m, 1 H, ꢀOCH2CH2ꢀ),
3.81 (s, 3 H, COOMe), 3.78 (d, 1 H, J4,5 10.5
Hz, H-5), 3.62–3.39 (m, 4 H, H-2,3,4,
ꢀOCH2CH2ꢀ), 3.49 (s, 3 H, OMe), 0.96–0.86
(m, 2 H, ꢀOCH2CH2ꢀ), −0.03 (s, 9 H,
Si(CH3)3); 13C NMR (CDCl3): l 169.1 (C-6),
103.4 (C-1), 83.7 (C-4), 81.6 (C-2), 81.1 (C-3),
75.5, 74.8 (2 PhCH2), 74.3 (C-5), 67.8
tion of 2-(trimethylsilyl)ethyl 2,3-di-O-benzyl-
b- -glucopyranoside (5) (1.7 g, 3.93 mmol) in
D
pyridine (15 mL) was added triphenylmethyl
chloride (2.2 g, 7.89 mmol). The reaction mix-
ture was stirred at 70 °C for 12 h, then diluted
with CHCl3 and washed with 5% HCl, aq
NaHCO3 and water, dried (Na2SO4), and con-
centrated. The product was purified by silica-
gel column chromatography using 100:1
benzene–acetone as eluent to give 2-(tri-

N. Hada et al. / Carbohydrate Research 334 (2001) 7–17
13
(ꢀOCH2CH2ꢀ), 60.6 (COOMe), 52.4 (OMe),
18.4 (ꢀOCH2CH2ꢀ), −1.5 (SiMe3). MALDI-
TOFMS: Calcd for C27H38O7Si: m/z 502.2.
Found: m/z 525.1 [M+Na]+.
(CDCl3): l 8.63 (s, 1 H, NH), 8.04–7.26 (m,
10 H, 2 Ph), 6.76 (d, 1 H, J1,2 3.3 Hz, H-1),
6.07 (t, 1 H, J2,3 =J3,4 9.5 Hz, H-3), 5.46 (dd,
1 H, H-2), 4.54 (d, 1 H, J4,5 99 Hz, H-S), 3.98
(t, 1 H, H-4), 3.84 (s, 3 H, COOMe), 3.45 (s,
3 H, OMe); MALDI-TOFMS: Calcd for
C24H22Cl3NO9: m/z 573.0. Found: m/z 596.1
[M+Na]+.
Methyl [2-(trimethylsilyl)ethyl 2,3-di-O-ben-
zoyl-4-O-methyl-i- -glucopyranosid]uronate
D
(8).—A solution of 7 (1.87 g, 3.73 mmol) in
MeOH (10 mL) and THF (10 mL) was hydro-
genated over 10% Pd–C (230 mg) for 3 h at
rt, filtered through Celite, and the residue was
washed with MeOH and concentrated. The
residue was benzoylated with BzCl (4 mL) in
pyridine (15 mL) for 10 h at rt. The reaction
mixture was poured into ice-water and ex-
tracted with CHCl3. The extract was washed
sequentially with 5% HCl, aq NaHCO3 and
water, dried (Na2SO4), and concentrated. The
product was purified by silica-gel column
chromatography using 5:1 hexane–EtOAc as
2-(Trimethylsilyl)ethyl [methyl (2,3-di-O-
benzoyl - 4 - O - methyl - i -
D
- glucopyranosyl)-
uronate] - (1“6) - 2,3,4 - tri - O - benzyl - i -
D
-
galactopyranoside (11).—To a solution of the
trichloroacetimidate 9 (636 mg, 1.11 mmol)
and 10 (300 mg, 0.55 mmol) in CH2Cl2 (2 mL)
,
were added 4 A MS (500 mg), and the mixture
was stirred for 3 h at rt, then cooled to 0 °C.
TMSOTf (32 mL, 160 mmol) was added, and
the mixture was stirred for 1 h at 0 °C, then
neutralized with Et3N. The solids were filtered
off and washed with CHCl3. The combined
filtrate and washings were successively washed
with water, dried (Na2SO4), and concentrated.
The product was purified by silica-gel column
chromatography using 2:1 hexane–EtOAc as
1
eluent to give 8 (1.84 g, 93.1%): H NMR: l
8.07–7.34 (10 H, m, 2 Ph), 5.68 (t, 1 H,
J2,3=J3,4 9.2 Hz, H-3), 5.43 (t, 1 H, J1,2 7.6
Hz, H-2), 4.82 (d, 1 H, H-1), 4.15 (d, 1 H, J4,5
9.6 Hz, H-5), 4.12-3.97 (m, 2 H, H-4,
ꢀOCH2CH2ꢀ), 3.92 (s, 3 H, COOMe), 3.69–
3.50 (m, 1 H, ꢀOCH2CH2ꢀ), 3.48 (s, 3 H,
OMe), 0.92–0.87 (m, 2 H, ꢀOCH2CH2), −
eluent to give 11 (429 mg, 80.8%): [h]D24
+
1
21.6° (c 1.1, CHCl3). H NMR (CDCl3): l
7.98–7.18 (m, 25 H, 5 Ph), 5.63 (t, 1 H, H-3%),
5.35 (t, 1 H, H-2%), 4.88–4.51 (m, 6 H, 3×
CH2Ph), 4.81 (d, 1 H, J1%,2% 7.9 Hz, H-1%), 4.24
(d, 1 H, J1,2 7.9 Hz, H-1), 4.05 (d, 1 H, H-5%),
3.97, 3.75 (dt, 2 H, H-6), 3.94 (m, 1 H, H-4%),
3.87, 3.46 (dt, 2 H, CH2CH2SiMe3), 3.78 (s, 3
H, COOMe), 3.77 (d, 1 H, H-4), 3.75 (m, 1 H,
H-5), 3.72 (t, 1 H, H-2), 3.40 (s, 3 H, OMe),
3.35, 3.34 (dd, 1 H, H-3), 0.96 (t, 2 H,
13
0.03 (s, 9 H, Si(CH3)3); C NMR (CDCl3): l
168.6 (C-6), 100.8 (C-1), 79.1 (C-4), 74.4 (C-
3,5), 72.0 (C-2), 67.8 (ꢀOCH2CH2ꢀ), 60.4
(COOMe), 52.7 (OMe), 17.8 (ꢀOCH2CH2ꢀ),
−1.5 (SiMe3). MALDI-TOFMS: Calcd for
C27H34O9Si: m/z 530.2. Found: m/z 553.3
[M+Na]+.
Methyl (2,3-di-O-benzoyl-4-O-methyl-h-
D
-
13
glucopyranosyl trichlornacetimidate)uronate
CH2SiMe3), −0.01 (s, 9 H, SiMe3); C NMR
(9).—To a solution of 8 (115 mg, 0.22 mmol)
in CH2Cl2 (2 mL) cooled to 0 °C was added
CF3COOH (1 mL), and the mixture was
stirred for 1 h at 0 °C and concentrated. Ethyl
acetate and toluene (1:2) were added, and the
solvent was evaporated to give the 1-hydroxy
compound. To a solution of the residue in
CH2Cl2 (1 mL), cooled at 0 °C were added
trichloroacetonitrile (0.7 mL, 7 mmol) and
DBU (60 mL, 0.4 mmol). The mixture was
stirred for 2 h at 0 °C. After completion of the
reaction, the mixture was concentrated.
Column chromatography (3:1 hexane–
EtOAc) of the residue on silica gel gave 9 (89
(CDCl3): l 168.4 (C-6%), 165.5, 165.1 (2 CꢁO),
103.3 (C-1), 101.2 (C-1%), 82.0 (C-3), 79.3 (C-
2), 79.1 (C-4%), 75.1 (C-4, 2PhCH2), 74.6 (C-5,
PhCH2), 74.3 (C-3%), 74.1 (C-5%), 72.0 (C-2%),
67.9 (C-6), 67.3 (ꢀOCH2CH2ꢀ), 60.5
(COOMe), 52.7 (OMe), 18.4 (ꢀOCH2CH2ꢀ),
−1.1 (SiMe3) MALDI-TOFMS: Calcd for
C54H62O14Si: m/z 962.2. Found: m/z 985.3
[M+Na]+.
2-(Trimethylsilyl)ethyl [methyl (2,3-di-O-
benzoyl - 4 - O - methyl - i - D - glucopyranosyl)-
uronate] - (1“6) - 2,3,4 - tri - O - benzoyl - i - -
D
galactopyranoside (12).—A solution of 11
(121 mg, 0.13 mmol) in MeOH (4 mL) and
THF (2 mL) was hydrogenated over 10%
1
mg, 70%) as an amorphous mass: H NMR

14
N. Hada et al. / Carbohydrate Research 334 (2001) 7–17
1 H, H-2), 5.57 (t, 1 H, H-3%), 5.33 (t, 1 H,
H-2%), 4.82 (d, 1 H, J1%,2% 7.3 Hz, H-1%), 4.05 (m,
1 H, H-5), 4.05 (d, 1 H, H-5%), 4.05, 3.80 (dt,
2 H, H-6), 3.89 (t, 1 H, H-4%), 3.72 (s, 3 H,
COOMe), 3.36 (s, 3 H, OMe)); 13C NMR
(CDCl3): l 168.4 (C-6%), 165.6, 165.4, 165.1,
160.4 (5 CꢁO), 100.9 (C-1%), 93.5 (C-1), 78.8
(C-4%), 74.2 (C-5), 74.2 (C-3%), 74.2 (C-5%), 71.7
(C-2%), 69.3 (C-3), 69.0 (C-2), 69.0 (C-4), 68.2
(C-6), 60.4 (COOMe), 52.7 (OMe). MALDI-
TOFMS: Calcd for C51H44Cl3NO17: m/z
1048.6. Found: m/z 1071.6 [M+Na]+.
Pd–C (100 mg) for 30 min at rt, then filtered
through Celite. The filtrate was concentrated,
and the residue was benzoylated with BzCl
(0.5 mL) in pyridine (1.5 mL) for 1 h at rt.
The reaction mixture was poured into ice-wa-
ter and extracted with CHCl3. The extract was
washed sequentially with 5% HCl, aq
NaHCO3 and water, dried (Na2SO4), and con-
centrated. The product was purified by silica-
gel column chromatography using 30:1
benzene–EtOAc as eluent to give 12 (102 mg,
80.7%): [h]2D5 +103.4° (c 1.8 CHCl3); 1H
NMR (CDCl3): l 8.06–7.19 (m, 25 H, 5 Ph),
5.84 (d, 1 H, H-4), 5.70 (t, 1 H, H-2), 5.62 (t,
1 H, H-3%), 5.48, 5.46 (dd, 1 H, H-3), 5.38 (t,
1 H, H-2%), 4.85 (d, 1 H, J1%,2% 6.7 Hz, H-1%),
4.67 (d, 1 H, J1,2 8.6 Hz, H-1), 4.10 (m, 1 H,
H-5), 4.06 (d, 1 H, H-5%), 4.05, 3.85 (dt, 2 H,
H-6), 3.93 (t, 1 H, H-4%), 3.87, 3.44 (dt, 2 H,
CH2CH2SiMe3), 3.72 (s, 3 H, COOMe), 3.38
(s, 3 H, OMe), 0.73 (t, 2 H, CH2SIMe3),
−0.12 (s, 9 H, SiMe3); 13C NMR (CDCl3): l
168.4 (C-6%), 165.5, 165.2, 165.1 (5 CꢁO),
101.1 (C-1%), 100.8 (C-1), 78.8 (C-4%), 73.2
(C-5), 74.2 (C-3%), 74.2 (C-5%), 71.9 (C-2%), 71.9
(C-3), 69.9 (C-2), 69.9 (C-6), 68.8 (C-4), 67.5
(ꢀOCH2CH2ꢀ), 60.4 (COOMe), 52.7 (OMe),
17.7 (ꢀOCH2CH2ꢀ), −1.4 (SiMe3). MALDI-
TOFMS: Calcd for C54H56O17Si: m/z 1004.3.
Found: m/z 1027.4 [M+Na]+.
2-(Trimethylsilyl)ethyl [methyl (2,3-di-O-
benzoyl - 4 - O - methyl - i -
D
- glucopyranosyl)-
uronate] - (1“6) - 2,3,4 - tri - O - benzoyl - i -
galactopyranosyl - (1“6) - 2,3,4 - tri - O - benzyl-
i- -galactopyranoside (14).—Compound 14
D
-
D
was prepared from 13 (700 mg, 0.67 mmol)
and 10 (180 mg, 0.33 mmol) as described in
the previous glycosylation procedure of disac-
charide 11, yielding 474 mg (84.5%) of a
1
syrup: [h]2D5 +58.2° (c 0.9 CHCl3); H NMR
(CDCl3): l 4.80 (d, 1 H, J1,2. 7.9 Hz, H-1%),
4.76 (d, 1 H, J1%,2% 9.1 Hz, H-1%%), 4.29 (d, 1 H,
J1,2 79 Hz, H-1), 3.55 (s, 3 H, COOMe), 3.39
(s, 3 H, OMe); 13C NMR (CDCl3): l 104.8
(C-1), 102.6 (C-1%), 102.3 (C-1%%). MALDI-
TOFMS: Calcd for C81H84O22Si: m/z 1436.6.
Found: m/z 1459.6 [M+Na)]+.
2-(Trimethylsilyl)ethyl [methyl (2,3-di-O-
benzoyl - 4 - O - methyl - i -
D
- glucopyranosyl)-
Methyl (2,3-di-O-benzoyl-4-O-methyl-i-
glucopyranosyl)uronate - (1“6) - 2,3,4 - tri - O-
benzoyl-h- -galactopyranosyl trichloroacetimi-
D
-
uronate] - (1“6) - 2,3,4 - tri - O - benzoyl - i -
galactopyranosyl-(1“6)-2,3,4-tri-O-benzoyl-
i- -galactopyranoside (15).—Compound 15
D
-
D
D
date (13).—To a solution of 12 (102 mg, 0.10
mmol) in CH2Cl2 (2 mL) cooled to 0 °C was
added CF3COOH (0.6 mL), and the mixture
was stirred for 1 h at rt and concentrated.
Ethyl acetate and toluene (1:2) were added,
and the solvents were evaporated to give the
1-hydroxy compound. To a solution of the
residue in CH2Cl2 (1 mL), cooled at 0 °C were
added trichloroacetonitrile (0.4 mL, 4 mmol)
and DBU (100 mL, 0.67 mmol). The mixture
was stirred for 1 h at 0 °C. After completion
of the reaction, the mixture was concentrated.
Column chromatography (10:1 benzene–
EtOAc) of the residue on silica gel gave 13 (87
was prepared from 14 (360 mg, 0.25 mmol) as
described for the preparation of 13, yielding
307 mg (83.0%) of a syrup: [h]2D5 +98.4° (c 0.7
CHCl3);1H NMR (CDCl3): l 4.81 (d, 1 H, J1,2
7.9 Hz, H-1), 4.74 (d, 1 H, J1%,2% 7.9 Hz, H-1%),
4.61 (d, 1 H, J1%%,2%% 7.3 Hz, H-1%%), 3.70 (s, 3 H,
COOMe), 3.40 (s, 3 H, OMe). 13C NMR
(CDCl3): l 103.0 (C-1%%), 102.9 (C-1%), 102.3
(C-1). MALDI-TOFMS: Calcd for C81H78O25
Si: m/z 1479.6. Found: m/z 1502.6 [M+Na]+.
Methyl (2,3-di-O-benzoyl-4-O-methyl-i-
glucopyranosyl)uronate - (1“6) - 2,3,4 - tri - O-
benzoyl-i- -galactopyranosyl-(1“6)-2,3,4-
tri-O-benzoyl-h- -galactopyranosyl trichloro-
D
-
D
D
1
mg, 83.0%) as an amorphous mass: H NMR
acetimidate (16).—Compound 16 was pre-
pared from 15 (100 mg, 67.6 mmo1) as de-
scribed for the preparation of 13, yielding 103
(CDCl3): l 8.32 (s, 1 H, NH), 8.08–7.22 (m,
25 H, 5 Ph), 6.73 (d, 1 H, J1,2 3.7 Hz, H-1),
6.00 (d, 1 H, H-4), 5.96 (m, 1 H, H-3), 5.84 (t,
1
mg (quant) of a syrup: H NMR (CDCl3): l

N. Hada et al. / Carbohydrate Research 334 (2001) 7–17
15
8.38 (s, 1 H, NH), 6.75 (d, 1 H, J1,2 3.7 Hz,
H-1), 4.71 (d, 1 H, J1%,2% 7.9 Hz, H-1%), 4.44 (d,
1 H, J1%%,2%% 7.3 Hz, H-1%), 3.62 (s, 3 H, COOMe),
3.36 (s, 3 H, OMe). 13C NMR (CDCl3): l
100.9 (C-1%%), 100.7 (C-1%), 93.7 (C-1). MALDI-
TOFMS: Calcd for C78H66Cl3NO25: m/z
1522.7. Found: m/z 1545.6 [M+Na]+.
tracted with CHCl3. The extract was succes-
sively washed with aq NaHCO3 and water,
dried (Na2SO4), and concentrated. This com-
pound was purified by column chromatogra-
phy (5:1 hexane–EtOAc) on silica gel to give
compound 17 (10.2 g, 47.7%): 1H NMR
(CDCl3): l 7.23–7.12 (m, 5 H, Ph), 4.44 (s, 2
H, CH2Ph), 3.59 (s, 3 H, COOMe); MALDI-
TOFMS: Calcd for C14H20O3: m/z 236.1.
Found: m/z 259.1 [M+Na]+.
2-(Trimethylsilyl)ethyl
4-O-methyl-i-
D
-
glucopyranosyluronic acid-(1“6)-i-
D
-galac-
topyranoside (1).—To a solution of 12 (26 mg,
25.6 mmol) in 2:1 MeOH–water (3 mL) was
added NaOMe (40 mg), and the mixture was
stirred for 2 h at rt, then neutralized with
Amberlite IR-120 (H+) resin. The resin was
filtered off and washed with MeOH. The
filtrate and washings were combined and con-
centrated. Column chromatography (3:1
MeOH–water) of the residue on Sephadex
LH-20 gave 1 (12.0 mg, quant): [h]2D5 −43.5°
(c 0.6 MeOH); MALDI-TOFMS: Calcd for
C18H34O12Si: m/z 470.5. Found: m/z 493.2
[M+Na]+, 515.2 [M+2Na−H]+.
5-(2-Aminoethyleneaminocarbonyl)pentyl
benzyl ether (18).—Compound 17 (1 g, 4.5
mmol) was treated with ethylenediamine (10
mL) at 70 °C for 18 h. After completion of the
reaction, the mixture was concentrated.
Column chromatography (2:1 CHCl3–MeOH)
of the residue on silica gel to give 18 (917 mg,
1
77.1%) as an amorphous mass: H NMR
(CD3OD): l 7.38–7.32 (m, 5 H, aromatic),
4.48 (s, 2 H, CH2Ph), 3.28 (br,s, 2 H,
CH2NHCOO), 2.79 (br,s, 2 H, CH2NH2);
MALDI-TOFMS: Calcd for C15H24N2O2: m/z
264.1. Found: m/z 287.1 [M+Na]+.
2-(Trimethylsilyl)ethyl
glucopyranosyluronic acid-(1“6)-i-
topyranosyl - (1“6) - i - - galactopyranoside
4-O-methyl-i-
D
-
D
-galac-
Benzyl 5 - [2 - (2,2,2 - trichloroethoxycar-
bonyl)aminoethyleneaminocarbonyl]pentyl ether
(19).—To a solution of 18 (37 mg, 0.14 mmol)
in pyridine (1 mL) cooled to 0 °C was added
TrocCl (58 mL, 0.42 mmol), and the mixture
was stirred for 30 min at rt and concentrated.
Column chromatography (90:1 CHCl3–
MeOH) of the residue on silica gel gave 19 (50
D
(2).—To a solution of 15 (27 mg, 17.9 mmol)
in 5:1 MeOH–water (2.4 mL) was added
NaOMe (40 mg), and the mixture was stirred
for 2 h at rt, then neutralized with Amberlite
IR-120 (H+) resin. The resin was filtered off
and washed with MeOH. The filtrate and
washings were combined and concentrated.
Column chromatography (3:1 MeOH–water)
of the residue on Sephadex LH-20 gave 2 (11.3
1
mg, 80.3 %); H NMR (CDCl3): l 7.34–7.27
(m, 5 H, Ph), 6.20 (br,s, 1 H, NHCO), 5.91
(br,s, 1 H, NHCOO), 4.71 (s, 2 H, CH2CCl3),
4.49 (s, 2 H, CH2Ph), 3.36 (t, 2 H,
CH2NHCO), 3.32 (t, 2 H, CH2NHCOO);
MALDI-TOFMS: Calcd for C18H25Cl3N2O4:
m/z 438.1. Found: m/z 461.0 [M+Na]+.
5-[2-(2,2,2-Trichloroethoxycarbonyl)amino-
ethyleneaminocarbonyl]pentanol (20).—A solu-
tion of 19 (340 mg, 0.77 mmol) in MeOH (4
mL) was hydrogenated over 10% Pd–C (60
mg) for 20 min at rt, then filtered through
Celite. The filtrate was concentrated, and the
residue was purified by silica-gel column chro-
matography using 10:1 CHCl3–MeOH as elu-
2
mg, quant): [h]D5 −32.6° (c 0.6 MeOH);
MALDI-TOFMS: Calcd for C24H44O17Si: m/z
632.3. Found: m/z 655.3 [M+Na]+, 677.3
[M+2Na−H]+.
Benzyl
5-methoxycarbonylpentyl
ether
(17).—To a solution of o-caprolactone (10
mL, 90.2 mmol) in MeOH (100 mL) was
added NaOMe (80 mg), and the mixture was
stirred for 3 h at rt, then neutralized with
Amberlite IR-120 (H+) resin. The resin was
filtered off and washed with 1:1 CHCl3–
MeOH. The filtrate and washings were com-
bined and concentrated. To a solution of this
compound in DMF (262 mL) was added NaH
in oil (19 g) and BnBr (56 mL). The mixture
was stirred for 4 h at rt, and then MeOH was
added to destroy excess NaH. The reaction
mixture was poured into ice-water and ex-
1
ent to give 20 (178 mg, 65.8%); H NMR
(CDCl3): l 6.73 (br,s, 1 H, NHCO), 6.32 (br,s,
1 H, NHCOO), 4.72 (s, 2 H, CH2CCl3), 3.39 (t,
2 H, CH2NHCO), 3.37 (t, 2 H, CH2-NHCOO);
MALDI-TOFMS: Calcd for C11H19Cl3N2O4:
m/z 348.0. Found: m/z 371.3 [M+Na]+.

16
N. Hada et al. / Carbohydrate Research 334 (2001) 7–17
5-(2-Aminoethyleneaminocarbonyl)pentyl
[methyl (2,3-di-O-benzoyl-4-O-methyl-i-
glucopyranosyl)uronate] - (1“6) - 2,3,4 - tri - O-
benzoyl-i- -galactopyranoside (23).—To a
5-[2-(2,2,2,-Trichloroethoxycarbonyl)amino-
D
-
ethyleneaminocarbonyl]pentyl [methyl (2,3-di-
O-benzoyl-4-O-methyl-i-
D
-glucopyranosyl)-
D
uronate] - (1“6) - 2,3,4 - tri - O - benzoyl - i -
D
-
solution of 21 (185 mg, 0.15 mmol) in AcOH
(10 mL) was added zinc powder (200 mg), and
the mixture was stirred for 12 h at rt. The
solids were filtered off and washed with
CHCl3. The combined filtrate and washings
were successively washed with water, dried
(Na2SO4), and concentrated. The product was
purified by silica-gel column chromatography
using 10:1 CHCl3–MeOH as eluent to give 23
(92 mg, 58.0%): [h]2D5 +74.8° (c 0.6 CHCl3);
1H NMR (CDCl3): l 8.06–7.21 (m, 25 H, 5
Ph), 5.99 (br,s, 1 H, NHCO), 5.80 (d, 1 H,
H-4), 5.65 (t, 1 H, H-2), 5.61 (t, 1 H, H-3%),
5.49, 5.47 (dd, 1 H, H-3), 5.38 (t, 1 H, H-2%),
4.84 (d, 1 H, J1%,2% 7.3 Hz, H-1%), 4.59 (d, 1 H,
J1,2 7.9 Hz, H-1), 4.10 (m, 1 H, H-5), 4.06 (d,
1 H, H-5%), 4.07, 3.80 (m, 2 H, H-6), 3.92 (t, 1
H, H-4%), 3.75 (s, 3 H, COOMe), 3.38 (s, 3 H,
OMe), 3.28 (m, 2 H, OCH2CH2), 3.28 (m, 2
H, CH2NHCOꢀ), 2.83 (br,d, 2 H, CH2NH2),
2.02 (br,s, 2 H, NH2), 1.88 (t, 2 H, CH2CO),
1.43 (m, 2 H, CH2CH2CO), 1.32 (m, 2 H,
OCH2CH2), 1.12 (t, 2 H, OCH2CH2CH2);
MALDI-TOFMS: Calcd for C57H60N2O18:
m/z 1060.4. Found: m/z 1083.4 [M+Na]+.
5-(2-Aminoethyleneaminocarbonyl)pentyl
galactopyranoside (21).—To a solution of the
trichloroacetimidate 13 (244 mg, 0.23 mmol)
and 20 (40 mg, 0.12 mmol) in CH2Cl2 (1 mL)
,
were added molecular sieves 4 A (300 mg),
and the mixture was stirred for 1 h at rt, then
cooled to 0 °C. TMSOTf (22 mL, 120 mmol)
was added, and the mixture was stirred for 1 h
at 0 °C, then neutralized with Et3N. The solids
were filtered off and washed with CHCl3. The
combined filtrate and washings were succes-
sively washed with water, dried (Na2SO4), and
concentrated. The product was purified by
silica-gel column chromatography using 1:3
benzene–EtOAc as eluent to give 21 (100 mg,
64.7%): [h]2D4 +74.2° (c 0.7, CHCl3). 1H NMR
(CDCl3): l 8.06–7.21 (m, 25 H, 5×Ph), 5.80
(d, 1 H, H-4), 5.64 (t, 1 H, H-2), 5.61 (t, 1 H,
H-3%), 5.48 (dd, 1 H, H-3), 5.37 (t, 1 H, H-2%),
4.85 (d, 1 H, J1%,2% 7.3 Hz, H-1%), 4.69 (q, 2 H,
OCH2CCl3), 4.58 (d, 1 H, J1,2 7.9 Hz, H-1),
4.10 (m, 1 H, H-5), 4.07 (d, 1 H, H-5%), 4.03,
3.91 (dt, 2 H, H-6), 3.91 (t, 1 H, H-4%), 3.78 (s,
3 H, COOMe), 3.38 (s, 3 H, OMe), 3.36 (m, 2
H, OCH2CH2), 3.36 (m, 2 H, CH2NHCOꢀ),
3.26 (m, 2 H, CH2NHCOO), 1.89 (t, 2 H,
CH2CO), 1.44 (m, 2 H, CH2CH2CO), 1.14 (m,
2 H, OCH2CH2), 0.88 (t, 2 H, OCH2CH2CH2);
MALDI-TOFMS: Calcd for C60H61Cl3N2O20:
m/z 1235.7 Found: m/z 1258.4 [M+Na]+.
5-[2-(2,2,2,-Trichloroethoxycarbonyl)amino-
ethyleneaminocarbonyl]pentyl [methyl (2,3-di-
[methyl (2,3-di-O-methyl-i-
uronate] - (1“6) - 2,3,4 - tri - O - benzoyl - i -
galactopyranosyl-(1“6)-2,3,4-tri-O-benzoyl-
i-
D
-glucopyranosyl)-
D
-
D
-galactopyranoside (24).—Compound 24
was prepared from 22 (56 mg) as described for
the preparation of 23, yielding 46 mg (91.5%)
of a syrup: [h]2D5 +73.3° (c 0.6 CHCl3). H
1
O-benzoyl-4-O-methyl-i-
uronate] - (1“6) - 2,3,4 - tri - O - benzoyl - i -
galactopyranosyl-(1“6)-2,3,4-tri-O-benzoyl-
i- -galactopyranoside (22).—Compound 22
D
-glucopyranosyl)-
D
-
NMR (CDCl3): l 4.78 (d, 1 H, J1%%,2%% 7.9 Hz,
H-1%%), 4.76 (d, 1 H, J1%,2% 7.9 Hz, H-1%), 4.64 (d,
1 H, J1,2 7.3 Hz, H-1), 3.70 (s, 3 H, COOMe),
3.40 (s, 3 H, OMe). 13C NMR (CDCl3): l
101.7 (C-1), 101.5 (C-1%), 101.4 (C-1%%);
MALDI-TOFMS: Calcd for C84H82N2O26:
m/z 1534.6. Found: m/z 1557.9 [M+Na]+.
N,N%,N%%-Tri-{5-[methyl (2,3-di-O-benzoyl-
D
was prepared from 16 (86 mg, 56.5 mmol) and
20 (10 mg, 28.7 mmol) as described in the
previous glycosylation procedure of 21, yield-
ing 29 mg (59.7%) of a syrup: [h]2D5 +72.1° (c
1.3 CHCl3); 1H NMR (CDCl3): l 4.77 (d, 1 H,
J1%%,2%% 7.9 Hz, H-1%%), 4.76 (d, 1 H, J1%,2% 7.9 Hz,
H-1%), 4.68 (d, 1 H, J1,2 7.9 Hz, H-1), 3.72 (s,
4 - O - methyl - i -
D
- glucopyranosyl)uronate]-
-galactopyran-
(1“6)-2,3,4-tri-O-benzoyl-i-
D
13
3 H, COOMe), 3.41 (s, 3 H, OMe). C NMR
osyloxypentylcarbonylaminoethyl} - 1,3,5 - ben-
zenetriamide (25).—To a solution of 23 (23
mg, 21.6 mmol) in CH2Cl2 (1 mL) were added
triethylamine (3 mL) and 1,3,5-benzenetricar-
bonyl trichloride (1.3 mg, 5.0 mmol). The mix-
(CDCl3): l 101.6 (C-1), 101.5 (C-1%%), 101.4
(C-1%);
MALDI-TOFMS:
Calcd
for
C87H83Cl3N2O28: m/z 1709.6. Found: m/z
1732.8 [M+Na]+.

N. Hada et al. / Carbohydrate Research 334 (2001) 7–17
17
syl - (1“6) - i -
D
- galactopyranosyloxy]pentyl-
ture was stirred for 5 min at rt. After comple-
tion of the reaction, the mixture was concen-
trated. Column chromatography (20:1
CHCl3–MeOH) of the residue on silica gel
carbonylaminoethyl}-1,3,5-benzenetriamide (4).
—To a solution of 26 (4.3 mg, 0.9 mmol) in 5:1
MeOH–water (0.6 mL) was added NaOMe
(16 mg), and the mixture was stirred for 4 h at
rt, then neutralized with Amberlite IR-120
(H+) resin. The resin was filtered off and
washed with MeOH. The filtrate and washings
were combinedand concentrated. Column
chromatography (3:1 MeOH–water) of the
residue on Sephadex LH-20 gave 4 (2.0 mg,
quant) [h]2D5 −23.2° (c 0.1 water); MALDI-
TOFMS: Calcd for C90H144N6O57: m/z 2220.8.
Found: m/z 2243.2 [M+Na]+, 2309.5 [M−
3H+4Na]+.
1
gave 25 (11 mg, 64.1%); H NMR (CDCl3): l
5.80 (d, 3 H, J3,4 3.0 Hz, H-4), 5.61 (t, 6 H, J
9.1 Hz, H-2, H-3%), 5.50 (dd, 3 H, J3,4 3.7, J2,3
10.4 Hz, H-3), 5.36 (t, 3 H, H-2%), 4.85 (d, 3 H,
J1%,2% 7.3 Hz, H-1%), 4.57 (d, 3 H, J1,2 7.9 Hz,
H-1), 4.10 (m, 3 H, H-5), 4.07 (d, 3 H, H-5%),
4.03, 3.91 (dt, 6 H, H-6), 3.91 (t, 3 H, H-4%),
3.73 (s, 9 H, COOMe), 3.37 (s, 9 H, OMe); 13C
NMR (CDCl3): 168.5, 132.6–128.3, 101.3,
101.1, 78.9, 76.8, 74.1, 73.3, 71.9, 71.6, 70.0,
69.8, 68.8, 68.6, 60.4, 52.7, 29.7–11.5.
MALDI-TOFMS: Calcd for C180H180N6O57:
m/z 3337.1. Found: m/z 3360.2 [M+Na]+.
N,N%,N%%-Tri-{5-[methyl (2,3-di-O-benzoyl-
Acknowledgements
4 - O - methyl - i -
(1“6)-2,3,4-tri-O-benzoyl-i-
syl - (1“6) - 2,3,4 - tri - O - i -
D
- glucopyranosyl)uronate]-
-galactopyrano-
- galactopyrano-
This work was supported by a Grant-in-Aid
for Scientific Research (No. 12672062) from
the Ministry of Education, Science, Sports and
Culture of Japan. We gratefully acknowledge
the financial support of Uehara Memorial
Foundation. The authors are grateful to Mrs
J. Hada for providing NMR and MS data.
D
D
syloxypentylcarbonylaminoethyl] - 1,3,5 - ben-
zenetriamide (26).—To a solution of 24 (26
mg, 16.8 mmol) in CH2Cl2 (1 mL) were added
triethylamine (2.4 mL) and 1,3,5-benzenetricar-
bonyl trichloride (1 mg, 3.9 mmol). The mix-
ture was stirred for 5 min at rt. After
completion of the reaction, the mixture was
concentrated. Column chromatography (20:1
CHCl3–MeOH) of the residue on silica gel
gave 26 (8.6 mg, 77.8%): MALDI-TOFMS:
Calcd for C261H246N6O81: m/z 4759.5. Found:
m/z 4782.0 [M+Na]+.
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syloxy]pentylcarbonylaminoethyl} - 1,3,5 - ben-
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N,N%,N%%-Tri-{5-[4-O-methyl-i-
D
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ranosyluronic acid (1“6)-i- -galactopyrano-
D