Synthesis and NMR assignments of galactosylgloboside and its β-d-GalNAc-(1→4)-α-D-Gal-linked positional isomer in a conjugatable form

Article abstract of DOI:10.1016/S0008-6215(99)00018-X

Two pentasaccharides suitable for conjugation, namely 3-aminopropyl glactosylgloboside and its β-D-GalNAc-(1→4)-α-D-Gal-linked positional isomer, were synthesized from 3(III),4(III)-di-O-unprotected globotrioside and the trichloroacetimidate of β-D-Gal-(1→3)-β-D-GalNPhth derivative. Glycosylation at both positions led to the formation of β-D-GalNPhth-(1→4)-α-D-Gal and β-D-GalNPhth-(1→3)-α-D-Gal-linked products in a ratio of 1:1 without selectivity. Complete NMR spectral assignments are also described. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00018-X

Carbohydrate Research 315 (1999) 251–261
Synthesis and NMR assignments of galactosylgloboside
and its b-
D
-GalNAc-(1“4)-a-
D
-Gal-linked positional
isomer in a conjugatable formꢀ
Wei Zou *, Jean-Robert Brisson, Suzon Larocque, Rebecca L. Gardner,
Harold J. Jennings
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada
Received 13 October 1998; accepted 11 January 1999
Abstract
Two pentasaccharides suitable for conjugation, namely 3-aminopropyl glactosylgloboside and its b-
(1“4)-a-
-Gal-linked positional isomer, were synthesized from 3III,4III-di-O-unprotected globotrioside and the
trichloroacetimidate of b- -Gal-(1“3)-b- -GalNPhth derivative. Glycosylation at both positions led to the forma-
tion of b- -GalNPhth-(1“4)-a- -Gal and b- -GalNPhth-(1“3)-a- -Gal-linked products in a ratio of 1:1 without
selectivity. Complete NMR spectral assignments are also described. © 1999 Elsevier Science Ltd. All rights reserved.
D-GalNAc-
D
D
D
D
D
D
D
Keywords: Synthesis; Galactosylgloboside; b-
D
-Gal-(1“3)-b-
D
-GalNPhth synthon; b-
D
-GalNAc-(1“3/4)-a-D-Gal; 3-
Aminopropyl spacer
galactosylgloboside also has the structure of
the defucosylated Globo-H antigen, which has
been clinically related to breast, pancreatic
and stomach cancers, and therefore is of inter-
est for therapeutic applications [8,9].
The syntheses of the glactosylgloboside
[10,11] and corresponding sialylated and sul-
fated forms [12] have been previously re-
ported. Here, we describe the synthesis of
1. Introduction
Galactosylgloboside, also called stage-spe-
cific embryonic antigen-3 (SSEA-3), was
found as glycosphingolipid (globopentaosyl
ceramide) in human [1] and green monkey [2]
kidney tissues, as well as in human teratocar-
cinoma [3] and seminoma cells [4]. Part of its
structure, globotrioside and globotetraoside,
are also epitopes recognized by antibodies of
the P blood-group system and by various bac-
terial adhesin proteins [5–7]. Furthermore,
galactosylgloboside and its b-
D
-GalNAc-(1“
4)-a- -Gal-linked positional isomer with an
D
aminopropyl spacer. These oligosaccharides
will eventually be conjugated to protein carri-
ers to investigate their immunological proper-
ties, such as antibody production and
specificity and/or their cross-reactivity with
other globosides, and as screening antigens to
find monoclonal antibodies useful as either
therapeutic or diagnosis reagents.
ꢀ This is NRCC Publication No. 42375.
* Corresponding author. Tel.: +1-613-990-0821; fax: +1-
613-941-1327.
E-mail address: wei.zou@nrc.ca (W. Zou)
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00018-X

252
W. Zou et al. / Carbohydrate Research 315 (1999) 251–261
disaccharide 10 from thiogalactoside 8 as
2. Results and discussion
donor [19] and GlcNPhth 9 as acceptor [20]
(NIS/TfOH) in excellent yield (70–80%). The
reason we chose 6-O-benzyl-protected 8 in-
stead of peracetylated thiogalactoside was be-
cause we wished eventually to synthesize
dimeric Lewis antigens (Lea–Lea, Leb–Lea) by
selective glycosylation at the 3-O-position of
the terminal galactosyl residue. Reductive
ring-opening of the benzylidene acetal of 10
(NaCNBH3/HCl) afforded 11 with a 4-OH in
The benzylidene derivative [PhCH(OMe)2/
p-TsOH/CH3CN] of 3-azidopropyl lactoside
(1) [13] was conventionally benzylated (BnBr/
NaH/DMF) to give 2 in 87% yield (two steps).
Removal of the benzylidene protecting group
(60% HOAc) to give 3, followed by selective
benzoylation at the 6-OH (BzCl/Py/CH2Cl2),
afforded 4 in 76% yield. Glycosylation of 4
with thiogalactoside 5 [14] (NIS/TfOH) pro-
vided the globotrioside derivative 6 in a mod-
erate yield (47%). The a-(1“4) glycosidic
linkage thus formed was confirmed by NMR
spectroscopy (lH 4.968 ppm, J1,2 3.2 Hz and
lC 100.07 ppm). Removal of the isopropyli-
dene group (1:9 90% TFA–CH2Cl2) furnished
7, an acceptor with 3III,4III-di-O-unprotected
hydroxyl groups, in 90% yield.
79% yield. The transformation of 11 into b-
D-
Gal-(1“3)-b- -GalNPhth derivative 12 (89%)
D
was then performed in two steps: (1) treatment
with triflic anhydride in CH2Cl2/pyridine fol-
lowed by (2) an SN2 reaction with Me4NOAc
in DMF. The chemical shift of the equatorial
H-4b in 12 was assigned at lH 5.48 (J3,4 3.0
Hz). Removal of the 2-(trimethylsilyl)ethyl
group in 12 (TFA/CH2Cl2) was quantitative,
and without purification the 1-OH derivative
b-
D
-Gal-(1“3)- -GalNPhth is among the
D
most difficult glycosidic linkages to be con-
structed [15]. The b-galactosylation at the 3-
O-position of GalNPhth resulted in an
extremely poor yield (B5%) of product [16],
likely due to the steric hindrance caused by
the neighboring phthaloyl group. As an alter-
native, 2-azido-2-deoxy-galactose derivatives
have commonly been used to avoid this prob-
lem [15]; however, unlike the phthaloyl group,
the 2-azido group is not good for neighboring
participation. As a result, further manipula-
tion, such as reduction and phthaloyl protec-
tion [15], was required to elaborate the
formation of b-linked GalNPhth glycosides
through thioglycoside or trichloroacetimidate
as glycosyl donors. Moreover, the synthesis of
the 2-azido derivative itself on a large-scale
was problematic [17]. In summary, the synthe-
was
transformed
(CNCCl3/DBU)
into
trichloroacetimidate 13 in 88% yield (two
steps).
The glycosylation reaction with N-ph-
thaloyl-protected trichloroacetimidate donors
have been systematically investigated and re-
viewed by Schmidt and Kinzy [21]. In the
reaction with 3,4-di-O-unprotected galactose
acceptors, higher reactivity was generally ob-
served at the 3-O-position. As a result highly
selective glycosylation was achieved by taking
advantage of the differences in reactivity.
However, lower regioselectivity in the glycosy-
lation was also observed. The reaction of the
3,4-di-O-unprotected
O-benzyl-galactose
derivative with GlcNPhth trichloroacetimidate
donor led to reaction at both positions [22];
thus b-(1“3)- and b-(1“4)-linked products
were formed in a ratio of 2–3:1.
Following the standard glycosylation proce-
dure [23], the reaction of 7 with 13
(Me3SiOTf/CH2Cl2) afforded two pentasac-
charide derivatives, namely a b-(1“3)-linked
14 and a b-(1“4)-linked 15 (see Scheme 2).
The structures were established by FABMS
and NMR spectroscopy. The two positional
isomers were formed in a ratio of ca. 1:1 as
estimated by TLC. However, the complete
separation of the two isomers by chromatog-
sis of b-
D
-Gal(1“3)- -GalNPhth takes many
D
steps and is very time consuming (see Scheme
1).
By contrast, b-
D
-Gal-(1“3)- -GlcNPhth
D
can be relatively easily synthesized. This disac-
charide is also a very important building block
in the synthesis of Lewis antigens (Lea and
Leb). The chemistry of converting Glc (GlcN)
to Gal (GalN) by an SN2 reaction has been
investigated [18]. Therefore, it should be possi-
ble to efficiently transform b-
D
-Gal-(1“3)-b-
-GlcNPhth (11) into b- -Gal-(1“3)-
-GalNPhth (12). We began by synthesizing
D
D
b-D

W. Zou et al. / Carbohydrate Research 315 (1999) 251–261
253
raphy was very difficult, and as repeated chro-
matography was required, the yield was greatly
sacrificed. To overcome this difficulty the mix-
ture was treated with hydrazine hydrate to
remove the N-phthaloyl group and the O-acetyl
groups, and the products obtained were then
fully acetylated (Ac2O/Py) to give a mixture of
16 and 17. The separation of 16 (34%) from 17
(32%) was then achieved by chromatography
on silica gel in 66% overall yield (two steps).
The structures of 16 and 17 were unambigu-
ously assigned by 2D NMR spectroscopic anal-
ysis. A characteristic chemical shift of H-4III, a
broad singlet at 5.35 ppm, was observed in 16
and that of H-3III, a broad doublet at 5.24 ppm,
in 17. The partial 2D 1H COSY spectrum of 17
is shown in Fig. 1. Removal of protecting
groups (O-acetyl groups and O-benzyl groups)
was then achieved by the treatment of 16 or 17
with 0.1% NaOMe/MeOH followed by deben-
zylation (H2–Pd/C) to obtain pentasaccharides
18 (79%) and 19 (75%), respectively (see Scheme
3).
Complete NMR assignments of galactosyl-
globoside 18 and its positional isomer 19 were
achieved based on various 2D NMR spectra
(COSY, NOESY, ROESY, TOCSY, HSQC
and HSQC–TOCSY); the data are listed in
1
Table 1. A comparison of both H and 13C
anomeric resonances shows that only slight
differences in chemical shifts are observed for
GalNAc-(1“3/4)-a- -Gal.
D
Scheme 1. Reagents and conditions: (a) i. PhCH(OMe)2/p-TsOH/CNCH3, ii. NaH/BnBr/DMF; (b) 60% HOAc; (c) BzCl/CH2Cl2/
Py at 0 °C; (d) NIS/TfOH/CH2Cl2 at −40 °C; (e) 10% TFA (aq 90%)/CH2Cl2 at 0 °C; (f) NaCNBH3/HCl/THF at 0 °C; (g) i.
(TfO)2O/Py at 0 °C, ii. Me4NOAc/DMF at rt; (h) i. 33% TFA/CH2Cl2, ii. CNCCl3/DBU at 0 °C.

254
W. Zou et al. / Carbohydrate Research 315 (1999) 251–261
Scheme 2. Reagents and conditions: (a) Me3SiOTf/CH2Cl2 at −40 °C; (b) i. NH2NH2H2O/95% EtOH reflux 16 h, ii. Ac2O/Py.
We also observed repeatedly the partial re-
duction of azido groups in azidopropyl gly-
coside derivatives to corresponding amines
during the FABMS analysis. The same observa-
tion has been previously reported by Peltier et
al. [24].
Column chromatography was performed on
Silica Gel 60 (E. Merck, 230–400 mesh), and
fractions were monitored by TLC on Silica Gel
60 F254 (E. Merck) unless otherwise noted.
Detection was performed by examination un-
der UV light and by charring with 5% H2SO4
in EtOH. Solutions were concentrated at or
below 40 °C and dried with anhydrous Na2SO4.
3-Azidopropyl 2,3-di-O-benzyl-4,6-di-O-be-
3. Experimental
nzylidene-i-
tri-O-benzyl-i-
D
-galactopyranosyl-(1“4)-2,3,6-
-glucopyranoside (2).—To a
General methods.—Optical rotations were
measured at room temperature (rt) with a
Perkin–Elmer 243 polarimeter, using a 10 cm,
D
solution of 3-azidopropyl lactoside (1) (2.0 g,
4.51 mmol) in dry MeCN (20 mL), Ph-
CH(OMe)2 (1.5 mL) and p-toluenesulfonic acid
(50 mg) were added. The mixture was stirred at
rt for 6 h, neutralized by the addition of
triethylamine (0.5 mL), and concentrated. The
residue obtained above was dissolved in
EtOAc–MeOH, and precipitated with the addi-
tion of hexane. The solid was collected and
dried (2.2 g). To a solution of the above product
in DMF (20 mL), NaH (50%, 1.5 g) was added.
The mixture was stirred at rt for 0.5 h. Benzyl
bromide (5 mL) was added to the mixture and
the stirring was continued for 3 h. Methanol (2
mL) was added to the mixture to destroy excess
1
1 mL cell. H and 13C NMR spectra were
recorded at 500 and 125 MHz, respectively,
with an INOVA-500 instrument at 300 K unless
otherwise noted. Chemical shifts are given in
ppm relative to the signal of internal Me4Si or
indirectly to solvent signals 7.25 (CDCl3) or
1
2.225 (acetone in D2O) for H NMR spectra,
and to the solvent signals 76.9 (CDCl3) or 31.07
(internal acetone) for 13C NMR spectra. The 1H
NMR resonances of oligosaccharides were as-
signed on the basis of 2D 1H COSY and 1H–13C
chemical shift correlated experiments. FABMS
analyses were performed with a Jeol JMS-
AX505H mass spectrometer.

W. Zou et al. / Carbohydrate Research 315 (1999) 251–261
255
NaH, and then cold water (100 mL) was
added. The emulsion was extracted with
EtOAc (150 mL). The organic phase was sub-
sequently washed with water, aq NaHCO3,
and water, dried and concentrated to a
residue. Purification by chromatography (1:3
EtOAc–hexane) gave 2 (3.8 g, 87%) as a wax:
[h]D +10.4° (c 2.2, MeOH); 1H NMR
(CDCl3) l 1.869 (m, 2 H, CH2CH2N3), 4.011
(d, 1 H, H-4%, J3,4 3.1 Hz), 4.364 (d, 1 H, H-1,
J1,2 8.8 Hz), 4.438 (d, 1 H, H-1%, J1,2 7.9 Hz),
5.443 (s, 1 H, PhCH), 7.152–7.495 (m, 30 H,
6 Ph) ppm. FABMS Calcd for C57H61N3O11:
964.1. Found: 963.5 (M). Anal. Calcd for
C57H61N3O11 (964.12): C, 71.0; H, 6.4; N, 4.4.
Found: C, 70.9; H, 6.2; N, 4.6.
1
Fig. 1. Partial H-COSY spectrum of pentasaccharide 17 recorded in CDCl3 at 300 K.

256
W. Zou et al. / Carbohydrate Research 315 (1999) 251–261
Scheme 3. Galactosyl globoside 18 and its positional isomer 19.
3-Azidopropyl 6-O-benzoyl-2,3-di-O-benzyl-
-galactopyranosyl-(1“4)-2,3,6-tri-O-ben-
-glucopyranoside (4).—A solution of 2
O-benzoyl-2,3-di-O-benzyl-i-
osyl-(1“4)-2,3,6-tri-O-benzyl-i-
D
-galactopyran-
-glucopyr-
i-
D
D
zyl-i-
D
anoside (6).—To a solution of 4 (1.0 g, 1.0
(2.0 g, 2.3 mmol) in 60% HOAc was stirred
at 60 °C for 1 h. The solvent was evaporated
under diminished pressure to yield compound
3 as a solid. To a solution of the above residue
in CH2Cl2 (20 mL), 2,6-lutidine (4 mL) and
benzoyl chloride (0.35 mL, 2.6 mmol) were
added at 0 °C. The solution was stirred for 6 h
when the starting material was completely
consumed, diluted with CH2Cl2 (100 mL),
washed subsequently with water, N HCl, and
water, dried and concentrated. Purification by
chromatography (1:4 EtOAc–hexane) gave 4
(1.7 g, 76%) as a solid: [h]D +25.6° (c 0.50,
mmol) and 5 (1.0 g, 2.3 mmol) in CH2Cl2 (30
˚
mL), powdered 4 A molecular sieves (3.0 g)
and NIS (0.6 g) were added. The mixture was
stirred at rt for 1 h, then cooled to −40 °C.
To the mixture TfOH (100 mL) was added and
the stirring was continued at −40 °C for 1.5
h. The mixture was neutralized by the addi-
tion of 1:20 Et3N–CH2Cl2 (50 mL), washed
subsequently with water, N HCl, aq Na2S2O3,
and water, dried and concentrated to a
residue. Purification by chromatography (1:3
EtOAc–hexane) afforded 6 (0.65 g, 47%) as a
1
glassy mass: [h]D +21.6° (c 0.58, MeOH); H
1
MeOH); H NMR (CDCl3) l 1.861 (m, 2 H,
NMR (CDCl3) l 1.270 and 1.277 (2s, 3 H
each, CMe2), 1.858 (m, 2 H, CH2CH2N3),
4.317 (d, 1 H, H-1I, J1,2 8.7 Hz), 4.415 (d, 1 H,
H-1II, J1,2 7.8 Hz), 4.968 (d, 1 H, H-1III, J1,2
CH2CH2N3), 4.268 (dd, 1 H, H-6%a, J5,6a 6.5
Hz, J6a,6b 11.0 Hz), 4.344 (d, 1 H, H-1, J1,2 8.8
Hz), 4.421 (d, 1 H, H-1%, J1,2 7.9 Hz), 4.510
(dd, 1 H, H-6%b, J5,6b 6.0 Hz), 7.150–7.690 (m,
30H, 6 Ph) ppm. HRFABMS Calcd for
C57H64NO12 [M−N2+3H]: 954.4429. Found:
954.4514. Anal. Calcd for C57H61N3O12
(980.12): C, 69.9; H, 6.3; N, 4.3. Found: C,
69.4; H, 6.5; N, 4.8.
13
3.2 Hz) ppm. C NMR (CDCl3) l 26.37 and
28.08 (CMe2), 29.31 (CH2CH2N3), 48.38
(CH2CH2N3), 100.07 (C-1III), 102.67 (C-1II),
103.58 (C-1I), 108.66 (CMe2), 166.15 (PhCO)
ppm. FABMS Calcd for C80H87N3O17: 1362.6.
Found: 1385.5 [M+Na], 1337.5 [M−N2+
3H].
3-Azidopropyl 2,6-di-O-benzyl-3,4-di-O-iso-
propylidene-h- -galactopyranosyl-(1“4)-6-
D

W. Zou et al. / Carbohydrate Research 315 (1999) 251–261
257
Table 1
NMR chemical shiftsa for galactosylgloboside 18 and its positional isomer 19
Residue
Glycose unit
Atom
Chemical shifts (l) in ppmb
18
19
1H
13C
1H
13C
GlcI
“4)-b-
D
-Glc(1“
1
4.507
102.93
4.507
102.91
J1,2
2
3
4
5
(8.1)
3.34
3.65
3.66
3.62
3.83
4.00
(8.1)
3.34
3.64
3.65
3.61
3.82
3.99
73.70
75.30
79.64
75.69
60.92
73.72
75.31
79.64
75.73
60.93
6
6%
GlcII
GlcIII
GlcIV
GlcV
“4)-b-
D
-Gal(1“
1
J1,2
2
3
4
5
6
6%
4.507
(8.1)
3.58
3.75
4.04
3.79
3.86
3.92
104.18
4.507
(8.1)
3.57
3.75
4.01
3.78
3.84
3.91
104.14
71.75
72.99
78.08
76.32
61.20
71.64
72.94
77.87
76.33
61.12
“3/4)-a-
D
-Gal(1“
1
J1,2
2
3
4
5
6
6%
4.919
(3.2)
3.92
3.96
4.26
4.38
3.70
3.70
101.29
4.895
(3.2)
3.71
4.01
4.19
4.38
3.66
3.80
101.13
68.46
79.52
69.79
71.17
61.27
69.70
69.90
77.68
70.98
60.81
“3)-b-
D
-GalNAc(1“
1
J1,2
2
3
4
5
6
6%
4.699
(9.3)
4.05
3.92
4.19
3.70
3.74
3.78
103.75
4.679
(9.3)
4.05
3.90
4.16
3.69
3.74
3.79
103.40
52.36
80.47
68.86
75.46
61.86
52.48
80.46
68.87
75.38
61.90
b-D
-Gal(1“
1
J1,2
2
3
4
5
6
6%
4.455
(8.1)
3.54
3.63
3.91
3.65
3.74
3.77
105.66
4.460
(8.1)
3.54
3.63
3.91
3.65
3.74
3.76
105.66
71.48
73.35
69.44
75.85
61.86
71.53
73.31
69.47
75.89
61.87
OCH2CH2CH2NH2
OCH2
CH2
CH2N
3.82, 4.06
2.03
3.16
68.68
27.55
38.41
3.82, 4.05
2.01
3.15
68.70
27.65
38.48
NHAc
2.03
23.27
2.05
23.25
a Recorded at 500 MHz at 300 K in D2O.
b First-order data.

258
W. Zou et al. / Carbohydrate Research 315 (1999) 251–261
Table 2
1H NMR chemical shiftsa (l) in ppm for oligosaccharides 7, 12, 16 and 17b
Saccharide
Glycose unit
H-1c
H-2
H-3
H-4
H-5
H-6, 6%
7
a- -Gal(1“
D
5.05
4.46
4.35
3.75
3.53
3.37
3.88
3.24
3.58
3.97
3.89
4.01
4.13
3.41
3.35
3.31, 3.38
4.60, 4.67
3.70, 3.80
“4)-b-
D
D
-Gal(1“
-Glc(1“
“4)-b-
OCH2CH2CH2N3
3.59
3.96
1.87
3.40
12
16
b-
D
-Gal(1“
-GalNphth(1“
4.35
5.09
4.96
4.50
4.74
4.66
5.32
5.48
3.87
3.33
3.56, 3.58
3.45, 3.68
“3)-b-
D
OCH2CH2SiMe3
3.46
3.93
0.765
−0.181
b-D
-Gal(1“
-GalNAc(1“
-Gal(1“
4.44
4.87
5.14
4.42
4.32
5.06
3.82
4.00
3.58
3.36
4.96
3.92
3.66
3.49
3.51
5.42
5.45
5.35
3.81
4.00
-
“3)-b-
D
“3)a-
“4)-b-
“4)-b-
D
D
-Gal(1“
-Glc(1“
D
OCH2CH2CH2N3
3.62
3.95
1.85
3.39
17
b-
D
-Gal(1“
-GalNAc(1“
-Gal(1“
4.55
4.88
5.11
4.40
4.33
5.07
3.22
3.90
3.51
3.38
4.93
4.73
5.24
3.44
3.54
5.43
5.45
4.15
3.79
3.93
“3)-b-
D
“4)a-
“4)-b-
“4)-b-
D
D
-Gal(1“
-Glc(1“
D
OCH2CH2CH2N3
3.62
3.95
1.85
3.39
a Recorded at 500 MHz at 300 K in CDCl3.
b The chemical shifts of protecting groups were not listed.
c First-order data.
copyranoside (10).—To a solution of 8 (1.0 g,
2.27 mmol) and 9 (1.0 g, 2.01 mmol) in
3-Azidopropyl 2,6-di-O-benzyl-h-
pyranosyl-(1“4)-6-O-benzoyl-2,3-di-O-benz-
yl-i- -galactopyranosyl-(1“4)-2,3,6-tri-O-
benzyl-i- -glucopyranoside (7).—To a solu-
D
-galacto-
˚
CH2Cl2 (30 mL), powdered 4 A molecular
D
sieves (2.5 g) and NIS (0.65 g) were added.
The mixture was stirred at rt for 1 h, then
cooled to −40 °C. To the mixture, TfOH
(100 mL) was added and the stirring was con-
tinued at −40 °C for 1 h. The mixture was
neutralized by the addition of 1:20 Et3N–
CH2Cl2 (50 mL), washed subsequently with
water, N HCl, aq Na2S2O3, and water, dried
and concentrated to a residue. Purification by
chromatography (1:2 EtOAc–hexane) af-
forded 10 (1.3 g, 74%) as a solid: [h]D +31.3°
D
tion of 6 (0.8 g, 0.59 mmol) in CH2Cl2 (45
mL), aq 90% TFA (5 mL) was added at 0 °C.
The solution was stirred for 0.5 h, after which
time the starting material had completely dis-
appeared. The solution was subsequently
washed with cold water, aq NaHCO3, and
water, dried and concentrated. Purification by
chromatography (1:2 EtOAc–hexane) af-
forded 7 (0.7 g, 90%) as a syrup: [h]D +37.0°
1
(c 0.63, MeOH); H NMR data are listed in
1
(c 0.80, MeOH); H NMR (CDCl3) l −0.159
13
Table 2 (BnO and BzO are not included). C
(s,
9
H, SiMe3), 0.735 (m,
2
H,
NMR (CDCl3) l 99.85 (C-1III), 102.02(C-1II),
103.10 (C-1I) ppm. FABMS Calcd for
C77H83N3O17: 1322.5. Found: 1345.5 [M+
Na], 1297.5 [M−N2 +3H].
CH2CH2SiMe3), 1.523, 1.814, 1.968 (3 s, 3 H
each, 3 OAc), 4.169, 4.365 (2 d, 1 H each,
CH2Ph, J 11.9 Hz), 4.276 (dd, 1 H, H-2I, J2,3
9.8 Hz), 4.525 (d, 1 H, H-1II, J1,2 7.9 Hz),
4.684 (dd, 1 H, H-3I, J3,4 9.1 Hz), 4.725 (dd, 1
H, H-3II, J2,3 10.3 Hz), 4.935 (dd, 1 H, H-2II),
5.147 (d, 1 H, H-1I, J1,2 8.5 Hz), 5.257 (d, 1 H,
2-Trimethylsilylethyl 2,3,4-tri-O-acetyl-6-O-
benzyl-i-
O-benzylidene-2-deoxy-2-phthalimido-i-
D
-galactopyranosyl-(1“3)-4,6-di-
-glu-
D

W. Zou et al. / Carbohydrate Research 315 (1999) 251–261
259
H-4II, J3,4 2.7 Hz), 5.480 (s, 1 H, PhCH),
7.159–7.850 (m, 14 H, 2 Ph and Phth) ppm;
13C NMR (CDCl3) l −0.846 (SiMe3), 18.53
(CH2Si), 20.39, 21.13, 21.30 (3×CH3CO),
55.56 (C-2I), 63.69, 67.88, 67.96, 68.39, 69.56,
71.50, 71.76, 73.12, 74.46, 76.06, 82.66 (11 C,
C-3I, 4I, 5I, 6I, 2II, 3II, 4II, 5II, 6II, CH2Ph,
and OCH2CH2Si), 98.42 (C-1I), 101.63 (C-
1II), 101.83 (PhCH), 124.20–137.69 (Ph,
Phth), 169.43, 170.64, 171.86 (3 OAc) ppm.
HRFABMS Calcd for C45H53NO15SiNa
[M+Na]: 898.3082. Found: 898.3054. Anal.
Calcd for C45H53NO15Si (875.99): C, 61.7; H,
6.1; N, 1.6. Found: C, 61.4; H, 5.9; N, 1.7.
2-Trimethylsilylethyl 2,3,4-tri-O-acetyl-6-O-
(1.4 g, 1.6 mmol) in 4:1 CH2Cl2–pyridine (20
mL), triflic anhydride (1 mL, 5.9 mmol) was
added at 0 °C. The solution was stirred at
0 °C, then at rt for 4 h, after which time
TLC indicated the completion of the reac-
tion. The yellowish solution was diluted with
CH2Cl2 (100 mL) and washed subsequently
with ice water, N HCl, and ice water again,
dried and concentrated to a residue. To a
solution of the above residue in DMF (10
mL), Me4NOAc (1.1 g, 8.3 mmol) was added
at rt. The mixture was stirred for 2 h, diluted
with EtOAc (100 mL), washed subsequently
with water, aq NaHCO3, and water, dried
and concentrated. Purification by chromatog-
raphy (1:2 EtOAc–hexane) gave 12 (1.3 g,
89%) as a solid: [h]D +4.3° (c 0.37, MeOH);
1H NMR data are listed in Table 2 (BnO
and AcO are not included). HRFABMS
benzyl - i -
benzyl-2-deoxy-2-phthalimido-i-
D
- galactopyranosyl - (1“3) - 6 - O-
-glucopyran-
D
oside (11).—To
a
mixture of sodium
˚
cyanoborohydride (0.55 g), powdered 3 A
molecular sieves (2.0 g) and compound 10
(1.0 g, 1.14 mmol) in THF (20 mL), a satu-
rated solution of HCl in ethyl ether was
added dropwise at 0 °C until the mixture be-
came acidic (pH 3). The mixture was further
stirred for another 2 h, when TLC indicated
completion of the reaction. The mixture was
diluted with EtOAc (80 mL) and filtered
through Celite. The filtrate was subsequently
washed with water, aq NaHCO3, and water,
dried and concentrated to a residue. Purifica-
tion by chromatography (2:3 EtOAc–hexane)
gave 11 (0.79 g, 79%) as a solid: [h]D −7.5°
Calcd
for
C47H57NO16SiNa
[M+Na]:
942.3344. Found: 942.3378. Anal. Calcd for
C47H57NO16Si (920.05): C, 61.4; H, 6.2; N,
1.5. Found: C, 61.2; H, 6.3; N, 2.0.
2,3,4-Tri-O-acetyl-6-O-benzyl-i-
pyranosyl-(1“3)-4-O-acetyl-6-O-benzyl-2-
deoxy-2-phthalimido-i- -galactopyranosyl tri-
D-galacto-
D
chloroacetimidate (13).—To a stirred solution
of 12 (1.2 g, 1.3 mmol) in CH2Cl2 (20 mL),
TFA (8 mL) was added. The stirring was
continued at rt for 1 h, and the solution was
then diluted with CH2Cl2 (50 mL), washed
subsequently with water, aq NaHCO3, and
water, dried and concentrated to a residue. To
1
(c 1.18, MeOH); H NMR (CDCl3) l −
0.241 (s, 9 H, SiMe3), 0.747 (m, 2 H,
CH2CH2SiMe3), 1.398, 1.967, 2.020 (3s, 3 H
each, 3 OAc), 4.213 (dd, 1 H, H-2I, J2,3 10.8
Hz), 4.394 (d, 1 H, H-1II, J1,2 8.1 Hz), 4.601
(s, 2 H, CH2Ph), 4.770 (dd, 1 H, H-3II, J2,3
10.3 Hz), 5.027 (d, 1 H, H-1I, J1,2 8.5 Hz),
5.089 (dd, 1 H, H-2II), 5.292 (d, 1 H, H-4II,
J3,4 3.2 Hz), 7.193–7.848 (m, 14 H, 2 Ph and
a
solution of the above residue and
trichloroacetonitrile (1.0 mL) in CH2Cl2 (15
mL) at 0 °C, 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU) (240 mL, 1.5 mmol) was added.
The mixture was stirred for 2 h at 0 °C, and
then concentrated. Purification by chro-
matography (2:3 EtOAc–hexane) gave 13 (1.1
g, 88%) as a solid: [h]D +11.8° (c 0.70,
MeOH); 1H NMR (CDCl3) l 4.371 (d,
1 H, H-1II, J1,2 8.5 Hz), 6.586 (d, 1 H, H-1I,
J1,2 9.0 Hz), 8.737 (s, 1 H, CꢀNH) ppm.
FABMS Calcd for C44H45Cl3N2O16: 964.2.
Found: 987.2, 985.2 [M+Na], 802.3 [M−
OCNHCCl3].
Phth)
ppm.
HRFABMS
Calcd
for
C45H55NO15SiNa [M+Na]: 900.3237. Found:
900.3265. Anal. Calcd for C45H55O15Si
(878.01): C, 61.6; H, 6.3; N, 1.6. Found: C,
61.2; H, 6.0; N, 1.4.
2-Trimethylsilylethyl 2,3,4-tri-O-acetyl-6-
O-benzyl-i-
acetyl-6-O-benzyl-2-deoxy-2-phthalimido-i-
galactopyranoside (12).—To a solution of 11
D
-galactopyranosyl-(1“3)-4-O-
3-Azidopropyl 2,3,4-tri-O-acetyl-6-O-benz-
D
-
yl-i-D
-galactopyranosyl-(1“3)-4-O-acetyl-6-
-galacto-
O-benzyl-2-deoxy-2-phthalimido-i-
D

260
W. Zou et al. / Carbohydrate Research 315 (1999) 251–261
pyranosyl-(1“3/4)-2,6-di-O-benzyl-h-
ctopyranosyl-(1“4)-6-O-benzoyl-2,3-di-O-
benzyl-i- -galactopyranosyl-(1“4)-2,3,6-tri-
O-benzyl-i- -glucopyranoside (14/15).—A
D
-gala-
1190.7 [M−N2+3H]. 17: [h]D +18.7° (c 2.0,
MeOH); 13C NMR (CDCl3) l 98.34 (C-1IV),
98.53 (C-1III), 99.63 (C-1V), 102.13 (C-1II),
103.07 (C-1I) ppm. FABMS Calcd for
C110H126N4O32: 2016.2. Found: 2038.7 [M+
Na], 1190.7 [M−N2+3H].
D
D
mixture of 7 (330 mg, 0.25 mmol), 13 (340 mg,
˚
0.35 mmol) and powdered 4 A molecular
sieves (1.3 g) in CH2Cl2 (5 mL) was stirred at
rt for 1 h. The mixture was cooled to −40 °C
and Me3SiOTf (50 mL) was added. The mix-
ture was stirred at −40 °C for 1.5 h and was
then neutralized with a solution of 2,6-lutidine
(0.5 mL) in CH2Cl2 (20 mL). The filtrate was
subsequently washed with water, N HCl, and
water, dried and concentrated to a residue.
Purification by chromatography (1:1 EtOAc–
hexane) gave 14 (10 mg), 15 (12 mg) and a
mixture of 14/15 (0.5 g). 14: 1H NMR
(CDCl3) l 4.824 (bs, 1 H, H-1III), 4.960 (dd, 1
H, H-2V, J2,3 9.0 Hz), 5.224 (d, 1 H, H-1IV, J1,2
8.5 Hz), 5.346 (bs, 1 H, H-4V), 5.438 (bs, 1 H,
3-Aminopropyl i-
2-acetamido-2 -deoxy-i-
(1“3/4)-h- -galactopyranosyl-(1“4)-i-
galactopyranosyl-(1“4)-i- -glucopyranoside
D-galactopyranosyl-(1“3)-
D
-galactopyranosyl-
D
D
-
D
(18 and 19).—A solution of 16 (or 17) (50 mg,
24.8 mmol) in 0.1% NaOMe/MeOH (3 mL) was
stirred at rt for 2 h. The solution was neutral-
ized with Dowex 50 (H+) ion-exchange resin,
and the filtrate was concentrated to a residue.
A solution of above residue and 10% Pd/C (50%
water, 30 mg) in 1:8:1 H2O–MeOH–AcOH (10
mL) was subjected to hydrogenation (40 psi) for
16 h. The filtrate was concentrated under di-
minished pressure to remove methanol and
then lyophilized to give crude products. Purifi-
cation by passage through a Sephadex G-10
column using water as eluent afforded, after
lyophilization, respectively 18 (18 mg, 79%) and
H-4IV)
ppm.
FABMS
Calcd
for
C119H126N4O32: 2124.3. Found: 2146.6 (M+
1
Na). 15: H NMR (CDCl3) l 4.819 (bs, 1 H,
H-1III), 4.947 (dd, 1 H, H-2V, J2,3 9.0 Hz),
5.152 (d, 1 H, H-1IV, J1,2 8.5 Hz), 5.314 (bs, 1
H, H-4V), 5.485 (bs, 1 H, H-4IV) ppm.
FABMS Calcd for C119H126N4O32: 2124.3.
Found: 2146.6 [M+Na].
1
19 (17 mg, 75%) as solids. Both H and 13C
NMR data for 18 and 19 are listed in Table 1.
18: [h]D +24.0° (c 0.78, H2O); FABMS Calcd
for C35H62N2O26: 926.9. Found: 949.3 [M+
Na], 927.3 [M+H]. 19: [h]D +31.2° (c 0.98,
H2O); FABMS Calcd for C35H62N2O26: 926.9.
Found: 949.3 [M+Na], 927.3 [M+H].
3-Azidopropyl 2,3,4-tri-O-acetyl-6-O-benz-
yl-i- -galactopyranosyl-(1“3)-2-acetamido-
D
4-O-acetyl-6-O-benzyl-2-deoxy-i-
pyranosyl - (1“3/4) - 4/3 - O - acetyl - 2,6 - di - O-
benzyl - h - - galactopyranosyl - (1“4) - 6 - O-
acetyl-2,3-di-O-benzyl-i- -galactopyranosyl-
(1“4)-2,3,6-tri-O-benzyl-i- -glucopyranoside
D
-galacto-
D
D
Acknowledgements
D
(16 and 17).—A mixture of 14/15 (0.5 g, 0.24
mmol), derived above, and hydrazine hydrate
(2.0 mL) in 95% EtOH (18 mL) was refluxed
for 16 h. Upon cooling, the solvent was evap-
orated and the residue was treated with 1:1
Ac2O–pyridine (10 mL) for 6 h. To the mix-
ture, ice water was added and the precipitate
was collected, which was then purified by
chromatography (3:2 EtOAc–hexane) to give
The authors thank Mr K. Chan for the
mass spectroscopic analysis and Ms A. Webb
for the elemental analysis.
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