Synthesis of a sialyl-α-(2→6)-lactosamine trisaccharide with a 5-amino-3-oxapentyl spacer group at C-1

Article abstract of DOI:10.1016/S0008-6215(99)00022-1

As part of a continuing study aimed to achieve improved monoclonal antibodies against carcinoembryonic antigen (CEA) carbohydrate fragments, the synthesis of a sialyl-(2→6)-lactosamine trisaccharide with a 5-amino-3-oxapentyl spacer group at C-1(I) has been developed. Two different routes to access this target are described. For this purpose 5-azido-3-oxapentyl 6-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (4) was selectively β-galactosylated in 81% yield using the crystalline 2,3-di-O-acetyl-4,6-O-benzylidene-α-D-galactopyranosyl trichloroacetimidate as the donor, taking advantage of the bulky phthalimido group at C-2 of 4. On the other hand, galactosylation of the suitable protected acceptor 5-azido-3-oxapentyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside with the crystalline 2,3-di-O-acetyl-4,6-O-benzylidene-α-D-galactosyl bromide renders the corresponding disaccharide in a moderate 58% yield. Despite the fact that the first strategy, unlike the second one, requires a hydrazinolysis-acetylation reaction at the disaccharide stage, it was found to be more convenient to access the disaccharide acceptor. Sialylation was performed using a thiophenyl donor under an NIS-TfOH activation procedure in acetonitrile to give a mixture of α and β trisaccharides in 49 and 16% yields, respectively. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(99)00022-1

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Carbohydrate Research 317 (1999) 29–38
Synthesis of a sialyl-a-(2“6)-lactosamine trisaccharide
with a 5-amino-3-oxapentyl spacer group at C-1Iꢀ
Santiago Figueroa-Pe´rez, Vicente Ve´rez-Bencomo *
Laboratory of Synthetic Antigens, Facultad de Qu´ımica, Uni6ersidad de La Habana, Ciudad, Ha6ana 10400, Cuba
Received 6 July 1998; accepted 2 January 1999
Abstract
As part of a continuing study aimed to achieve improved monoclonal antibodies against carcinoembryonic antigen
(CEA) carbohydrate fragments, the synthesis of a sialyl-(2“6)-lactosamine trisaccharide with a 5-amino-3-oxapentyl
spacer group at C-1I has been developed. Two different routes to access this target are described. For this purpose
5-azido-3-oxapentyl 6-O-benzyl-2-deoxy-2-phthalimido-b-
81% yield using the crystalline 2,3-di-O-acetyl-4,6-O-benzylidene-a-
donor, taking advantage of the bulky phthalimido group at C-2 of 4. On the other hand, galactosylation of the
suitable protected acceptor 5-azido-3-oxapentyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-b- -glucopyranoside with the
crystalline 2,3-di-O-acetyl-4,6-O-benzylidene-a- -galactosyl bromide renders the corresponding disaccharide in a
D
-glucopyranoside (4) was selectively b-galactosylated in
D
-galactopyranosyl trichloroacetimidate as the
D
D
moderate 58% yield. Despite the fact that the first strategy, unlike the second one, requires a hydrazinolysis–acety-
lation reaction at the disaccharide stage, it was found to be more convenient to access the disaccharide acceptor.
Sialylation was performed using a thiophenyl donor under an NIS–TfOH activation procedure in acetonitrile to give
a mixture of a and b trisaccharides in 49 and 16% yields, respectively. © 1999 Elsevier Science Ltd. All rights
reserved.
Keywords: Selective glycosylation; Trichloroacetimidates; Carcinoembryonic antigen; Sialyllactosamine
1. Introduction
CEA monoclonal antibodies by their protein
specificity [5]. Two other proteins, non-specific
cross-reacting antigen-2 (NCA-2) [6], and nor-
mal fecal antigen-2 (NFA-2) [7], have been
found to be the same gene products as CEA,
and largely cross-reacted with it.
A comparison between all these proteins [4]
revealed that, among other distinctive features,
in all the examined CEA samples were found
the determinants Lewisy, a-Neu5Ac-(1“6)-b-
Notwithstanding the discovery of many new
tumour markers, carcinoembryonic antigen
(CEA) is still one of the most widely used [1].
Its serum levels, currently measured by sand-
wich immunoassay, are used for monitoring
surgery-treated patients with several types of
adenocarcinomas [2].
CEA is a highly glycosylated protein contain-
ing an average of 25 N-linked oligosaccharides
[3,4]. The high heterogeneity observed for all
these chains prompted the selection of anti-
D
-Gal-(1“4)-
-GlcNAc6S, as common chain termini. Lewisy
-Gal-(1“4)- -Glc-
D
-GlcNAc and b-
D
-Gal-(1“4)-
D
and a-Neu5Ac-(1“6)-b-
D
D
NAc haptens have already been employed for
the detection of CEA, using either a mono-
clonal antibody [8] or the lectin [9,10] Tri-
chosanthes japonica agglutinin-I.
ꢀ Presented at the IXth European Carbohydrate Sympo-
sium, Utrecht, The Netherlands, 1997.
* Corresponding author.
E-mail address: vicente@fq.oc.uh.cu (V. Ve´rez-Bencomo)
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00022-1

30
S. Figueroa-Pe´rez, V. Ve´rez-Bencomo / Carbohydrate Research 317 (1999) 29–38
As part of our ongoing study to develop an
On the other hand, benzylation of the readily
accessible benzylidene glycoside 5 (85%; C-3,
77.6 ppm) followed by reductive benzylidene
opening gave acceptor 7 in moderate 60% yield.
The shielding of the C-4 signal (“75.1 ppm),
and the presence of the C-6 signal at 68.7 ppm,
confirmed the expected transformation.
improved anti-CEA sandwich immunoassay by
using monoclonal antibodies against synthetic
carbohydrates as a complement for anti-protein
antibodies, we have focused on these three
distinct structures. The synthesis of Lewisy
neoglycoproteins [11] was the subject of a
previous paper, whereas in the present study
sialyllactosamine neoglycoprotein was chosen
as the target.
Two different strategies were evaluated for
the synthesis of the lactosamine acceptor 16,
which was further sialylated, deprotected, and
hydrogenated to afford the trisaccharide 21
with a 5-amino-3-oxapentyl spacer group at
C-1I. The amino group on the spacer linkage of
21 was used to attach the trisaccharide to
bovine serum albumin (BSA) following a very
efficient procedure previously described by us
[12].
1,2,3-Tri-O-acetyl-4,6-O-benzylidene- -gal-
D
actopyranose (8) [11] was used as the starting
material for the construction of the galactosyl
donor. Selective deacetylation of 8 using 2-
aminoethanol in ethyl acetate [14] afforded the
anomeric mixture 9 (55%), which on treatment
overnight with potassium carbonate and
trichloroacetonitrile in dichloromethane [15],
gave the crystalline a-imidate 10 in good 71%
2. Results and discussion
1,3,4,6-Tetra-O-acetyl-2-deoxy-2-phthal-
imido-b- -glucopyranose (1) was condensed
D
with 1-azido-3-oxapentanol in the presence of
stannic chloride [13] to afford the glycoside 2
in an acceptable 62% yield. The signal for C-1
appeared at 98.0 ppm in the 13C NMR spec-
tra, while in the 1H NMR spectra, H-1
showed a doublet at 5.46 ppm. The presence
of the spacer arm was confirmed here and in
all the following steps by the signal at 50.2
ppm in the 13C NMR spectra, corresponding
to the methylene group attached to the azido
group. The diol acceptor 4 was obtained in
63% yield after deacetylation of 2 (“3) and
further selective benzylation of the primary
position using bis-tributyltin oxide. The 13C
NMR spectrum of compound 4 showed a
deshielding for C-6 (61.1“68.7 ppm), that in
conjunction with the unchanged shift of the
signals for C-4 and C-3 was the main evidence
that position 6 was selectively benzylated.
1
yield. The H NMR spectra shows singlets at
8.65 and 5.57 ppm, corresponding to imidate
NH and benzylidene protons, respectively, the
doublet of the a-anomeric proton appeared at
6.71 ppm with a coupling constant of 3.5 Hz.
Selective galactosylation [16] of the diol 4
with donor 10, in the presence of trimethylsilyl
trifluoromethanesulfonate as catalyst, pro-
ceeded smoothly, giving disaccharide 13 in an
excellent 81% yield, whereby no (1“3)-linked

S. Figueroa-Pe´rez, V. Ve´rez-Bencomo / Carbohydrate Research 317 (1999) 29–38
31
and (3) the relative stability of imidate 10 (it
can be stored at 4 °C for at least 4 months)
while bromide 12 is particularly unstable.
Sialylation of 17 was conducted using the
thiophenyl glycoside 18 [19] as the donor and
using NIS/triflic acid as the activating system,
in acetonitrile at −40 °C [20]. These condi-
tions led to an a:b mixture (3:1) in a 67%
overall yield; the a isomer 19 was recovered in
50.5% yield after a simple chromatographic
purification. The stereochemistry of the glyco-
sidic linkage was determined by the long-
range coupling constant between C-1 and
H-3ax [21]. These values were 6.6 Hz for the a
isomer 19 and 1.5 Hz for the b isomer 20.
product was detected. The regioselectivity of
the reaction was confirmed in the 13C NMR
spectra by the deshielding of the signal as-
signed to C-4 (72.9“80.9 ppm), and in the 1H
NMR spectrum of the acetylated product 14
by the particular deshielding of the signal
assigned to H-3 at 5.62 ppm. The galactose
anomeric proton doublet at 4.48 ppm in the
1H NMR spectrum of 13 had a J value of 8
Hz, thus proving the b-stereochemistry of the
formed bond.
Galactosylation of acceptor 7 with donor 10
using boron trifluoride–diethyl ether complex
as a catalyst [17] was not feasible in our
hands. So, we decided to prepare the crys-
talline a-bromide 12 from the readily available
thioglycoside 11 by reaction with bromine in
dichloromethane [18].
1
Other differences in the H chemical shifts
between the b and the a isomers are reported
in Table 1.
Galactosylation of 7 with 12 was then per-
formed under silver triflate promotion in
dichloromethane, using Hu¨nig’s base to neu-
tralize the acid, to afford the desired disaccha-
ride 15 in a moderate 59% yield. In the 13C
NMR spectrum, the signal for C-4 was
deshielded to 76.1 ppm owing to the substitu-
tion by the galactosyl moiety; the doublet at
Compound 19 was deacetylated and sa-
ponified in one step [22], then hydrogenolysed
to give the free trisaccharide 21 in 91% overall
yield. The triplet located at 3.19 ppm in the 1H
NMR spectrum and the signal at 41.5 ppm in
the 13C NMR spectrum, which represent the
CH2NH2 group, are indicative of the transfor-
mation of the azido into an amino group.
Finally, compound 21 was treated with an
excess of N-succinimidoxyl b-maleimidopropi-
onate in N,N-dimethylformamide/PBS to
functionalize the trisaccharide for conjugation
to BSA. Compound 22 was obtained together
with a small quantity of b-maleimidopropi-
onic acid as a consequence of the partial
1
4.46 ppm in the H NMR with J 8.1 Hz
corresponding to H-1% ascertained the b-
configuration at the galactose anomeric
center.
At this point it seems to be clear that the
selective glycosylation strategy was more ad-
vantageous mainly due to the shorter route to
the acceptor and the faster, cheaper and
higher-yielding galactosylation reaction. How-
ever, the phthalimido group of the disac-
charide derivative has to be removed before
sialylation, owing the incompatibility of the
phthalimido and methyl ester group at the
deprotection stage. Therefore, hydrazinolysis
of compound 13 followed by acetylation and
hydrolysis of the benzylidene ring afforded the
disaccharide acceptor 17 in 73% yield, which
represents an overall yield of 59% starting
from diol acceptor 4.
Though the two strategies could afford
comparable yields of disaccharide acceptors,
the selective galactosylation route is more con-
venient owing to: (1) the faster and cleaner
galactosylation reaction; (2) the more compli-
cated route to donor 12 and to acceptor 7;
1
hydrolysis of the active ester. H NMR analy-
sis of the mixture showed a deshielding of the
triplet initially located at 3.19 to 3.32 ppm
owing to acylation of the amino function. The
singlet at 6.89 ppm and the triplet at 2.52 ppm
confirmed the presence of the maleimidopro-
pionamide moiety.
Crude compound 22 was directly condensed
with previously thiolated BSA in an excellent
80% oligosaccharide-based yield affording a
(trisaccharide)14-BSA molecule. The use of
this neoglycoprotein for the elucidation of the
epitopes recognized by anti-CEA carbohy-
drate-specific monoclonal antibodies and also
as immunogens for the preparation of mono-
clonal antibodies is now in progress (Scheme
1).

32
S. Figueroa-Pe´rez, V. Ve´rez-Bencomo / Carbohydrate Research 317 (1999) 29–38
Scheme 1.
define the NMR signals: % for Gal, and neu
for neuramic acid unit.
3. Experimental
General procedures.—Optical rotations
were measured at 25 °C with a POLAMAT A
automatic polarimeter, using a 5-cm 5-mL
cell. NMR spectra were recorded at 25 °C
with a Bruker AC-250F spectrometer. Chem-
ical shifts (l) are given in ppm relative to
All compounds were purified by column
chromatography on Kieselgel 60 (Fluka, B
230 mesh ASTM) and fractions were moni-
tored by TLC on Kieselgel 60F254 (E.
Merck). Detection was effected by charring
with aq 20% H2SO4 after examination under
UV light. Evaporations were conducted un-
der reduced pressure at 50 °C (bath).
1
the signal for internal Me4Si for H; and
13
1
indirectly to CDCl3 (l 77.03) for C. H, and
13C assignments were made on the basis of
homo- and heteronuclear correlation experi-
ments. The following notation was used to
5-Azido-3-oxapentyl 3,4,6-tri-O-acetyl-2-
deoxy-2-phthalimido-i- -glucopyranoside (2).
D
—To a stirred solution of 1 (1 g, 2.1 mmol)

S. Figueroa-Pe´rez, V. Ve´rez-Bencomo / Carbohydrate Research 317 (1999) 29–38
33
Table 1
1H NMR data for a and b trisaccharrides 19 and 20
GlcNAc unit
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
NH
J1,2
J2,3
J3,4
JNH,2
19
20
4.53
(7.7)
4.51
(7.6)
4.06
4.05
5.02
(9.8)
5.02
(9.5)
3.92
(8.7)
3.88
(8.3)
3.50
3.50
3.72
3.72
3.9
4
3.9
3
6.08
(9.2)
6.15
(9.5)
Gal unit
J1,2
J2,3
J3,4
19
20
4.46
(7.9)
4.45
(7.9)
5.14
(10.2)
5.13
4.79
(3.1)
4.82
(3.0)
4.03
4.16
3.73
3.56
3.51
3.43
3.92
3.78
(10.2)
Neu5Ac unit
H-3a
H-3e
H-4
H-5
H-6
H-7
H-8
H-9a
H-9b
NH
J3a,3e
J3e,4
J4,3
J5,6
J6,7
J7.8
J8,9a
J9a,9b
JNH,5
19
20
1.97
(12.8)
1.82
2.57
(4.7)
2.48
(4.8)
4.90
(11.9)
5.31
4.05
(9.8)
3.81
(9.8)
4.15
5.31
5.32
4.37
(2.5)
4.73
(2.0)
4.15
(12.7)
4.18
5.42
(9.2)
6.00
(9.4)
4.25
(10.5)
5.34
(2.0)
5.23
(2.6)
(12.8)
(12.3)
5-Azido-3-oxapentyl 6-O-benzyl-2-deoxy-2-
phthalimido-i- -glucopyranoside (4).—To a
and 5-azido-3-oxapentanol (0.8 mL, 4.2 mmol)
in dry CH2Cl2 (5 mL) was added dropwise
SnCl4 (0.5 mL, 4.2 mmol). After being stirred
overnight at room temperature (rt), CH2Cl2
was added (20 mL) and the solution was
poured into ice-satd NaHCO3 (40 mL). The
resulting emulsion was filtered through Celite
and the solid thoroughly rinsed with CH2Cl2.
The organic layer was collected from the
filtrate and washed with satd NaCl (20 mL),
then dried and concentrated. Column chro-
matography of the residue (1:1 hexane–
EtOAc) afforded pure 2 as a colorless syrup
(690 mg, 62%); [h]D +41° (c 0.96, CHCl3); Rf
D
stirred solution of 2 (0.69 g, 1.3 mmol) in a
mixture of 1:1 dry MeOH–CH2Cl2 (10 mL),
was added a freshly prepared solution of
methanolic sodium methoxide (0.5 M, 1 mL).
The reaction was monitored by TLC (1:1 hex-
ane–EtOAc). When all the starting material
was transformed (ꢀ30 min), the solution was
neutralized with Amberlite IR-120 (H+) resin,
filtered, and evaporated to dryness to afford a
slightly yellow syrup 3 (500 mg, 96%). Bis-(tri-
n-butyl)tin oxide (0.64 mL) was added to a
solution of compound 3 (0.8 g, 2 mmol) in
toluene (10 mL) and the mixture was stirred
with continuous azeotropic removal of H2O.
After 4 h, the mixture was concentrated to a
volume of 3 mL, then benzyl bromide (0.8
mL, 6.7 mmol) and tetrabutylammonium bro-
mide (0.32 g, 1 mmol) were added, and the
mixture was stirred at 100 °C for 4 h. The
solution was then diluted with EtOAc (15
mL), and aq 10% NaF (5 mL) was added to
the solution. After 20 min of vigorous stirring,
the insoluble material was removed by filtra-
tion over Celite, and the organic phase was
collected from the filtrate, washed with satd
1
0.35 (2:1 hexane–EtOAc); NMR (CDCl3): H,
l 7.85 and 7.75 (Phth), 5.83 (dd, 1 H, J2,3 10.7,
J3,4 9.2 Hz, H-3), 5.46 (d, 1 H, J1,2 8.5 Hz, H-1),
5.19 (d, 1 H, J4,5 5.9 Hz, H-4), 4.20 (dd, 1 H,
J6a,6b 12.3, J5,6a 2.3 Hz, H-6a), 3.33 (t, 2 H, J
4.2 Hz, CH2N3), 2.20–2.01 (9 H, CH3COO);
13C, l 170.6, 170.0 and 169.4 (CꢀO), 134.2,
131.3 and 123.4 (Phth), 98.0 (C-1), 71.7 (C-3),
70.6 (C-5), 69.9, 69.7 (CH2O), 68.9 (C-4 and
CH2O), 61.9 (C-6), 54.5 (C-2), 50.2 (CH2N3),
20.6, 20.5 and 20.3 (CH3COO). Anal. Calcd
for C28H43N4O11: C, 54.98; H, 7.09; N, 9.16.
Found: C, 54.87; H, 7.18; N, 9.24.

34
S. Figueroa-Pe´rez, V. Ve´rez-Bencomo / Carbohydrate Research 317 (1999) 29–38
5-Azido-3-oxapentyl 2-acetamido-3,6-di-O-
benzyl-2-deoxy-i- -glucopyranoside (7).—A
aq NaHCO3 (5 mL) and H2O (5 mL), then
dried (Na2SO4), filtered, and concentrated.
The syrupy residue was chromatographed (1:1
toluene–EtOAc) to give 4 as a colorless syrup
(0.61 g, 63%); [h]D −22.7° (c 0.96, CHCl3); Rf
0.37 (1:1 toluene–EtOAc); NMR (CDCl3):
1H, l 7.85 and 7.75 (Phth), 7.30 (Ph), 5.21 (d,
1 H, J1,2 8.4 Hz, H-1), 4.58 (s, 2 H, CH2Ph),
4.30 (dd, 1 H, J2,3 10.6, J3,4 7.9 Hz, H-3), 4.10
(dd, 1 H, H-2), 3.85 (m, 1 H, H-6a), 3.61 (m,
1 H, H-6b), 3.59 (m, 1 H, H-5), 3.53 (m, 1 H,
H-4), 3.34 (t, 2 H, CH2N3); 13C, l 170.6 and
170.0 (CꢀO), 134.2, 131.3 and 123.4 (Phth),
128.9–127.7 (Ph), 98.2 (C-1), 74.3 (C-5), 72.9
(C-4), 71.4 (C-3), 69.9 and 69.6 (CH2O), 68.7
(C-6), 56.3 (C-2), 50.3 (CH2N3). Anal. Calcd
for C29H43N4O8: C, 60.51; H, 7.53; N, 9.73.
Found: C, 60.35; H, 7.59; N, 9.65.
D
mixture of 6 (1 g, 1.7 mmol), NaBH3CN (1.07
,
g, 17 mmol) and 3 A molecular sieves (2 g) in
dry THF (20 mL) was stirred for 15 min at rt.
Then, the mixture was cooled to 0 °C, and dry
Et2O saturated with HCl was added at 0 °C
until the evolution of gas stopped. The cooling
bath was then removed and the mixture was
further stirred for 20 min. Cold H2O (5 mL)
was then added, and the suspension was di-
luted with CH2Cl2 (50 mL) and filtered
through Celite. The filtrate was washed with
aq 1% KMnO4 (3×20 mL), satd aq NaHCO3
(20 mL) and H2O (20 mL), dried, and concen-
trated.
Column
chromatography
(5:1
CH2Cl2–acetone) of the residue afforded 7
(600 mg, 60%) as a colorless solid. An analyt-
ical sample was obtained by recrystallization
from EtOAc: mp 92–94 °C; [h]D −12.3° (c
1.0, CHCl3); Rf 0.48 (4:1 CH2Cl2–acetone); 1H
NMR (C6D6): l 7.45–7.35 (m, 10 H, Ph), 6.62
(d, 1 H, J 7.7 Hz, NH), 4.97 (AB pattern, 2 H,
CH2Ph), 4.69 (d, 1 H, J1,2 8.3 Hz, H-1), 4.41
(s, 2 H, PhCH2), 4.15 (m, 1 H, H-2), 3.88 (m,
3 H, H-3,4,6a), 3.70 (m, 1 H, H-5), 3.45 (m, 1
H, H-6b), 3.23 (t, 2 H, CH2CH2N3), 2.87 (t, 2
H, J 4.9 Hz, CH2N3) and 1.82 (s, 3 H,
CH3CON); 13C NMR (CDCl3): l 170.4
(CꢀO), 129.6-127.7 (Ph), 101.7 (C-1), 83.0 (C-
3), 75.1 (C-4), 73.6 and 72.8 (CH2Ph), 72.3
(C-5), 68.7 (C-6), 55.6 (C-2), 50.7 (CH2N3),
23.3 (CH3CON). Anal. Calcd for C27H38N4O7:
C, 61.12; H, 7.22; N, 10.56. Found: C, 60.98;
H, 7.30; N, 10.35.
5-Azido-3-oxapentyl 2-acetamido-3-O-benz-
yl-4,6-O-benzylidene-2-deoxy-i- -glucopyran-
D
oside (6).—To a solution of 5 (2.5 g, 5.35
mmol), BaO (4.1 g, 26.75 mmol) and
Ba(OH)2·8H2O (740 mg, 2.3 mmol) in
Me2NCHO (20 mL) was added benzyl bro-
mide (1.83 mL, 12.6 mmol) and the mixture
was stirred for 15 min at rt. Then, CH2Cl2 (50
mL) was added and the resulting mixture was
boiled under reflux for 1 h, and then filtered
through Celite. The filtrate was washed with
2% HCl (25 mL), satd aq NaHCO3 (25 mL)
and H2O (25 mL), then dried and concen-
trated. After the addition of toluene (10 mL)
the solid was filtered, rinsed thoroughly with
toluene and recrystallized from EtOAc to af-
ford 6 (2.6 g, 85%): mp 203–205 °C; [h]D
−18° (c 1.0, CHCl3); Rf1 0.69 (5:1 CH2Cl2–
acetone); NMR (CDCl3): H, l 7.5–7.2 (m, 10
H, Ph), 6.35 (d, 1 H, J 8.5 Hz, NH), 5.65 (s, 1
H, PhCH), 4.95 (d, 1 H, J1,2 8.3 Hz, H-1), 4.36
(dd, 1 H, J5,6a 4.1, J6a,6b 10.2 Hz, H-6a), 4.10
(t, 1 H, J2,3 9.3 Hz, H-3), 3.95 (m, 1 H, H-2),
3.73 (m, 2 H, H-4,6b), 3.50 (m, 1 H, H-5),
3.38 (t, 2 H, J 4.9 Hz, CH2N3), 1.95 (s, 3 H,
CH3CON); 13C, ( 170.4 (CꢀO), 127.4–129.3
(Ph), 101.2 (PhCH), 100.9 (C-1), 82.1 (C-4),
77.6 (C-3), 72.0 (CH2Ph), 68.5 (C-6), 65.8
(C-5), 56.2 (C-2), 50.1 (CH2N3), 23.2
(CH3CON). Anal. Calcd for C27H36N4O7: C,
61.35; H, 6.86; N, 10.60. Found: C, 61.24; H,
6.98; N, 10.51.
2,3-Di-O-acetyl-4,6-O-benzylidene- -galac-
D
topyranose (9).—To a solution of 8 (1 g, 2.5
mmol) in EtOAc (25 mL) was added 2-
aminoethanol (0.3 mL, 5 mmol) and the mix-
ture was stirred overnight at rt. Then the
solution was washed with H2O (5 mL) and
brine (5 mL), dried (Na2SO4), filtered, and
concentrated. Column chromatography (2:1
hexane–EtOAc) of the residue afford the
anomeric mixture 9 as a white foam (0.49 g,
1
55%); Rf 0.36 (3:2 hexane–EtOAc); H NMR
[23]; 13C NMR (CDCl3): l 170.6 and 170.4
(CꢀO), 137.4 (ipso Ph), 128.9–125.9 (Ph),
100.4 (PhCH), 95.3 (C-1b), 90.5 (C-1a), 20.8–
20.4 (CH3COO). Anal. Calcd for C17H20O8: C,
57.95; H, 5.72. Found: C, 57.79; H, 5.85.

S. Figueroa-Pe´rez, V. Ve´rez-Bencomo / Carbohydrate Research 317 (1999) 29–38
35
5-Azido-3-oxapentyl (2,3-di-O-acetyl-4,6-
O-benzylidene-i- -galactopyranosyl)-(1“4)-
6-O-benzyl-2-deoxy-2-phthalimido-i- -glu-
2,3-Di-O-acetyl-4,6-O-benzylidene-h- -gal-
D
actopyranosyl trichloroacetimidate (10).—To a
solution of 9 (500 mg, 1.42 mmol) and
trichloroacetonitrile (0.7 mL, 7.09 mmol) in
dry CH2Cl2 (2 mL) was added anhyd K2CO3
(500 mg, 3.55 mmol), and the mixture was
stirred overnight at rt. Then, diethyl ether (2
mL) was added, the mixture was filtered over
Celite, and the filtrate was concentrated. The
syrupy residue was crystallized from Et2O to
give 10 as a white powder (503 mg, 71%): mp
174–175 °C; [h]D +176° (c 1.02, CHCl3), lit
+152.5° [23]; Rf1 0.46 (3:2 hexane–EtOAc);
NMR (CDCl3): H, l 8.65 (s, 1 H, NH),
7.55–7.45 (m, 5 H, Ph), 6.71 (d, 1 H, J 3.5 Hz,
H-1), 5.60 (dd, 1 H, J2,3 10.9 Hz, H-2), 5.57 (s,
1 H, PhCH), 5.39 (dd, 1 H, J3,4 3.3 Hz, H-3),
4.62 (d, 1 H, H-4), 4.35 (dd, 1 H, J5,6a 1.3,
J6a,6b 11.2 Hz, H-6a), 4.11 (m, 2 H, H-5,6b),
D
D
copyranoside (13).—A solution of 4 (600 mg,
1.7 mmol) and 10 (640 mg, 1.28 mmol) in dry
,
CH2Cl2 (5 mL) containing 4 A molecular
sieves (1 g) was stirred under dry N2 for 1 h at
rt. Then the mixture was cooled to −25 °C
and a solution of 0.1 M trimethylsilyl trifl-
uoromethanesulfonate in CH2Cl2 (0.2 mL,
0.02 mmol) was added. When TLC (2:1
toluene–EtOAc) showed the complete trans-
formation of the starting material, the mixture
was diluted with CH2Cl2 (10 mL) and filtered
over Celite. The filtrate was washed with satd
aq NaHCO3 (3 mL) and H2O (3 mL), then
dried (Na2SO4), filtered, and concentrated.
Column chromatography (2:1 toluene–
EtOAc) of the residue afforded 13 as a color-
less syrup (803 mg, 81%); [h]D +15° (c 1.30,
CHCl3); Rf 0.33 (2:1 toluene–EtOAc); NMR
13
2.10 and 2.05 (2 s, each 3H, 2CH3COO); C,
l 171.1–170.6 (CꢀO), 137.4 (ipso Ph), 128.9–
126.1 (Ph), 100.8 (PhCH), 94.4 (C-1), 20.9 and
1
(CDCl3): H, l 7.84 and 7.73 (Phth), 7.39
20.5
(2CH3COO).
Anal.
Calcd
for
(Ph), 5.43 (s, 1 H, PhCH), 5.33 (dd, 1 H, J1%,2%
8, J2%,3% 10.5 Hz, H-2%), 5.29 (d, 1 H, J1,2 8.4 Hz,
H-1), 4.85 (dd, 1 H, J3%,4% 3.6, H-3), 4.69 (AB,
2 H, J 12 Hz, CH2Ph), 4.48 (d, 1 H, J1%,2% 8 Hz,
H-1%), 4.45 (dd, 1 H, J2,3 10.4, J3,4 8.1 Hz,
H-3), 4.32 (d, 1 H, J3%,4 3.6 Hz, H-4%), 4.22 (m,
1 H, H-6%a), 4.18 (dd, 1 H, H-2), 3.97 (m, 1 H,
H-6%b), 3.91 (m, 1 H, H-6a), 3.75 (m, 1 H,
H-4), 3.66 (m, 1 H, H-6b), 3.55 (m, 1 H, H-5),
3.45 (m, 1 H, H-5%), 3.05 (t, 2 H, CH2N3), 2.01
and 1.98 (2CH3COO); 13C, l 170.6, 170.5,
170.0 and 168.9 (CꢀO), 137.9 and 137.1 (ipso
Ar), 134.2, 131.3 and 123.4 (Phth), 128.9-127.7
(Ph), 101.0 (C-1%), 100.8 (CHPh), 98.1 (C-1),
80.9 (C-4), 74.1 (C-5), 73.5 (CH2), 72.9 (C-4%),
71.5 (C-3%), 69.9 and 69.7 (CH2O), 69.3 (C-3),
68.7 (C-6), 68.5 (C-2%), 68.2 (C-6%), 66.5 (C-5%),
55.9 (C-2), 50.3 (CH2N3), 20.7 and 20.3
(2CH3COO). Anal. Calcd for C47H64N4O15: C,
61.03; H, 6.97; N, 6.06. Found: C, 60.89; H,
7.06; N, 5.97.
C18H20Cl3NO8: C, 44.60; H, 4.16; N, 2.89.
Found: C, 44.47; H, 4.26; N, 2.79.
2,3-Di-O-acetyl-4,6-O-benzylidene-h- -gal-
D
actopyranosyl bromide (12).—A solution of
bromine (61 mL, 1.1 mmol) in dry CH2Cl2 (0.6
mL) was added at 0 °C to a stirred solution of
11 (500 mg, 1.09 mmol) in dry CH2Cl2 (5 mL).
After 20 min, tetraethylammonium bromide
(100 mg) was added, and the mixture was
further stirred for 3 h at rt. Allyl bromide
(0.33 mL) was added to destroy the excess
bromine. The decolorized mixture was succes-
sively washed with cold aq satd NaHCO3 and
H2O, dried, and concentrated. Crystallization
of the residue from Et2O–hexane afforded 12
as a white powder (325 mg, 72%): mp 145–
147 °C; [h]D+285° (c 1, CHCl3); Rf 0.47 (3:2
1
hexane–EtOAc); NMR (CDCl3): H, l 7.58–
7.44 (m, 5 H, Ph), 6.82 (d, 1 H, J1,2 2.1 Hz,
H-1), 5.53 (s, 1 H, PhCH), 5.45 (dd, 1 H, J2,3
10.4 Hz, H-2), 5.26 (dd, 1 H, J3,4 3.7 Hz, H-3),
4.61 (d, 1 H, H-4), 4.34 (d, 1 H, J6a,6b 11.2 Hz,
H-6a), 4.13 (m, 1 H, H-6b), 2.10 and 2.05 (2 s,
each 3H, 2CH3COO); 13C, l 171.1–170.6
(CꢀO), 137.4 (ipso Ph), 128.9–126.1 (Ph),
100.8 (PhCH), 89.8 (C-1), 20.9 and 20.5
(CH3COO). Anal. Calcd for C17H19BrO7: C,
49.17; H, 4.61. Found: C, 49.00; H, 4.67.
5-Azido-3-oxapentyl (2,3-di-O-acetyl-4,6-
O-benzylidene-i-
3-O-acetyl-6-O-benzyl-2-deoxy-2-phthalimido-
i- -glucopyranoside (14).—Compound 13
D
-galactopyranosyl)-(1“4)-
D
(114 mg, 0.14 mmol) was treated with 1:1
Ac2O–pyridine (3 mL) for 24 h, then con-
centrated and co-concentrated with toluene to

36
S. Figueroa-Pe´rez, V. Ve´rez-Bencomo / Carbohydrate Research 317 (1999) 29–38
(CH3COO); 13C, l 170.3 and 169.4 (CꢀO),
137.7 and 137.3 (ipso Ar), 129.3–127.9 (Ph),
101.3 (C-1), 101.0 (CHPh), 100.2 (C-1%), 79.1
(C-3), 76.1 (C-4), 75.1 (C-5), 73.4 and 72.7
(CH2Ph), 73.3 (C-4%), 71.4 (C-3%), 69.9 and
69.5 (CH2O), 69.6 (C-2%), 68.5 (C-6), 68.2 (C-
6%), 66.1 (C-5%), 52.8 (C-2), 50.4 (CH2N3), 22.7
(CH2CON), 20.5 and 20.0 (CH3COO). Anal.
Calcd for C45H59N4O14: C, 61.42; H, 6.76; N,
6.37. Found: C, 61.54; H, 6.83; N, 6.21.
give 120 mg of 14: mp 212–213 °C (EtOAc–
hexane); [h]D +32.5° (c 2.4, EtOAc); Rf 0.45
1
(2:1 toluene–EtOAc); NMR (CDCl3): H, l
7.84 and 7.73 (Phth), 7.39 (Ph), 5.62 (dd, 1 H,
J2,3 9.7, J3,4 10.6 Hz, H-3), 5.42 (s, 1 H,
PhCH), 5.40 (d, 1 H, J1,2 9.7 Hz, H-1), 5.18
(dd, 1 H, J1%,2% 8, J2%,3% 10.5 Hz, H-2%), 4.77 (dd,
1 H, J3%,4% 3.6 Hz, H-3%), 4.65 (AB, 2 H, J 12
Hz, CH2Ph), 4.51 (d, 1 H, J1%,2% 8 Hz, H-1),
4.32 (d, 1 H, J3%,4% 3.6 Hz, H-4%), 3.66 (m, 1 H,
H-5), 3.28 (s, 1 H, H-5%), 3.10 (t, 2 H, CH2N3),
5-Azido-3-oxapentyl (2,3-di-O-acetyl-4,6-
13
2.01, 1.99 and 1.98 (3CH3COO); C, l 170.6,
O-benzylidene-i-
2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-i-
-glucopyranoside (16).—To a solution of 13
D
-galactopyranosyl)-(1“4)-
170.5, 170.0 and 168.9 (CꢀO), 137.9 and 137.1
(ipso Ar), 134.2, 131.3 and 123.4 (Phth),
128.9–127.7 (Ph), 101.0 (CHPh), 99.9 (C-1%),
97.1 (C-1), 75.0 (C-4), 74.5 (C-5), 73.4 (CH2),
73.0 (C-4%), 71.9 (C-3%), 70.5 (C-3), 69.9 (C-6),
69.7 (CH2O), 68.9 (C-2%), 68.3 (C-6%), 65.9
(C-5%), 54.7 (C-2), 50.3 (CH2N3), 20.6, 20.5
and 20.3 (3CH3COO). Anal. Calcd for
C49H66N4O16: C, 60.86; H, 6.88; N, 5.79.
Found: C, 60.79; H, 7.00; N, 5.61.
D
(237 mg, 0.31 mmol) in EtOH (25 mL) was
added hydrazine hydrate (1 mL, 20 mmol),
and the solution was stirred for 12 h at 90 °C.
Then, the solution was concentrated, and the
residue was treated with 1:1 Ac2O–pyridine (8
mL). After being stirred for 24 h at rt, the
mixture was concentrated and dissolved in
CH2Cl2 (10 mL), the resulting solution was
successively washed with 3% HCl (2×3 mL),
satd aq NaHCO3 (5 mL) and H2O (5 mL),
dried (Na2SO4), and concentrated. Column
chromatography of the residue (3:2 toluene–
EtOAc) afforded 16 as a yellow syrup (177
mg, 72.8%); [h]D +13° (c 2.80, CHCl3); Rf
0.33 (3:2 toluene–EtOAc); NMR (CDCl3):
1H, l 7.39 (Ph), 6.55 (d, 1 H, J 9.5 Hz, NH),
5.43 (s, 1 H, PhCH), 5.13 (dd, 1 H, J1%,2% 8, J2%,3%
5-Azido-3-oxapentyl (2,3-di-O-acetyl-4,6-
O-benzylidene-i-
D
-galactopyranosyl)-(1“4)-
-glu-
2-acetamido-3,6-di-O-benzyl-2-deoxy-i-
D
copyranoside (15).—A solution of 7 (100 mg,
0.17 mmol), silver trifuoromethanesulfonate
(150 mg, 0.85 mmol) and ethyldiisopropy-
lamine (32 mL, 0.17 mmol) in dry CH2Cl2 (1.5
,
mL) containing 4 A molecular sieves (200 mg)
was stirred under dry N2 for 1 h at rt. Then, a
solution of 12 (331 mg, 0.8 mmol) in dry
CH2Cl2 was added and the mixture was stirred
overnight. The mixture was diluted with
CH2Cl2 (10 mL) and filtered over Celite. The
filtrate was washed with aq 10% Na2S2O3 (5
mL), satd aq NaHCO3 (5 mL), and H2O (5
mL), then dried (Na2SO4) and concentrated.
The residue was chromatographed (6:1
CH2Cl2 –acetone) to afford 15 (95 mg, 58%) as
a slightly yellow syrup; [h]D+22° (c 1.0,
CHCl3); Rf 0.35 (5:1 CH2Cl2–acetone); NMR
10.2 Hz, H-2%), 5.05 (dd, 1 H, J2,3 10.1, J
9
3,4
Hz, H-3), 4.76 (dd, 1 H, J3%,4% 4 Hz, H-3%), 4.61
(AB, 2 H, J 12.3 Hz, CH2Ph), 4.52 (d, 1 H,
J1,2 8.1 Hz, H-1), 4.45 (d, 1 H, J1%,2% 8 Hz,
H-1%), 4.32 (m, 2 H, H-4%,6%a), 4.14 (dd, 1 H,
H-2), 3.95 (m, 3 H, H-6%b,6a,4), 3.70 (dd, 1 H,
H-6b), 3.58 (t, 6 H, CH2O), 3.46 (m, 1 H,
H-5), 3.32 (t, 2 H, CH2N3), 3.24 (s, 1 H, H-5%),
2.21 (CH2CON), 2.00 and 1.98 (CH3COO);
13C, l 170.0 and 168.9 (CꢀO), 137.7 and 137.4
(ipso Ar), 128.9–127.7 (Ph), 101.4 (C-1), 100.9
(CHPh), 100.0 (C-1%), 74.6 (C-4), 74.6 (C-5),
73.4 (CH2Ph), 73.0 (C-4%), 72.9 (C-3), 71.6
(C-3%), 70.2 and 69.7 (CH2O), 68.8 (C-2%), 68.3
(C-6), 68.3 (C-6%), 65.8 (C-5%), 53.1 (C-2), 50.6
(CH2N3), 22.9 (CH2CON), 20.7 and 20.3
(CH3COO). Anal. Calcd for C39H51N4O15: C,
57.42; H, 6.30; N, 6.87. Found: C, 57.58; H,
6.45; N, 6.79.
1
(CDCl3): H, l 7.38–7.29 (Ph), 6.21 (d, 1 H, J
8.9 Hz, NH), 5.41 (s, 1 H, PhCH), 5.35 (dd, 1
H, J1%,2% 8, J2%,3% 10.5 Hz, H-2%), 4.87 (dd, 1 H,
J3%,4% 3.8 Hz, H-3%), 4.77 (d, 1 H, J1,2 8.4 Hz,
H-1), 4.46 (d, 1 H, J1%,2% 8.1 Hz, H-1%), 4.43 (m,
1 H, H-2), 4.32 (m, 2 H, H-4,4%), 3.96 (m, 3 H,
H-6a,3,6%b), 3.91 (m, 1 H, H-6a), 3.66 (m, 1 H,
H-6b), 3.43 (m, 2 H, H-5,5%), 3.07 (t, 2 H,
CH2N3), 2.01 (s, 3 H, CH3CON), 1.98

S. Figueroa-Pe´rez, V. Ve´rez-Bencomo / Carbohydrate Research 317 (1999) 29–38
37
5-Azido-3-oxapentyl (2,3-di-O-acetyl-i-
galactopyranosyl) - (1“4) - 2 - acetamido - 3 - O-
acetyl-6-O-benzyl-2-deoxy-i- -glucopyranos-
D
-
(Na2SO4), and concentrated. Column chro-
matography of the residue (50:1 CHCl3–
MeOH) afforded 19 (50.5 mg, 45%);
[h]D +35° (c 0.6, CHCl3); Rf 0.35 (20:1
D
ide (17).—A solution of 16 (177 mg, 0.22
mmol) in aq 60% HOAc (2 mL) was stirred at
100 °C until TLC (2:1 CH2Cl2–acetone)
showed the complete transformation of the
starting material into a slower migrating
product (ꢀ30 min). Then, the mixture was
concentrated and the remaining acid was co-
evaporated with toluene (3×3 mL). The
residue was filtered through a short column of
silica gel (1 g) using EtOAc as the solvent to
give 17 as a foam (150 mg, 94%); NMR
13
CHCl3–MeOH); C NMR (CDCl3): l 171.1–
169.3 (6CꢀO), 167.8 (JC-1neu,H-3 6.6 Hz, C-
a
1neu), 137.7 (ipso Ar), 128.9–127.7 (Ph),
101.2 (C-1), 100.4 (C-1%), 98.8 (C-2neu), 62.3
and 62.1 (C-9neu,6%), 53.3 (C-2), 53.0 (OCH3),
50.8 (CH2N3), 49.3 (C-5neu), 37.0 (C-3neu),
23.2 and 23.1 (CH3CON), 20.9–20.7
(CH3COO). Anal. Calcd for C53H76N5O26: C,
53.08; H, 6.39; N, 5.84. Found: C, 52.99; H,
6.51; N, 5.80 and 20 (16 mg, 16.5%); Rf 0.37
(20:1 CHCl3–MeOH); 13C NMR (CDCl3): l
1
(CDCl3): H, l 7.39 (Ph), 6.47 (d, 1 H, J 9.2
Hz, NH), 5.11 (dd, 1 H, J1%,2% 8, J2%,3% 10.2 Hz,
171.7–169.4 (6CꢀO), 166.9 (JC-1neu,H-3 1.7 Hz,
a
H-2%), 5.01 (dd, 1 H, J2,3 10.1, J 9 Hz, H-3),
3,4
C-1neu), 137.7 (ipso Ar), 128.9–127.7 (Ph),
101.2 (C-1), 100.5 (C-1%), 98.3 (C-2neu), 62.5
and 60.7 (C-9neu,6%), 53.2 (C-2), 52.9 (OCH3),
50.8 (CH2N3), 49.6 (C-5neu), 37.3 (C-3neu),
23.1 (2CH3CON), 21.0–20.8 (CH3COO).
Anal. Calcd for C53H76N5O26: C, 53.08; H,
6.39; N, 5.84. Found: C, 52.97; H, 6.53; N,
5.78.
4.69 (dd, 1 H, J3%,4% 4 Hz, H-3%), 4.63 (AB, 2 H,
J 12.3 Hz, CH2Ph), 4.51 (d, 1 H, J1,2 8.1 Hz,
H-1), 4.41 (d, 1 H, J1%,2% 8 Hz, H-1%), 3.35 (t, 2
H, CH2N3), 2.21 (s, 3 H, CH3CON), 2.00 and
1.98 (2 s, 6 H, CH3COO); 13C, l 170.0 and
168.9 (CꢀO), 137.7 (ipso Ar), 128.9–127.7
(Ph), 101.1 (C-1), 99.9 (C-1%), 61.1 (C-6%), 55.1
(C-2), 50.4 (CH2N3), 22.9 (CH2CON), 20.7
and 20.3 (CH3COO). Anal. Calcd for
C32H47N4O15: C, 52.81; H, 6.51; N, 7.70.
Found: C, 52.68; H, 6.65; N, 7.57.
5-Amino-3-oxapentyl (sodium [5-acetamido-
3,5-dideoxy-
pyranosyl]onate)-(2“6)-(i-
syl)-(1“4)-2-acetamido-2-deoxy-i-
D
-glycero-h-
D
-galacto-2-nonulo-
-galactopyrano-
-gluco-
D
D
5-Azido-3-oxapentyl (methyl [5-acetamido-
pyranoside (21).—Compound 19 (60 mg, 51.9
mmol) was dissolved in a solution of methanolic
NaOMe (0.05 M, 2 mL), and stirred overnight
at rt, then H2O (1 mL) was added and the
mixture was stirred for 12 h at rt. Then, the pH
was brought to 7 with Amberlite IR 120 (H+)
resin, and after filtration, aq 0.1 M NaOH (0.5
mL) was added to the solution. The solvents
were evaporated in vacuo and the residue was
chromatographed on a LH-20 column slurred
with MeOH. Hydrogenolysis of the resulting
foam in 2:1 MeOH–H2O in the presence of 10%
Pd/C under H2 gas rendered after lyophiliza-
tion 21 as a white powder (35.5 mg, 91%); Rf
0.17 (3:3:1:0.1 EtOAc–MeOH–H2O–HOAc);
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
D
-glycero-h-
D
-galacto-2-nonulopyranosyl]onate)-(2“6)-(2,-
3-di-O-acetyl-i- -galactopyranosyl)-(1“4)-2-
D
acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-i-
D
-glucopyranoside (19) and 5-azido-3-oxa-
pentyl (methyl [5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy- -glycero-i- -galacto-2-
nonulopyranosyl]onate)-(2“6)-(2,3-di-O-acet-
yl-i- -galactopyranosyl)-(1“4)-2-acetam-
ido-3-O-acetyl-2-deoxy-6-O-benzyl-i- -glu-
D
D
D
D
copyranoside (20).—A solution of 17 (62 mg,
0.09 mmol) and 18 [19] (103 mg, 0.18 mmol) in
,
dry CH3CN (1 mL) containing 3 A molecu-
lar sieves (200 mg) was stirred for 1 h at rt.
Then, the mixture was cooled to −40 °C, and
N-iodosuccinimide (50 mg, 0.22 mmol) and a
solution of trifluoromethanesulfonic acid in
CH3CN (0.22 M, 0.1 mL) were added under
dry N2. After being stirred for 4 h at −40 °C,
the mixture was diluted with CH2Cl2 (10 mL),
filtered through Celite, and the filtrate was
washed with aq 10% Na2S2O3 (3 mL), satd aq
NaHCO3 (3 mL) and H2O (4 mL), then dried
1
NMR (D2O): H, l 4.48 (d, 1 H, J1,2 7.9 Hz,
H-1), 4.45 (d, 1 H, J1%,2% 8.2 Hz, H-1%), 3.19 (t,
2 H, CH2NH2), 2.69 (dd, 1 H, Jgem 13.7, J3e,4
4.5 Hz, H-3eneu), 2.04 and 2.02 (2 s, 6 H,
CH3CON), 1.73 (t, 1 H, H-3aneu); 13C, l 105.1,
103.4 and 100.4 (C-1, 1%, 2neu), 78.4, 78.0 and
77.8, (C-4,3%,6neu), 64.7, 64.0 and 62.0 (C-
6,6%,9neu), 57.9 and 54.0 (C-2,5neu), 42.4 (C-

38
S. Figueroa-Pe´rez, V. Ve´rez-Bencomo / Carbohydrate Research 317 (1999) 29–38
[2] D.M. Goldenberg, A.M. Neville, A.C. Carter, V.L.W.
Go, E.D. Holyoke, K.J. Isselbacher, P.S. Schein, M.
Schwartz, K.R. McIntire, L.P. Greenberg, Cancer Res.,
41 (1981) 2017–2018.
[3] K. Yamashita, K. Totani, M. Kuroki, Y. Matsuoka, I.
Ueda, A. Kobata, Cancer Res., 47 (1987) 3451–3459.
[4] K. Yamashita, A. Kobata, Carcinoembryonic antigen
and related normal antigens, in J. Montreuil, J.F.G.
Vliegenthart, H. Schachter (Eds.), Glycoproteins and Dis-
ease, Elsevier, 1996, pp. 229–238.
[5] A. Matsunaga, M. Kuroki, H. Higushi, F. Arakawa, K.
Takakura, N. Okamoto, Y. Matsuoka, Cancer Res., 47
(1987) 56–61.
[6] K. Yamashita, K. Totani, Y. Iwaki, M. Kuroki, Y.
Matsuoka, Y. Endo, A. Kobata, J. Biol. Chem., 264
(1989) 17873–17881.
[7] K. Fukushima, T. Ohkura, M. Kanai, M. Kuroki, Y.
Matsuoka, A. Kobata, K. Yamashita, Glycobiology, 5
(1995) 105–115.
[8] E.J. Nichols, R. Kannagi, S. Hakomori, M.J. Krantz, A.
Fucks, J. Immunol., 135 (1985) 11911–11913.
[9] K. Yamashita, K. Umetsu, T. Susuki, T. Ohkura, Bio-
chemistry, 31 (1992) 11647–11650.
[10] K. Yamashita, K. Fukushima, T. Sakiyama, F. Muruta,
M. Kuroki, Y. Matsuoka, Cancer Res., 55 (1995) 1675–
1679.
[11] S. Figueroa Pe´rez, R. Gonza´les Lio, V. Ferna´ndez San-
tana, V Verez-Bencomo, J. Carbohydr. Chem., 17 (1998)
835–850.
3neu), 41.5 (CH2NH2), 24.8 and 24.2
(CH3CON). Anal. Calcd for C31H55N3NaO19:
C, 46.73; H, 6.96; N, 5.27. Found: C, 46.85;
H, 7.09; N, 5.15.
5-(i-Maleimidopropionamido)-3-oxapentyl
(sodium [5-acetamido-3,5-dideoxy-
h-
(i-
2-deoxy-i-
D
-glycero-
D
-galacto-2-nonulopyranosyl]onate)-(2“6)-
-galactopyranosyl)-(1“4)-2-acetamido-
-glucopyranoside (22).—To a so-
D
D
lution of the free oligosaccharide 21 (5.2 mg, 4
mmol) in Me2NCHO/PBS (0.5 mL) was added
the N-hydroxysuccinimide derivative of b-
maleimidopropanoic acid [24] (2.6 mg, 10
mmol). After 2 h, the solution was concen-
trated and dried in vacuo, the residue was
resuspended in D2O (0.5 mL, for the NMR
analysis) and centrifuged to remove the excess
of reagent. The maleimido derivative 22 could
be stored in a lyophilized form at 0 °C or used
1
directly in the coupling reaction; H NMR
(D2O): l 6.90 (s, 0.3 H, HCꢀCH b-maleimido-
propionic acid), 6.89 (s, 2 H, HCꢀCH), 3.32 (t,
2 H, CH2NH), 3.05 and 2.56 (2 t, 0.3 H each,
CH2 b-maleimido-propionic acid), 2.48 (t, 2
H, CH2CO).
[12] V. Ferna´ndez Santana, R. Gonza´les Lio, J. Sarracent
Pe´rez, V. Verez-Bencomo, Glycoconjugate J., 15 (1998)
549–553.
[13] M.T. Campos-Valdes, J.R. Marin˜o-Albernas, V. Verez-
Bencomo, J. Carbohydr. Chem., 6 (1987) 509–512.
[14] G. Grynkiewicz, I. Fokt, W. Szeja, H. Fitak, J. Chem.
Res. (S), (1989) 152–154.
Coupling reaction between 22 and thiolated
BSA.—Compound 22 was added to a solu-
tion [12] of BSA-SH30 in PBS (pH 7.2, 0.4
mL). After 2 h, the process was complete as
evidenced by a negative Ellman test [25]. The
resulting solution was dyafiltrated against PBS
(pH 7.4). The protein and carbohydrate con-
tents were determined by the Lowry [26] and
phenol–sulfuric acid methods [27], respec-
tively. Several assays gave a carbohydrate to
protein ratio of 13–15 oligosaccharide units
per BSA molecule.
[15] R.R. Schmidt, J. Michel, M. Roos, Liebigs Ann. Chem.,
(1984) 1343–1357.
[16] (a) T. Ehara, A. Kameyama, Y. Yamada, H. Ishida, M.
Kiso, A. Hasegawa, Carbohydr. Res., 281 (1996) 237–
252. (b) S. Nunomura, M. Iida, M. Numata, M. Sugi-
moto, T. Ogawa, Carbohydr. Res., 263 (1994) C1–C6. (c)
L. Lay, L. Manzoni, R.R. Schmidt, Abstracts of Papers,
XVIII International Carbohydrate Symposium, Milan,
Italy, July 1996, BP166.
[17] W. Stahl, U. Sprengard, G. Kretzmar, H. Kunz, Angew.
Chem., Int. Ed. Engl., 33 (1994) 2096–2098.
[18] M. Nilsson, T. Norberg, Carbohydr. Res., 183 (1988)
71–82.
[19] E. Kirchner, F. Thiem, R. Dernick, J. Heukeshoven, J.
Thiem, J. Carbohydr. Chem., 7 (1988) 453–486.
[20] H. Prabhanan, K. Aoyama, M. Kiso, A. Hasegawa,
Carbohydr. Res., 233 (1992) 87–99.
Acknowledgements
[21] H. Hori, T. Nakajima, Y. Nishida, H. Meguro, Tetrahe-
dron Lett., 29 (1988) 6317–6320.
[22] T. Murase, H. Ishida, M. Kiso, A. Hasegawa, Carbo-
hydr. Res., 188 (1989) 71–80.
The authors wish to thank the Ministry of
Health for financial support.
[23] U. Greilich, P. Zimmermann, K.-H. Jung, R.R. Schmidt,
Liebigs Ann. Chem., (1993) 859–864.
[24] O. Keller, J. Rudinger, Hel6. Chim. Acta, 58 (1975)
531–541.
References
[1] D.E. Henson, L.P. Fielding, D.J. Grignon, D.L. Page,
M.E. Hammond, G. Nash, N.M. Pettigrew, F. Gorstein,
R.V.P. Hutter, Arch. Pathol. Lab. Med., 119 (1995)
1109–1112.
[25] G.L. Ellman, Arch. Biochem. Biophys., 74 (1958) 443.
[26] C. Capla, Methods Enzymol., 73 (1957) 448–450.
[27] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F.
Smith, Anal. Biochem., 28 (1956) 350–356.