Synthesis of 3-O-sulfoglucuronyl lacto-N-neotetraose 2-aminoethyl glycoside and biotinylated neoglycoconjugates thereof

Article abstract of DOI:10.1016/S0008-6215(00)00258-5

The 2-aminoethyl glycoside of pentasaccharide 3-O-sulfo-GlcA(β-1→3)Gal(β-1→4)GlcNAc(β-1→3) Gal(β-1→4)Glc(β (1) and its conjugates with biotin and biotinylated polyacrylic acid were synthesized as molecular probes to investigate the recognition of the HNK-1 epitope containing carbohydrates by proteins. Key steps in the first of two investigated schemes for the preparation of the target compound 1 were (a) assembling of the pentasaccharide backbone (compound 10) by glycosylation of selectively substituted allyl glycoside of the trisaccharide GlcNAc(β-1→3)Gal(β-1→4)Glc(β with glucuronyl-galactose glycosyl donor, (b) transformation of the allyl aglycon in 10 into 2-azidoethyl one (to give 11), (c) selective deprotection of the OH group at C-3 of the GlcA residue in 11 via saponification, intramolecular formation of 6,3-lacton (13) and its methanolysis, and (d) subsequent O-sulfation. The alternative scheme with the use of 2-azido-ethyl glycoside of the trisaccharide GlcNAc(β-1→3)Gal(β-1→4)Glc(β instead of the allyl glycoside 6 was less effective due to smaller yield at the step of pentasaccharide synthesis. Additionally to 1 the 2-aminoethyl glycosides of the oligosaccharides GlcA(β-1→3)Gal(β-1→4)GlcNAc(β-1→3)Gal(β -1→4)Glc(β, 3-O-sulfo-GlcA(β-1→3)Gal(β, and GlcA(β-1→3)Gal(β were also synthesized.

Full text of DOI:10.1016/S0008-6215(00)00258-5

www.elsevier.nl/locate/carres
Carbohydrate Research 329 (2000) 717–730
Synthesis of 3-O-sulfoglucuronyl lacto-N-neotetraose
2-aminoethyl glycoside and biotinylated
neoglycoconjugates thereofꢀ
Andrei V. Kornilov, Andrey A. Sherman, Leonid O. Kononov,
Alexander S. Shashkov, Nikolay E. Nifant’ev *
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47,
Moscow 117913, Russia
Received 14 April 2000; accepted 15 August 2000
Abstract
The 2-aminoethyl glycoside of pentasaccharide 3-O-sulfo-GlcA(b-1“3)Gal(b-1“4)GlcNAc(b-1“3)Gal(b-1“
4)Glc(b (1) and its conjugates with biotin and biotinylated polyacrylic acid were synthesized as molecular probes to
investigate the recognition of the HNK-1 epitope containing carbohydrates by proteins. Key steps in the first of two
investigated schemes for the preparation of the target compound 1 were (a) assembling of the pentasaccharide
backbone (compound 10) by glycosylation of selectively substituted allyl glycoside of the trisaccharide GlcNAc(b-
1“3)Gal(b-1“4)Glc(b with glucuronyl-galactose glycosyl donor, (b) transformation of the allyl aglycon in 10 into
2-azidoethyl one (to give 11), (c) selective deprotection of the OH group at C-3 of the GlcA residue in 11 via
saponification, intramolecular formation of 6,3-lacton (13) and its methanolysis, and (d) subsequent O-sulfation. The
alternative scheme with the use of 2-azido-ethyl glycoside of the trisaccharide GlcNAc(b-1“3)Gal(b-1“4)Glc(b
instead of the allyl glycoside 6 was less effective due to smaller yield at the step of pentasaccharide synthesis.
Additionally to 1 the 2-aminoethyl glycosides of the oligosaccharides GlcA(b-1“3)Gal(b-1“4)GlcNAc(b-1“
3)Gal(b-1“4)Glc(b, 3-O-sulfo-GlcA(b-1“3)Gal(b, and GlcA(b-1“3)Gal(b were also synthesized. © 2000 Elsevier
Science Ltd. All rights reserved.
Keywords: HNK-1; Neoglycoconjugates; Glucuronic acid; Lactonization
1. Introduction
5]). This epitope is presented in glycolipids [6]
with penta- or heptasaccharide chains 3-O-
sulfo-GlcA(b1-[3)Gal(b-1“4)GlcNAc(b1-]n3)-
Gal(b-1“4)Glc(b (n=1, 2) and on several
glycoproteins and proteoglycans [5] of neural
tissues. In the case of glycoproteins the anti-
genic structure is variable and has been pre-
cisely described only for the carbohydrate
chains of bovine peripheral nerve glycoprotein
P0 [7] in which the trisaccharide fragment
3-O-sulfo-GlcA(b-1“3)Gal(b-1“4)GlcNAc
is attached to bisected mannose core.
Natural carbohydrates that have the HNK-
1 epitope participate in neurite outgrowth and
in neural cells adhesion. They are the ligands
for L- and P-selectins, and play important
roles in other developmental processes of
mammalian nervous system (see reviews [2–
ꢀ Synthesis of oligosaccharides related to HNK-1 antigen,
Part 3. For Part 2, see Ref. [1].
* Corresponding author. Fax: +7-95-1358784.
E-mail address: nen@ioc.ac.ru (N.E. Nifant’ev).
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00258-5

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A.V. Kornilo6 et al. / Carbohydrate Research 329 (2000) 717–730
In order to prepare the tools for investiga-
The second distinct feature in the synthesis
of target compound 1 was the use of the allyl
aglycon as the pre-spacer which provided
good flexibility during assembling of the pen-
tasaccharide backbone and permitted intro-
duction and removal of necessary protecting
groups (Scheme 1). The 2-azidoethyl group is
known [12] to be a direct precursor of the
2-aminoethyl spacer. To transform the allyl
group into 2-azidoethyl we used a convenient
procedure what included sequences ozonoly-
sis“reduction“sulfonylation [13] and sulfo-
nate“azide [14] according to known
procedures which we adopted to the cases of
glucuronic and sialic acid derivatives [15].
Along these lines we investigated two ways
(Routes A and B in the Scheme 1) for the
preparation of substituted pentasaccharide 2-
azidoethyl glycoside 11 which was a direct
precursor of target compound 1. In Route A
the pentasaccharide allyl glycoside 10 was pre-
pared first and then transformed into 11. In
Route B functionalization of allyl aglycon was
performed at an earlier stage during the syn-
thesis of trisaccharide block 8.
tion of proteins which recognize HNK-1 car-
bohydrates, we have performed systematic
synthesis of related oligosaccharides and neo-
glycoconjugates. In this paper we describe the
synthesis of di- and pentasaccharide 2-
aminoethyl glycosides 1–4, and of the conju-
gates of 1 with biotin (24) and biotinylated
polyacrylic acid (27). The pentasaccharide 3-
O-sulfo-GlcA(b-1“3)Gal(b-1“4)GlcNAc(b-
1“3)Gal(b-1“4)Glc was previously synthe-
sized as ceramide [8–10] and propyl [1] gly-
cosides.
[3 - O - sulfo - GlcA(b - 1“3)Gal(b - 1“4)Glc-
NAc(b - 1“3)Gal(b - 1“4)Glc(b1 - O(CH2)2-
NH2]−Na+2
1
[GlcA(b-1“3)Gal(b-1“4)GlcNAc(b-1“3)-
Gal(b-1“4)Glc(b1-O(CH2)2NH2]−Na+
2
[3 - O - sulfo - GlcA(b - 1“3)Gal(b1 - O(CH2)2-
NH2]−Na+2
3
[GlcA(b-1“3)Gal(b1-O(CH2)2NH2]−Na+
4
Ozonolysis of trisaccharide allyl glycoside 5
[1] and subsequent reduction of ozonides with
excess of sodium borohydride afforded 2-hy-
droxyethyl glycoside 7. Its mesylation, treat-
ment with NaN3 and O-deacetylation afforded
to the corresponding 2-azidoethyl glycoside 8
in the overall yield of 77%.
2. Results and discussion
Preparation of the target pentasaccharide 1
was performed by two synthetic schemes with
the use of disaccharide glycosyl donor 9 [1]
and trisaccharide glycosyl acceptor blocks 6
[1] and 8 as main precursors (Scheme 1). They
were selected according to retrosynthetic anal-
ysis of structure 1 which revealed the effi-
ciency of the [2+3]. The strategy used to
build the pentasaccharide backbone was also
used by Hasegawa and co-workers [9].
The synthesis of pentasaccharide 1 (Scheme
1) included the selective deprotection of the
OH group at C-3 of the glucuronic acid
(GlcA) residue via saponification and the 6,3-
lactonization–methanolysis procedure [1,11]
(12“13“14) prior to O-sulfation. This key
feature of the strategy allowed us to use the
readily available disaccharide trichloroacetimi-
date 9 [1] which does not contain any tempo-
rary protecting group at O-3 of the GlcA
residue (instead of much less available selec-
tively protected derivatives which were used in
first syntheses of HNK-1 related oligosaccha-
rides [8,9]).
Coupling of the trichloroacetimidate 9 with
the trisaccharides 6 [1] and 8 in dichloro-
methane under promotion with trimethylsilyl-
triflate (TMSOTf) afforded the pentasaccha-
ride allyl and 2-azidoethyl glycosides 10
(identical to the previously obtained com-
pound [1]) and 11 in 82 and 62% yields,
respectively. The b configuration of the newly
formed glycosidic bonds was deduced from
the characteristic values of the respective
spin–spin coupling constants J1,2 (8.0 and 7.4
1
Hz) in the H NMR spectra of 10 and 11.
Before transforming the allyl aglycon in 10
into 2-azidoethyl group, the CO2Me group in
pentasaccharide 10 was converted into its
lithium salt under treatment with LiI in Py
[16] in order to avoid the undesired reduction
of C-6 in GlcA residue during the treatment of
ozonides with sodium borohydride (Scheme

A.V. Kornilo6 et al. / Carbohydrate Research 329 (2000) 717–730
719
1). The salt was subjected to ozonolysis, re-
duction, O-methylation with CH2N2, tosyla-
tion, and subsequent treatment with NaN3 to
give 11 in 53% yield over nine steps along
Route A. A shorter Route B gave a compara-
ble but slightly lower overall yield (49%) of 11
for six steps from 5. The diminished yield in
the latter case is due to less effective glycosyla-
tion by imidate 9 of 2-azidoethyl glycoside 8
than of respective allyl glycoside 6.
Selective deprotection of the OH group at
C-3 of the GlcA residue in pentasaccharide 11
was the next step of the preparation of target
compound 1. It was achieved using saponifica-
tion of 11 and further 6,3-lactonization,
exhaustive O-acetylation and subsequent
Scheme 1. Reagents and conditions: (a) MeONa in MeOH; (b) O3, −78 °C, CH2Cl2–MeOH; (c) NaBH4, −78 °C; (d) MsCl,
,
Et3N; (e) NaN3, 18-crown-6, DMF; (f) TMSOTf, CH2Cl2, MS 4 A, −30 °C, 100 h; (g) LiI, Py, reflux; (h) treatment with 1 M
H2SO4; (i) CH2N2, 0 °C, CH2Cl2; (j) TsCl, Py; (k) H2, PdO/C; (l) CF3COOEt, Et3N; (m) LiOH in aq THF; (n) Ac2O, 80 °C; (o)
Ac2O, Py, DMAP, rt; (p) MeOH, anhyd AcONa; (r) SO3·Py, DMF; (s) MeONa in aq MeOH.

720
A.V. Kornilo6 et al. / Carbohydrate Research 329 (2000) 717–730
Scheme 2. Reagents and conditions: (a) LiI, Py, reflux; (b) O3, −78 °C, CH2Cl2–MeOH; (c) NaBH4, −78 °C; (d) treatment with
1 M H2SO4; (e) CH2N2, 0 °C, CH2Cl2; (f) MsCl, Et3N; (g) NaN3, 18-crown-6, DMF, rt; (h) LiOH in aq THF, NaOH in aq
MeOH; (i) Ac2O, 80 °C; (j) Ac2O, Py, DMAP, rt; (k) MeOH, AcONa; (l) SO3·Py, DMF; (m) H2, PdO/C.
opening of lacton ring by methanolysis under
treatment with sodium acetate and methanol
(11“12“13“14). For this sequence of reac-
tions we used an experimental procedure,
which was previously applied [1] for the trans-
formation of pentasaccharide allyl glycoside
10 and gave its 3¨-hydroxy derivative in 74%
overall yield [1].
Treatment of the monohydroxy derivative
14 with SO3·Py in DMF and subsequent gel
filtration on an LH-20 column in methanol
gave the O-sulfated compound 15 in 95% yield
(Scheme 1). Location of the sulfate at C-3 of
the GlcA residue in 15 was confirmed by
characteristic low-field shift of the H-3 reso-
1
nance (l 3.70“4.62 ppm) in the H NMR
Thus pentasaccharide 11 was saponified
first to give the acid 12, which was lactonized
under treatment with acetic anhydride at
70 °C and then O-acetylated by addition of Py
and a catalytic amount of 4-(N,N-dimethyl-
amino)pyridine added to the reaction mixture.
The thus formed crude lactone 13 was sub-
jected to methanolysis to give the target
monohydroxy derivative 14 in 35% overall
yield from 11. The presence of the free OH
group at C-3 of GlcA residue in 14 followed
from the high-field position of H-3¨ reso-
nance (l 3.70 ppm) and the low-field values of
the chemical shifts of H-2¨ and H-4¨ protons
spectrum when compared with the data for
alcohol 14. The sulfate 15 was subjected to
hydrogenolysis and subsequent saponification
to give the target pentasaccharide 1 (67%)
which was purified by gel chromatography.
Characteristic low-field positions of H-3¨ res-
1
onance (l 4.33 ppm) in the H NMR and of
13
C-3¨ resonance (l 84.8 ppm) in the C NMR
spectra of 1 confirmed the location of the
sulfate group at C-3 of the GlcA residue.
In a similar way to the preparation of 11, 15
and 1, the disaccharide allyl glycoside 16 [17]
was transformed into its 2-azidoethyl gly-
coside 18 in 65% overall yield in six steps,
monohydroxy derivative 21 (53%), its sulfate
22 (85%) and target 2-aminoethyl glycosides 3
1
(l 4.81 and 5.14 ppm, respectively) in the H
NMR spectrum.

A.V. Kornilo6 et al. / Carbohydrate Research 329 (2000) 717–730
721
and 4 (Scheme 2). Compound 4 as the Na salt
has been previously obtained [18].
The structure of conjugates 24 and 27 was
1 13
confirmed by H and C NMR spectroscopy.
Particularly, the location of the sulfate group
at C-3 of the GlcA residue was confirmed by
the characteristic values of H-3¨ resonances
Aiming to obtain 2 we tried to perform
hydrogenolysis of the pentasaccharide 11. It
was unsuccessful in different mixtures of sol-
vents (THF–AcOH (1%); EtOH–AcOH
(1%)) and even under heating to 35–45 °C.
This problem could be connected with catalyst
passivation due to interaction with the
aminoethyl fragment formed. Finally the hy-
drogenolysis was carried out in two steps:
after the completing of the reduction of N3
group in 11 the mixture was treated with ethyl
trifluoroacetate. The N-trifluoroacetylated
product formed was separated by flash
column chromatography and subjected to fur-
ther hydrogenolysis and subsequent saponifi-
cation to give spacered pentasaccharide 2
which was purified by gel chromatography
(Scheme 1).
Condensation of the spacer-armed penta-
saccharide 1 with the p-nitrophenyl ester of
v-N-biotinyl capronic acid 23 gave quantita-
tively the monovalent biotinylated glycoconju-
gate 24 (Scheme 3) which was purified by gel
chromatography. The polyvalent biotinylated
conjugate (27) of spacered pentasaccharide 1
was synthesized according to the original pro-
cedure by Bovin et al. [19] by treatment of
poly(p-nitrophenyl acrylate) 25 with 0.2 equiv
of 1 and 0.05 equiv of N-biotinyl hexam-
ethylendiamine 26 followed by capping the
unreacted activated carboxylic groups of the
polyacrylic backbone with an excess of 2-
aminoethanol (Scheme 3).
1
in H NMR spectra (l 4.26 and 4.28 ppm for
compounds 24 and 27, respectively). The sac-
charide patterns in the 13C NMR spectra of
conjugates 24 and 27 agreed well with those of
the 13C NMR spectra of starting material 1.
The only marked difference occurred in the
case of conjugate 27 for C-1 of the Glc residue
which was observed as a broad signal due to
the complex surrounding the matrix.
In conclusion, we have accomplished the
first synthesis of a spacer-armed oligosaccha-
rides 1–4 together with mono- and polyvalent
biotinylated neoglycoconjugates 24 and 27.
These compounds were used in the investiga-
tions of a mechanism of long-term regulation
of perisomatic inhibition in the hyppocampus
and assessment of selectins [20] and anti-CD57
Abs [21] carbohydrate specificities.
3. Experimental
NMR spectra were recorded with Bruker
DRX-500 and Bruker AM-300 instruments,
assignments were made by 2D correlation
spectroscopy and polarization-transfer experi-
ments, and only characteristic signals are in-
cluded in Section 3. For solutions in D2O,
tert-BuOH served as the reference at 1.23 and
Scheme 3. Reagents and conditions: (a) DMSO, Et3N; (b) 1 (0.2 equiv), R1CH2NH2 (0.05 equiv), Et3N, 1:1 DMF–DMSO (v/v),
40 °C, 24 h; (c) HO(CH2)2NH2 (excess), Et3N, 24 h.

722
A.V. Kornilo6 et al. / Carbohydrate Research 329 (2000) 717–730
31.1 ppm for H and 13C NMR, respectively.
Mass spectra were recorded using matrix-as-
sisted laser-desorption ionization time-of-
flight (MALDI-TOF) on a Vision 2000 mass
spectrometer. The optical rotation values were
measured on a JASCO DIP-360 digital polar-
imeter at 22–26 °C. TLC was performed on
Kieselgel 60 plates (Merck, Germany); the
spots were visualized by spraying with 10%
H3PO4 in EtOH and subsequent heating to
ꢀ150 °C. Column chromatography was car-
ried out on a Silica Woelm 32–63 mm chro-
matograph (Woelm Pharma) using the solvent
system specified. Ion-exchange chromatogra-
phy was performed on a DEAE-Spheron 25–
40 mm (Chemapol) column (2×15 cm) using a
gradient elution from water to 20% AcOH at
a flow rate of 3 mL/min. Gel filtration was
performed on a TSK-HW40s (TOYO SODA)
column (1.5×90 cm) by elution with 0.1 M
aq AcOH at a flow rate of 0.8 mL/min, a
Sephadex G-10 column (2×100 cm) by elu-
tion with water at a flow rate 2 mL/min, and
a Sephadex LH-20 column (2×40 cm) by
elution with either MeOH or 1:1 CH3CN–wa-
ter at a flow rate of 1 mL/min. HPLC was
performed on a 7 mm Silica Gel column (1×
20 cm) at a flow rate of 4 mL/min. The
hydrogenolysis was performed over 10% PdO/
C (E. Merck) at atmospheric pressure and
35 °C. Biotin derivatives 23 and 26 and
poly(p-nitrophenyl acrylate) 25 were obtained
from Syntesome GmbH (Muenchen).
1
ton wool and concd. Column chromatography
(3:2 EtOAc–petroleum ether) of the residue
gave 7 as a white foam (423 mg, 90%): [h]D
1
+6° (c 1, CHCl3); H NMR (CDCl3): l 5.07
(dd, 1 H, J3¦,4¦, J4¦,5¦ 10.0 Hz, H-4¦), 5.02 (d, 1
H, J1¦,2¦ 8.9 Hz, H-1¦), 4.39 (d, 1 H, J1,2 9.2 Hz,
H-1), 4.33 (d, 1 H, J1%,2% 7.9 Hz, H-1%), 4.06 (dd,
1 H, J2¦,3¦, J3¦,4¦ 10.0 Hz, H-3¦), 4.00 (d, 1 H,
J3%,4% 3.2 Hz, H-4%), 3.89 (dd, 1 H, J3,4, J4,5 9.2
Hz, H-4), 3.71 (dd, 1 H, J1%,2% 9.3 Hz, J2%,3% 10.5
Hz, H-2%), 3.71 (dd, 1 H, H-5¦), 3.69 (m, 2 H,
H-6a, H-6b), 3.61 (dd, 1 H, H-3%), 3.56 (b dd,
1 H, H-2¦), 3.55 (dd, 1 H, H-3, J2,3 10.1 Hz,
H-3), 3.43 (dd, 1 H, H-2), 3.36 (m, 1 H, H-5);
13C NMR (CDCl3): l 62.41 (CH2OH), 57.02
(C-2¦), 23.08 (CH3CONH), 20.88 (CH3COO);
MALDI-TOF-MS: Calcd for [M+Na]+:
1374.6. Found 1375.2. Anal. Calcd for
C80H89NO18: C, 71.04; H, 6.63. Found: C,
71.25; H, 6.81.
2-Azidoethyl (2-acetamido-3,6-di-O-benzyl-
2-deoxy-i-
tri-O-benzyl-i-
2,3,6-tri-O-benzyl-i-
D
-glucopyranosyl)-(1“3)-(2,4,6-
D
-galactopyranosyl)-(1“4)-
-glucopyranoside (8).—
D
To a solution of 7 (737 mg, 0.55 mmol) and
Et3N (0.4 mL, 5.50 mmol) in anhyd CH2Cl2
(10 mL) was added dropwise methanesulfonyl
chloride (MsCl) (0.09 mL, 1.12 mmol) at
−10 °C. The reaction mixture was diluted with
CH2Cl2 and washed with satd aq NaHCO3 (20
mL) and brine (20 mL), filtered through cot-
ton wool, concd and filtered through a pad of
Silica Gel (EtOAc–petroleum ether, 1:1). To
the solution of the residue (744 mg) in anhyd
DMF (5 mL), 18-crown-6 (150 mg, 0.56
mmol) and NaN3 (432 mg, 6.60 mmol) were
added and the reaction mixture was stirred for
50 h at rt. EA (50 mL) was added and the
solution was washed with water (2×30 mL),
filtered through cotton wool, concd and
filtered through a pad of Silica Gel (EtOAc–
petroleum ether, 1:1). To the solution of the
crude mesylate thus obtained (720 mg) in an-
hyd MeOH (10 mL) MeONa (80 mg, 1.48
mmol) was added and the mixture was kept
for 2 h at rt, neutralized with KU-2 (H+)
cation-exchange resin, filtered and concd.
Column chromatography (2:3 EtOAc–
petroleum ether) gave amorphous 8 (545 mg,
79%): [h]D −6° (c 3, CHCl3); 1H NMR
(CDCl3): l 4.87 (d, 1 H, J1¦,2¦ 8.1 Hz, H-1¦),
4.45 (d, 1 H, J1%,2% 7.9 Hz, H-1%), 4.41 (d, 1 H,
2-Hydroxyethyl (2-acetamido-4-O-acetyl-
3,6-di-O-benzyl-2-deoxy-i-
(1“3)-(2,4,6-tri-O-benzyl-i-
osyl)-(1“4)-2,3,6-tri-O-benzyl-i-
D
-glucopyranosyl)-
-galactopyran-
-gluco-
D
D
pyranoside (7).—Ozone was bubbled through
a stirred solution of 6 [1] (476 mg, 0.35 mmol)
in 2:1 anhyd MeOH–CH2Cl2 (30 mL) at
−78 °C until a blue color persisted, the excess
of ozone was removed with a stream of Ar for
15 min at −78 °C, and NaBH4 (200 mg, 13.3
mmol) was added. The solution was allowed
to attain −5 °C over a period of 1.5 h, and
the next portion of NaBH4 (133 mg, 3.5 mmol)
was added. The mixture was stirred for 1 h at
5–10 °C, neutralized with AcOH (1 mL), and
concd to dryness. The solution of the residue
in CH2Cl2 was washed with 1 M H2SO4 (15
mL) and brine (20 mL), filtered through cot-

A.V. Kornilo6 et al. / Carbohydrate Research 329 (2000) 717–730
723
for 1 h and the reaction mixture was coevapo-
rated with toluene (5 mL). Chromatography
on a short column of Silica Gel (1:1 benzene–
EtOAc) gave the crude tosylate (65 mg, 52%)
and the corresponding 2-hydroxyethyl gly-
coside (21 mg, 18%), which was recovered and
recycled. The tosylate thus obtained (86 mg)
was stirred with NaN3 (32 mg, 0.5 mmol) and
18-crown-6 (20 mg) in DMF (2 mL) for 72 h
at rt, then diluted with EtOAc (15 mL),
washed with water (10 mL), concd and co-
evaporated with toluene (10 mL). Column
chromatography of the residue (1:1 benzene–
EtOAc) gave the syrup 11 (74 mg, 64%).
J1,2 7.5 Hz, H-1), 4.03 (m, 1 H, OCH2CH2N3),
3.99 (dd, 1 H, J3,4, J4,5 9.5 Hz, H-4), 3.97 (b d,
1 H, J3%,4% 3.5 Hz, H-4%), 3.74 (b dd, 1 H, J2¦,3¦
10.5 Hz, H-2¦), 3.73 (dd, 1 H, J1%,2% 8.9 Hz, J2%,3%
10.0 Hz, H-2%), 3.68-3.79 (m, 2 H, H-6a, H-
6b), 3.71 (m, 1 H, OCH2CH2N3), 3.65 (dd, 1
H, H-3%), 3.58 (dd, 1 H, J2,3 9.5 Hz, H-3),
3.55–3.41 (m, 2 H, OCH2CH2N3), 3.46 (dd, 1
H, H-2), 3.35 (m, 1 H, H-5); 13C NMR
(CDCl3): l 55.69 (C-2¦), 50.90 (CH2N3), 23.07
(CH3CONH); MALDI-TOF-MS: Calcd for
[C78H86N4O16+Na]+: 1357.6. Found 1358.6.
2-Azidoethyl (methyl 2,3,4-tri-O-pi6aloyl-i-
D
- glucopyranosyluronate) - (1“3) - (4,6 - di - O-
acetyl - 2 - O - benzoyl - i - - galactopyranosyl)-
(1“4)-(2-acetamido-3,6-di-O-benzyl-2-deoxy-
i - - glucopyranosyl) - (1“3) - (2,4,6 - tri - O-
benzyl - i - - galactopyranosyl) - (1“4) - 2,3,6-
tri-O-benzyl-i- -glucopyranoside (11)
D
Method B. A solution of 9 [1] (130 mg, 0.10
mmol) and 8 (135 mg, 0.14 mmol) in anhyd
CH2Cl2 (3 mL) was stirred over powdered
molecular sieves (800 mg) under dry Ar at rt
for 1 h and was then cooled to −30 °C.
Trimethylsilyl triflate (8 mL, 44 mmol) was
added and the reaction mixture was kept at
−30 °C for 80 h. Triethylamine (0.1 mL) was
added, the reaction mixture was diluted with
CH2Cl2 (20 mL), filtered through a pad of
Celite, washed with satd aq NaHCO3 (15 mL)
and water, filtered through a cotton wool and
concd. HPLC (1:20 acetone–toluene) afforded
11 (132 mg, 62%) as a white foam: [h]D −11°
D
D
D
Method A. A solution of 10 [1] (116 mg,
0.05 mmol) and anhyd LiI (50 mg, 0.37 mmol)
in dry Py (4 mL) was refluxed for 2 h under
dry Ar and concd. A solution of the residue in
CH2Cl2 (20 mL) was washed with 1 M H2SO4
(10 mL), satd aq NaHCO3 (10 mL), and satd
aq NaCl (10 mL), filtered through cotton
wool and concd. The crude salt thus obtained
was dissolved in a 2:1 mixture of anhyd
MeOH–CH2Cl2 (7.5 mL) and ozone was bub-
bled through the solution at −78 °C until a
blue color persisted. The excess of ozone was
removed with a stream of Ar for 15 min at
−78 °C, NaBH4 (76 mg, 2.0 mmol) was
added, and the mixture was allowed to attain
−5 °C over a 1 h period. Another portion of
NaBH4 (38 mg, 1.0 mmol) was added, the
mixture was kept for 1 h at −5 °C, neutral-
ized with AcOH, and concd to dryness. A
solution of the residue in CH2Cl2 (10 mL) was
filtered, diluted with CH2Cl2 (10 mL), washed
with 1 M H2SO4 (15 mL), satd aq NaCl (20
mL), filtered through cotton wool and concd.
The residue was dissolved in 1:5 anhyd
MeOH–CH2Cl2 (6 mL) and a solution of
CH2N2 in Et2O was added at 0 °C until a
yellow color persisted. The mixture was neu-
tralized with AcOH (1 mL), concd and dried
in vacuo. The monohydroxy derivative thus
obtained was dissolved in dry Py, TosCl (95
mg, 0.5 mmol) was added, the mixture was
kept for 20 h at rt, MeOH (3 mL) was added
1
(c 1, CHCl3); H NMR (CDCl3): l 5.45 (d, 1
H, J3§,4§ 3.5 Hz, H-4§), 5.35 (dd, 1 H, J1§,2§
8.2 Hz, J2§,3§ 10.3 Hz, H-2§), 5.26 (dd, 1 H,
J3¨,4¨, J4¨,5¨ 9.7 Hz, H-4¨), 5.16 (dd, 1 H,
J2¨,3¨ 9.7 Hz, H-3¨), 4.92 (dd, 1 H, J1¨,2¨ 7.3
Hz, H-2¨), 4.70 (d, 1 H, H-1¨), 4.54 (d, 1 H,
J1§,2§ 8.2 Hz, H-1§), 4.51 (d, 1 H, J1¦,2¦ 7.8 Hz,
H-1¦), 4.38 (d, 1 H, J1,2 7.0 Hz, H-1), 4.36 (d,
1 H, J1%,2% 8.6 Hz, H-1%), 4.12 (dd, 1 H, J2§,3§
7.9 Hz, H-3§), 4.00 (d, 1 H, H-5¨), 3.89 (dd,
1 H, J3,4, J4,5 9.6 Hz, H-4), 3.80 (s, 3 H,
COOCH3), 3.79 (d, 1 H, J3%,4% 2.8 Hz, H-4%),
3.62 (dd, 1 H, J2%,3% 9.6 Hz, H-2%), 3.54 (dd, 1
H, J2,3 9.6 Hz, H-3), 3.42 (dd, 1 H, H-2), 3.11
(dd, 1 H, H-3%); 13C NMR (CDCl3): l 61.44
(C-6§), 53.65 (C-2¦), 52.74 (COOCH3), 50.93
(CH2N3); MALDI-TOF-MS: Calcd for [M+
Na]+: 2149.9. Found: 2151.6. Anal. Calcd for
C117H138N4O33: C, 66.03; H, 6.54; N, 2.63.
Found: C, 66.09; H, 6.56; N, 2.61.
2-Azidoethyl
(i-
D
-glucopyranosyluronic
acid)-(1“3)-(i-
D
-galactopyranosyl)-(1“4)-
(2-acetamido-3,6-di-O-benzyl-2-deoxy-i- -
D

724
A.V. Kornilo6 et al. / Carbohydrate Research 329 (2000) 717–730
Hz, H-1¦), 4.81 (dd, 1 H, J1¨,2¨ 9.2 Hz, H-2¨),
4.67 (d, 1 H, H-1¨), 4.48 (d, 1 H, J1§,2§ 8.6
Hz, H-1§), 4.43 (d, 1 H, J1%,2% 7.6 Hz, H-1%),
4.37 (d, 1 H, J1,2 8.2 Hz, H-1), 4.03 (m, 1 H,
OCH2CH2N3), 4.01 (1 H, H-4¦), 3.97 (d, 1 H,
H-5¨), 3.95 (d, 1 H, J3%,4% 2.8 Hz, H-4%), 3.94
(dd, 1 H, J3,4=J4,5 8.6 Hz, H-4), 3.91 (1 H,
H-2¦), 3.89 (m, 1 H, OCH2CH2N3), 3.80 (s, 3
H, COOCH3), 3.77 (1 H, H-3§), 3.77 (2 H,
H-6a¦, H-6b¦), 3.72 (1 H, H-6b), 3.71 (1 H,
H-2%), 3.70 (1 H, H-3¨), 3.70 (1 H, H-3¦), 3.63
(m, 2 H, OCH2CH2N3), 3.62 (1 H, H-5§), 3.62
(1 H, H-6a), 3.60 (dd, 1 H, J2%,3% 9.4 Hz, H-3%),
3.59 (1 H, H-5¦), 3.53 (dd, 1 H, J2,3 8.6 Hz,
H-3), 3.41 (dd, 1 H, H-2), 3.30 (b dd, 1 H,
H-5); 13C NMR (CDCl3): l 61.53 (C-6§),
54.27 (C-2¦), 52.82 (COOCH3), 50.90
(CH2N3). MALDI-TOF-MS: Calcd for [M+
Na]+: 1919.8. Found: 1920.0. Anal. Calcd for
C101H116N4O32: C, 63.91; H, 6.16; N, 2.95.
Found: C, 64.27; H, 6.74; N, 2.50.
glucopyranosyl)-(1“3)-(2,4,6-tri-O-benzyl-i-
-galactopyranosyl)-(1“4)-2,3,6-tri-O-benzyl-
i- -glucopyranoside (12).—Lithium hydrox-
D
D
ide (1 M aq, 0.34 mL) was added to a solution
of 11 (101 mg, 47 mmol) in 8:1 THF–water (9
mL) at −10 °C, the mixture was allowed to
attain slowly rt, and was kept overnight. Ace-
tic acid (0.02 mL) was added, and the reaction
mixture was concd. The residue was coevapo-
rated with toluene (3×10 mL), dissolved in
MeOH (3 mL), MeONa (40 mg, 0.74 mmol)
was added, and the mixture was kept for 100
h at rt, neutralized with AcOH (0.1 mL) and
concd. Toluene (20 mL) was added, the solids
were filtered off through a pad of Celite and
the pad was washed with toluene (20 mL).
The filtrate was concd and the resulting
sodium salt of 12 was converted into the free
acid by treatment with 90% aq TFA (0.3 mL)
in CH2Cl2 (7 mL) for 1 h at rt. Coevaporation
with toluene (3×20 mL) and MeOH (3×15
mL) followed by gel filtration (Sephadex, LH-
20) in MeOH gave 12 (79 mg, 99%) as a white
solid: [h]D −5° (c 1, MeOH); 13C NMR
(CDCl3 +two drops CD3OD): l 104.0, 103.3,
102.2, 102.0 and 102.2 (five anomeric car-
bons), 50.8 (CH2N3), 22.4 (NHCOCH3). Anal.
Calcd for C90H104N4O27·3 H2O: C, 62.56; H,
6.42; N, 3.24. Found: C, 62.24; H, 6.08; N,
2.97.
2-Azidoethyl (methyl 2,4-di-O-acetyl-3-O-
sulfo - i -
(2,4,6 - tri - O - acetyl - i -
(1“4)-(2-acetamido-3,6-di-O-benzyl-2-deoxy-
i - - glucopyranosyl) - (1“3) - (2,4,6 - tri - O-
benzyl - i - - galactopyranosyl) - (1“4) - 2,3,6-
tri-O-benzyl-i- -glucopyranoside, sodium salt
D
- glucopyranosyluronate) - (1“3)-
D
- galactopyranosyl)-
D
D
D
(15).—A solution of 14 (37 mg, 0.02 mmol) in
DMF (0.5 mL) was treated with SO3·Py (17
mg, 0.11 mmol) for 1 h at rt, then quenched
with NaHCO3 (80 mg), and stirred for 1 h.
The solid was filtered and washed with MeOH
(10 mL). The filtrate was treated with KU-2
(Na+) cation-exchange resin for 20 min, the
resin was filtered off, and the filtrate was
concd. Gel chromatography on the Sephadex
LH-20 column in MeOH afforded amorphous
15 (36 mg, 95%): [h]D −17° (c 1.5, MeOH);
1H NMR (CD3OD): l 5.33 (d, 1 H, J3§,4§ 3.4
Hz, H-4§), 5.06 (dd, 1 H, J3¨,4¨ 9.3 Hz, J4¨,5¨
10.1 Hz, H-4¨), 5.06 (dd, 1 H, J1§,2§ 8.4 Hz,
2-Azidoethyl (methyl 2,4-di-O-acetyl-i-
glucopyranosyluronate) - (1“3) - (2,4,6 - tri - O-
acetyl-i- -galactopyranosyl)-(1“4)-(2-ace-
tamido-3,6-di-O-benzyl-2-deoxy-i- -gluco-
pyranosyl)-(1“3)-(2,4,6-tri-O-benzyl-i-
galactopyranosyl)-(1“4)-2,3,6-tri-O-benzyl-
i- -glucopyranoside (14).—A solution of 12
D-
D
D
D-
D
(63 mg, 38 mmol) in Ac2O (6 mL) was kept at
70 °C for 2 h, then cooled to rt, and Py (2 mL)
together with a catalytic amount of DMAP
were added. After 48 h the reaction mixture
was coevaporated with toluene (2×5 mL) at
30 °C. MeOH (10 mL) was added and the
mixture was kept overnight at rt, quenched
with AcOH (1 mL) and concd at rt. Column
chromatography (1:4 acetone–toluene) gave
14 (25 mg, 35%) as a white solid: [h]D −7° (c
J2§,3§ 9.5 Hz, H-2§), 4.85 (dd, 1 H, J1¨,2¨
,
J2¨,3¨ 9.3 Hz, H-2¨), 4.79 (d, 1 H, H-1¦), 4.76
(d, 1 H, J1¨,2¨ 9.3 Hz, H-1¨), 4.62 (dd, 1 H,
H-3¨), 4.56 (d, 1 H, J1§,2§ 8.8 Hz, H-1§), 4.38
(d, 1 H, H-1%), 4.37 (d, 1 H, J1,2 8.7 Hz, H-1),
4.21 (d, 1 H, H-5¨), 3.74 (s, 3 H, COOCH3).
13C NMR (CD3OD): l 63.02 (C-6§), 56.87
(C-2¦), 53.23 (COOCH3), 52.17 (CH2N3);
1
0.6, CHCl3); H NMR (CDCl3): l 5.35 (d, 1
H, J3§,4§ 3.1 Hz, H-4§), 5.14 (dd, 1 H, J3¨,4¨
,
J4¨,5¨ 9.4 Hz, H-4¨), 5.13 (dd, 1 H, J1§,2§ 8.4
Hz, J2§,3§ 9.5 Hz, H-2§), 4.85 (d, 1 H, J1¦,2¦ 7.1

A.V. Kornilo6 et al. / Carbohydrate Research 329 (2000) 717–730
725
(CH2OH), 62.01 (C-6), 52.76 (COOCH3);
MALDI-TOF-MS: Calcd for [M+Na]+:
877.4. Found 876.9, 878.0. Anal. Calcd for
C41H58O19: C, 57.60; H, 6.84. Found: C, 57.68;
H, 7.06.
MALDI-TOF-MS: Calcd for [C101H115N4-
NaO35S+Na]+: 2021.7. Found: 2023.6. Calcd
for [M+K]+: 2038.6. Found: 2039.0.
2-Hydroxyethyl (methyl 2,3,4-tri-O-pi6al-
oyl-i-
D
-glucopyranosyluronate)-(1“3)-4,6-
-galactopyranoside
2-Azidoethyl (methyl 2,3,4-tri-O-pi6aloyl-i-
di-O-acetyl-2-O-benzoyl-i-
D
D
- glucopyranosyluronate) - (1“3) - 4,6 - di - O-
(17).—A solution of 16 [1] (851 mg, 1.0
mmol) and anhyd LiI (1.0 g, 5.82 mmol) in 20
mL of dry Py was refluxed for 5 h under dry
Ar, and then concd. A solution of the residue
in CH2Cl2 (30 mL) was washed with 1 M
H2SO4 (15 mL), satd aq NaHCO3 (15 mL),
and satd aq NaCl (15 mL), filtered through
cotton wool and concd. The salt thus obtained
(908 mg) was dissolved in 2:1 anhyd MeOH–
CH2Cl2 (30 mL) and ozone was bubbled
through this solution at −78 °C until a blue
color persisted. The excess of ozone was re-
moved with a stream of Ar for 15 min at
−78 °C, and NaBH4 (784 mg, 20.8 mmol)
was added. The mixture was then allowed to
attain −5 °C over a 1 h period. Another
portion of NaBH4 (340 mg, 8.95 mmol) was
added, the mixture was kept for 1 h at −5 °C,
neutralized with AcOH (3 mL), and concd to
dryness. A solution of the residue in CH2Cl2
(15 mL) was filtered, diluted with CH2Cl2 (50
mL), washed with 1 M H2SO4 (20 mL), satd
aq NaCl (30 mL), filtered through cotton
wool and concd. The residue was dissolved in
2:5 anhyd MeOH–CH2Cl2 (7 mL) and a solu-
tion of CH2N2 in Et2O was added at 0 °C until
the yellow color persisted. The mixture was
neutralized with AcOH (1 mL), and concd to
dryness. Column chromatography (3:2
EtOAc–petroleum ether) of the residue on
Silica Gel gave 17 (813 mg, 95%) as a white
acetyl - 2 - O - benzoyl - i -
D
- galactopyranoside
(18).—To a solution of 17 (737 mg, 0.55
mmol) and Et3N (0.4 mL, 5.50 mmol) in
anhyd CH2Cl2 (10 mL) was added dropwise
MsCl (0.09 mL, 1.12 mmol) at −10 °C over a
10 min period. The reaction mixture was di-
luted with CH2Cl2, washed with satd aq
NaHCO3 (20 mL) and satd aq NaCl (20 mL),
filtered through cotton wool, concd, and
filtered through a pad of Silica Gel (1:1
EtOAc–petroleum ether). To the solution of
the crude mesylate thus obtained (744 mg) in
anhyd DMF (5 mL), 18-crown-6 (150 mg,
0.56 mmol) and NaN3 (432 mg, 6.60 mmol)
were added and the reaction mixture was
stirred for 50 h at rt, then diluted with EtOAc
(50 mL), washed with water (2×30 mL),
filtered through cotton wool and concd.
Column chromatography (3:2 EtOAc–
petroleum ether) of the residue gave 18 (423
mg, 90%) as a white foam, [h]D −1° (c 1,
CHCl3); 1H NMR (CDCl3): l 5.49 (d, 1 H, J3,4
3.2 Hz, H-4), 5.41 (dd, 1 H, J1,2 8.0 Hz, J2,3 9.0
Hz, H-2), 5.22 (dd, 1 H, J3%,4%, J4%,5% 9.5 Hz,
H-4%), 5.10 (dd, 1 H, J2%,3% 9.5 Hz, H-3%), 4.92
(dd, 1 H, J1%,2% 7.6 Hz, H-2%), 4.70 (d, 1 H,
H-1%), 4.59 (d, 1 H, H-1), 4.26 (dd, 1 H, H-3),
4.18 (dd, 1 H, J5,6b 5.7 Hz, J6a,6b 11.5 Hz,
H-6b), 4.12 (dd, 1 H, J5,6a 6.9 Hz, H-6a), 3.93
(m, 1 H, H-5), 3.93 (d, 1 H, H-5%), 3.92 (m, 1
H, OCH2CH2N3), 3.75 (s, 3 H, COOCH3),
3.61 (m, 1 H, OCH2CH2N3), 3.35 (m, 1 H,
OCH2CH2N3), 3.28 (m, 1 H, OCH2CH2N3);
13C NMR (CDCl3): l 62.04 (C-6), 52.70
(COOCH3), 50.71 (CH2N3); MALDI-TOF-
MS: Calcd for [M+Na]+: 902.4. Found
902.2. Anal. Calcd for C41H57N3O18: C, 55.97;
H, 6.53. Found: C, 55.83; H, 6.39.
1
foam, [h]D +12° (c 1, CHCl3); H NMR
(CDCl3): l 5.49 (d, 1 H, J3,4 3.2 Hz, H-4), 5.41
(dd, 1 H, J1,2 8.1 Hz, J2,3 9.8 Hz, H-2), 5.23
(dd, 1 H, J3%,4%, J4%,5% 9.5 Hz, H-4%), 5.13 (dd, 1
H, J2%,3% 9.5 Hz, H-3%), 4.93 (dd, 1 H, J1%,2% 7.5
Hz, H-2%), 4.72 (d, 1 H, H-1%), 4.55 (d, 1 H,
H-1), 4.27 (dd, 1 H, H-3), 4.22 (dd, 1 H, J5,6b
4.8 Hz, J6a,6b 11.4 Hz, H-6b), 4.10 (dd, 1 H,
J5,6a 7.8 Hz, H-6a), 3.97 (m, 1 H, H-5), 3.96
(d, 1 H, H-5%), 3.79 (m, 2 H, OCH2CH2OH),
3.76 (s, 3 H, COOCH3), 3.63 (m, 2 H,
OCH2CH2OH), 2.15 and 2.09 (2s, 2×3 H,
2×COCH3), 1.10, 1.09 and 0.99 (3s, 3×9 H,
3×COC(CH3)3); 13C NMR (CDCl3): l 62.31
2-Azidoethyl
acid)-(1“3)-i-
(i-
D
-glucopyranosyluronic
D
-galactopyranoside (19).—To
a solution of 18 (162 mg, 0.18 mmol) in a 10:1
mixture of THF–water (7.5 mL), LiOH (1 M
aq, 0.5 mL) was added at −15 °C and the
mixture was allowed to attain rt slowly and
was kept overnight. The reaction mixture was

Table 1
1H NMR data for compounds 1–4 and 19 (l, ppm; J, Hz; D2O, 293–315 K) a
Compound
Residue
H-1
H-2
H-3
H-4
H-5
H-6a
H-6b
J1,2
J2,3
J3,4
J4,5
J5,6a
J5,6b
J6a,6b
1
Glc
Gal
GlcNAc
Gal *
GlcA
4.54
4.44
4.72
4.54
4.74
3.38
3.62
3.79
3.70
3.60
3.68
3.72
3.74–3.76
3.82
4.33
3.66
4.15
3.74–3.76
4.18
3.71
3.64
3.60
3.61
3.74
3.77
3.98
3.86
3.94
3.73–3.75
3.80
3.74
3.86
3.73–3.75
7.6
7.6
8.1
7.6
7.7
7.4
2.8
2.8
9.0
/
9.0
9.3
2
Glc
Gal
GlcNAc
Gal *
GlcA
4.52
4.41
4.68
4.51
4.65
3.36
3.56
3.79
3.67
3.39
3.63
3.71
3.72
3.79
3.62
4.14
3.73
4.17
3.61
3.62
3.58
3.72
3.70
3.79
3.70–3.75
3.94
3.96
3.70–3.75
3.82
7.7
7.9
8.3
7.7
7.8
12.1
12.4
3.2
3.1
4.7
3.70–3.75
3.70–3.75
3.49–3.51
3.49–3.51
8.2
9.8
9.0
3
Gal
GlcA
4.51
4.76
3.71
3.62
3.83
4.33
4.19
3.72
3.72
3.80
3.75
3.75
7.9
8.0
9.5
8.5
3.0
9.0
329
(
4
Gal
GlcA
4.47
4.67
3.71
3.41
3.79
3.50–3.53
4.18
3.50–3.53
3.71
3.75
3.75–3.78
3.76
3.75–3.78
3.69
7.8
7.8
9.9
2.8
1
19
Gal
GlcA
4.47
4.75
3.71
3.44
3.79
3.55
4.13
3.61
3.68
4.01
7.9
7.9
9.5
8.3
2.7
9.0
730
9.4
a Other signals: OCH2CH2NH2 m, 1 H, l 4.11–4.12 and m, 1 H, l 3.92–3.97; OCH2CH2NH2 t, 2 H, l 3.25–3.27 ppm (for 1–4). OCH2CH2N3 m, 1 H, l 3.76 and m, 1
H, l 3.95; OCH2CH2N3 t, l 3.46 ppm (for 19).
* Gal residue from the GlcA-Gal block.

A.V. Kornilo6 et al. / Carbohydrate Research 329 (2000) 717–730
727
Table 2
13C NMR data for compounds 1–4 and 19 (l, ppm; D2O, 293–315 K) a
Compound
Residue
C-1
C-2
C-3
C-4
C-5
C-6
61.1
1
Glc
Gal
GlcNAc
Gal *
GlcA
103.0
104.0
103.7
103.6
104.2
73.8
71.1
56.3
71.2
73.1
75.3
83.1
73.3
83.3
84.8
79.5
69.4
79.5
69.2
71.5
75.9
76.0
75.7
76.2
77.2
b
62.1
61.1
62.0 b
175.8
2
Glc
Gal
GlcNAc
Gal *
GlcA
102.6
103.6
103.2
103.3
104.2
73.3
70.6
55.9
70.8
73.7
74.9
82.7
72.8
82.8
75.9
79.0
68.9
79.0
68.8
72.3
75.5
60.7
61.6
60.7
61.6
175.4
75.7 c
75.3
75.9 c
76.3
3
Gal
GlcA
103.0
103.8
70.4
72.7
83.2
84.3
68.8
71.1
75.6
76.8
61.7
176.0
4
Gal
GlcA
103.0
104.2
70.4
73.8
83.1
75.9
68.7
72.3
75.6
76.6
61.7
176.0
19
Gal
GlcA
103.9
104.9
71.1
74.4
83.8
76.1
69.5
72.8
76.1
76.5
62.2
a Other signals: OCH2CH2NH2 66.4–67.0; OCH2CH2NH2 40.1–40.6 (for compounds 1–4). OCH2CH2N3 69.5; OCH2CH2N3
51.8 (for 19).
b Signals can be interchanged.
c Signals can be interchanged.
* Gal residue from the GlcA-Gal block.
concd, dissolved in MeOH (10 mL) and
treated with aq NaOH (2.5 M, 0.8 mL) at
−5 °C for 48 h. The reaction mixture was
then neutralized with KU-2 (H+) cation-ex-
change resin, the resin was filtered off, and the
filtrate was evaporated. Ion-exchange chro-
matography and subsequent freeze-drying af-
forded amorphous 19 (63 mg, 82%), [h]D
mg, 0.24 mmol) in anhyd MeOH (5 mL)
overnight at rt, then quenched with KU-2
(H+) cation-exchange resin. The resin was
then filtered off, and the filtrate was concd.
Column chromatography (1:3 acetone–tolu-
ene) of the residue on Silica Gel gave 21 (85
mg, 65%) as a white solid, [h]D −27° (c 1,
1
CHCl3); H NMR (CDCl3): l 5.37 (sd, 1 H,
1
−24° (c 1, MeOH); for H NMR data see
J3,4 3.5 Hz, H-4), 5.21 (dd, 1 H, J1,2 9.5 Hz,
J2,3 11.0 Hz, H-2), 5.09 (t, 1 H, J3%,4%=J4%,5% 10.9
Hz, H-4%), 4.70 (dd, 1 H, J1%,2% 9.2 Hz, J2%,3% 10.5
Hz, H-2%), 4.59 (d, 1 H, H-1%), 4.45 (d, 1 H,
H-1), 4.16 (dd, 1 H, J5,6b 6.8 Hz, J6a,6b 13.6
Hz, H-6b), 4.05 (dd, 1 H, J5,6a 8.5 Hz, H-6a),
4.00 (m, 1 H, OCH2CH2N3), 3.91 (d, 1 H,
H-5%), 3.89 (dd, 1 H, H-3), 3.84 (m, 1 H, H-5),
3.76 (s, 3 H, COOCH3), 3.69 (dd, 1 H, H-3%),
3.66 (m, 1 H, OCH2CH2N3), 3.48 (m, 1 H,
OCH2CH2N3), 3.29 (m, 1 H, OCH2CH2N3);
13C NMR (CDCl3): l 100.64 (C-1), 100.28
(C-1%), 75.71 (C-3), 61.96 (C-6), 52.63
(COOCH3), 50.42 (CH2N3); MALDI-TOF-
MS: Calcd for [M+Na]+: 672.2. Found
671.9. Anal. Calcd for C25H35N3O17: C, 46.23;
H, 5.43. Found: C, 46.52; H, 5.31.
Table 1, and for 13C NMR data see Table 2;
MALDI-TOF-MS: Calcd for [C14H23N3O12+
Na]+: 448.1. Found 448.2.
2-Azidoethyl (methyl 2,4-di-O-acetyl-i-
glucopyranosyluronate) - (1“3) - 2,4,6 - tri - O-
acetyl-i- -galactopyranoside (21).—A sus-
D-
D
pension of 19 (83 mg, 0.20 mmol) in Ac2O (5
mL) was kept at 80 °C for 1 h, then cooled to
rt, and Py (3 mL) and a catalytic amount of
DMAP (10 mg) were added. After 72 h the
reaction mixture was coevaporated with tolu-
ene (2×5 mL) at 30 °C, dissolved in benzene,
washed with satd aq NaCl and water, filtered
through cotton wool, concd, and dried in
vacuo. A solution of the crude lactone thus
obtained was treated with anhyd AcONa (16

728
A.V. Kornilo6 et al. / Carbohydrate Research 329 (2000) 717–730
1
mg, 67%), [h]D −8° (c 0.4, water); for H
NMR data see Table 1, and for 13C NMR data
see Table 2; MALDI-TOF-MS (negative
mode): Calcd for [C34H56Na2N2O30S−2 Na+
H]−: 1005.3. Found 1005.8.
2-Azidoethyl (methyl 2,4-di-O-acetyl-3-O-
sulfo - i - - glucopyranosyluronate) - (1“3)-
2,4,6 - tri - O - acetyl - i - - galactopyranoside,
D
D
sodium salt (22).—A solution of 21 (76 mg,
0.12 mmol) in DMF (1 mL) was treated with
SO3·Py (90 mg, 0.57 mmol) for 2 h at rt, then
quenched with NaHCO3 (80 mg) and stirred
for 1 h. The solid was filtered off, washed with
MeOH (10 mL), and the filtrate was treated
with KU-2 (Na+) cation-exchange resin for 20
min. The resin was filtered off, and the filtrate
was concd. Column chromatography (1:10
MeOH–CH2Cl2) of the residue on Silica Gel
afforded amorphous 22 (75 mg, 85%), [h]D
2-Aminoethyl
(i-
-galactopyranosyl)-(1“4)-
-glucopyranosyl)-
- galactopyranosyl) - (1“4) - i-
-glucopyranoside, sodium salt (2).—A solu-
D
-glucopyranosyluronic
acid)-(1“3)-(i-
D
(2-acetamido-2-deoxy-i-
D
(1“3) - (i -
D
D
tion of 11 (92 mg, 0.04 mmol) in MeOH (2 mL)
and 0.01 mL AcOH was hydrogenated for 2 h
over Pd–C. The catalyst was filtered through a
pad of Celite, the pad was washed thoroughly
with MeOH (20 mL), and the filtrate was concd
and dried in vacuo. The residue was dissolved
in anhyd MeOH (2 mL), and CF3COOEt (25
mL, 21 mmol) and Et3N (20 mL) were added.
The mixture was evaporated and the residue
was filtered through a short column of Silica
Gel by elution with EtOAc to give a mixture
(60 mg) of partially debenzylated 2-trifluoro-
acetamidoethyl glycosides. This mixture was
hydrogenolyzed in EtOH (1.5 mL) for 72 h
over Pd–C. The catalyst was filtered off
through a pad of Celite, the pad was washed
with EtOH (20 mL), and the filtrate was concd.
A solution of the residue in 10:1 THF–water
(11 mL) was treated with 1 M aq LiOH (0.2
mL) at −10 °C for 2 h, stirred overnight at rt,
and then concd. The residue was dissolved in
5:3 MeOH–water (8 mL) and treated with 2.5
M aq NaOH (0.4 mL) for 48 h at rt. The
solution was neutralized with AcOH (0.1 mL),
concd, followed by gel filtration (Sephadex
G-10) and subsequent freeze-drying yielded
amorphous 2 (20 mg, 49%), [h]D −5° (c 1,
1
−8° (c 1, CHCl3); H NMR (CD3OD): l 5.44
(d, 1 H, J3,4 3.3 Hz, H-4), 5.10 (dd, 1 H, J1,2 8.3
Hz, J2,3 9.9 Hz, H-2), 5.02 (dd, 1 H, J3%,4%, J4%,5%
9.7 Hz, H-4%), 4.85 (d, 1 H, J1%,2% 8.2 Hz, H-1%),
4.82 (dd, 1 H, J2%,3% 8.2 Hz, H-2%), 4.62 (d, 1 H,
H-1), 4.61 (dd, 1 H, H-3%), 4.21 (d, 1 H, H-5%),
4.16 (dd, 1 H, J5,6b 8.7 Hz, J6a,6b 14.4 Hz, H-6b),
4.12 (dd, 1 H, H-3), 4.03 (m, 2 H, H-6a, H-5),
3.98 (m, 1 H, OCH2CH2N3), 3.74 (s, 3 H,
COOCH3), 3.71 (m, 1 H, OCH2CH2N3), 3.45
(m, 1 H, OCH2CH2N3), 3.33 (m, 1 H,
OCH2CH2N3), 2.12–2.03 (5 s, 15 H, 5×
COCH3); 13C NMR (CD3OD): l 78.9 (C-3%),
78.2 (C-3), 63.4 (C-6), 53.2 (COOCH3), 51.8
(CH2N3). Anal. Calcd for C25H34N3NaO20S: C,
39.95; H, 4.56. Found: C, 40.02; H, 4.50.
2-Aminoethyl (3-O-sulfo-i-
osyluronic acid)-(1“3)-(i- -galactopyran-
osyl)-(1“4)-(2-acetamido-2-deoxy-i- -gluco-
pyranosyl) - (1“3) - (i - - galactopyranosyl)-
(1“4)-i- -glucopyranoside, disodium salt
D-glucopyran-
D
D
D
D
(1).—A solution of 15 (40 mg, 0.02 mmol) in
10:1 MeOH–water (8 mL) and 0.1 mL AcOH
was hydrogenolyzed for 19 h over Pd–C. The
catalyst was filtered using a pad of Celite, the
pad was washed with a linear gradient from
MeOH to water (50 mL), and the filtrate was
concd to a volume of 3 mL. 1 M aq LiOH (2.2
mL) was added at −10 °C, and the mixture
was allowed to attain rt over a 4 h period, then
neutralized with AcOH. The reaction mixture
was concd, diluted with water (1.5 mL) and
MeONa (12 mg, 0.20 mmol) was added. The
mixture was kept for 48 h at rt, neutralized with
AcOH (0.1 mL) and concd to a volume of 0.5
mL. Gel filtration (TSK-HW40s) and subse-
quent freeze-drying yielded amorphous 1 (14
1
water); for H NMR data see Table 1, and for
13C NMR data see Table 2; MALDI-TOF-MS:
Calcd
for
[C34H57NaN2-
O27+H]+: 949.3. Found 949.5. Calcd for
[M+Na]+: 971.3. Found 971.4. Calcd for
[M+K]+: 988.2. Found 987.2.
2-Aminoethyl (3-O-sulfo-i-
D
-glucopyranos-
yluronic acid)-(1“3)-i- -galactopyranoside,
D
disodium salt (3).—2 M aq LiOH (0.2 mL) was
added to a solution of 22 (59 mg, 0.08 mmol)
in 10:1 THF–water (5.5 mL) at −10 °C. The
mixture was allowed to attain rt over a 2 h
period, it was neutralized with AcOH, and then
it was concd. To a solution of the residue in
MeOH (1.5 mL) and water (0.5 mL) MeONa

A.V. Kornilo6 et al. / Carbohydrate Research 329 (2000) 717–730
729
J10,11 6.6 Hz, H-10a, H-10b), 3.12 (q, 12 H,
2×NH(CH2CH3)3), 3.01 (dd, 1 H, J5a,5 13.0
Hz, H-5a), 2.80 (d, 1 H, H-5b), 2.27 (m, 4 H,
H-9a, H-9b, H-14a, H-14b), 1.73 (m, 1 H,
H-6a), 1.66 (m, 2 H, H-8a, H-8b), 1.62 (m, 2
H, H-13a, H-13b), 1.60 (m, 1 H, H-6b), 1.53 (m,
2 H, H-11a, H-11b), 1.43 (m, 2 H, H-7a, H-7b),
1.34 (m, 2 H, H-12a, H-12b), 1.20 (t, 18 H,
2×NH(CH2CH3)3). Other signals were very
(35 mg, 0.65 mmol) was added. The mixture
was kept at rt for 48 h, neutralized with AcOH
and concd. A solution of the residue in water
(2 mL) was hydrogenated for 1 h over Pd–C.
The catalyst was filtered off through a pad of
Celite, the pad was washed with water (20 mL),
and the filtrate was concd to a volume of 0.5
mL. Gel-filtration (TSK HW-40s) and subse-
quent freeze-drying yielded amorphous 3 (27
mg, 67%), [h]D −7° (c 0.8, water); for 1H NMR
data see Table 1, and for 13C NMR data see
Table 2; MALDI-TOF-MS (negative mode):
Calcd for [C14H23NNa2O15S−2 Na+H]−:
478.1. Found 478.4; Calcd for [M−Na]−:
500.1. Found 500.5.
13
similar to those for 1 (see Table 1). C NMR
(D2O, see Scheme 3 for the numeration of
atoms in the aglycon): l (for aglycon) 62.9
(C-3), 61.1 (C-4), 56.2 (C-2), 48.0
(NH(CH2CH3)3), 40.5 (C-5), 39.9 (C-10), 36.4
(C-9, C-14), 28.8 (C-11), 28.6 (C-7), 28.5 (C-6),
26.4 (C-12), 26.0 (C-8), 25.8 (C-13), 9.5
(NH(CH2CH3)3). Other signals were very simi-
lar to those for 1 (see Table 2). MALDI-
TOF-MS (negative mode): Calcd for
[C62H113N7O33S2−2(Et3NH)+H]−: 1344.4.
Found 1344.8. Calcd for [M−2(Et3NH)+
Na]−: 1366.4. Found 1366.8.
2-Aminoethyl
acid)-(1“3)-i-
(i-
D
-glucopyranosyluronic
D
-galactopyranoside (4).—A
solution of 19 (48 mg, 0.11 mmol) in water (2
mL) was hydrogenated for 19 h over Pd–C. The
catalyst was filtered off through a pad of
Celite, the pad was washed with water (20 mL),
and the filtrate was concd to a volume of
0.5 mL. Gel-filtration (Sephadex G-10) and
subsequent freeze-drying yielded amorphous 4
Synthesis of biotinylated PAA-neoglycocon-
jugate (27).—To a solution A of 1 (4.0 mg, 3.7
mmol) in Me2SO (0.2 mL), solution B (178 mL,
18.4 mmol of 25), solution C (42 mL, 0.92 mmol
of 26) and Et3N (13 mL) were added. Solution
B: poly(p-nitrophenyl acrylate) 25 (14.2 mg) in
DMF (1.0 mL). Solution C: 6-(biotinyl-
amido)hexamethylenamine 26 (1.0 mg) in
Me2SO (0.1 mL). The reaction mixture was
kept at 40 °C for 24 h then 2-ethanolamine (43
mL) was added and the mixture was kept at rt
for 24 h. Gel chromatography (Sephadex LH-
20) in 1:1 CH3CN–water and subsequent con-
centration and freeze-drying gave 25 as a white
powder (6.2 mg, 90%).
1
(40 mg, 89%), [h]D −15° (c 2, water); for H
NMR data see Table 1, and for 13C NMR
data see Table 2; MALDI-TOF-MS: Calcd
for
[C14H25NO12+H]+
400.1.
Found
399.9; Calcd. for [M+Na]+: 422.1. Found
421.9.
2-(6-N-Biotinylamido)hexanamidoethyl (3-
O - sulfo - i -
(1“3)-(i- -galactopyranosyl)-(1“4)-(2-ace-
tamido-2-deoxy-i- -glucopyranosyl)-(1“3)-
(i - - galactopyranosyl) - (1“4) - i - - gluco-
D
- glucopyranosyluronic acid)-
D
D
D
D
pyranoside, bis-triethylammonium salt (23).—
To a solution of 1 (6.5 mg, 6.2 mmol) and
p-nitrophenyl(6-N-biotinylamido)capronoate
(3.6 mg, 7.5 mmol) in 0.5 mL DMSO was added
Et3N (0.01 mL) and the reaction mixture was
kept at rt for 24 h. Gel-filtration (Sephadex
LH-20) in 1:1 CH3CN–water and subsequent
concentration and freeze-drying gave 23 (9.2
mg, 94%) as a white powder, [h]D 9° (c 0.6,
Acknowledgements
This work was supported, in part, by Synte-
some GmbH, Muenchen and the Russian
Foundation for Basic Research (Project No.
00-03-32815a). We thank E.A. Gordeeva
(M.M. Shemyakin and Y.A. Ovchinnikov Insti-
tute of Bioorganic Chemistry, RAS) for the
kind assistance in the preparation of the poly-
meric conjugate 27.
1
water); H NMR (D2O, see Scheme 3 for the
numeration of atoms in the aglycon): l (for
aglycon) 4.62 (dd, 1 H, J3,4 7.7 Hz, J4,5a 5.1 Hz,
H-4), 4.43 (dd, 1 H, J2,3 4.6 Hz, H-3), 3.99 and
3.81 (2m, 2 H, OCH2CH2NH), 3.45 (m, 2 H,
OCH2CH2N), 3.35 (m, 1 H, H-2), 3.19 (t, 2 H,

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A.V. Kornilo6 et al. / Carbohydrate Research 329 (2000) 717–730
[12] A.Ya. Chernyak, G.V.M. Sharma, L.O. Kononov, P.
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