The synthesis and NMR and conformational studies of fucoidan fragments: VI. Fragments with an α-(1 → 2)-linked fucobioside unit

Article abstract of DOI:10.1023/B:RUBI.0000023099.48598.9a

A series of selectively sulfated di- and trisaccharide derivatives corresponding to the potential fragments of fucoidans with a α-(1 → 2)-linked fucobioside unit were synthesized and studied by ¹H and ¹³C NMR spectroscopy. NOE experi

Full text of DOI:10.1023/B:RUBI.0000023099.48598.9a

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 2, 2004, pp. 137–147. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 2, 2004, pp. 156–167.
Original Russian Text Copyright © 2004 by Gerbst, Grachev, Ustyuzhanina, Khatuntseva, Tsvetkov, Usov, Shashkov, Preobrazhenskaya, Ushakova, Nifantiev.
The Synthesis and NMR
and Conformational Studies of Fucoidan Fragments:
VI.1 Fragments with an a-(1 2)-Linked Fucobioside Unit
A. G. Gerbst*, A. A. Grachev*, N. E. Ustyuzhanina*, E. A. Khatuntseva*,
D. E. Tsvetkov*, A. I. Usov*, A. S. Shashkov*, M. E. Preobrazhenskaya**,
2
N. A. Ushakova**, and N. E. Nifantiev*
*Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii pr. 47, Moscow, 119991 Russia
**Orekhovich Institute of Biomedicinal Chemistry, Russian Academy of Medical Sciences,
Pogodinskaya ul. 10, Moscow, 119832 Russia
Received May 22, 2003; in final form, September 10, 2003
Abstract—A series of selectively sulfated di- and trisaccharide derivatives corresponding to the potential frag-
ments of fucoidans with a α-(1
2)-linked fucobioside unit were synthesized and studied by 1ç and 13
C
NMR spectroscopy. NOE experiments and molecular modeling were used for a conformational analysis of the
compounds synthesized. In the case of disaccharides, the experimental NOE values were found to agree with
those obtained using modeling with the use of density functional theory (DFT) and differ from those resulting
from modeling by the molecular mechanics MM3 force field. Trisaccharide fragments partially or completely
sulfated in position 4 turned out to be correctly described by both MM3 force field and DFT computation.
Key words: conformational analysis; density functional theory; fucoidans, synthesis of fragments; molecular
mechanics; NOE
21
INTRODUCTION
linked fucotrioside [1], its nonsulfated analogue, and
the constituent disaccharides [2, 4], as well as 2,3- and
3,4-branched trisaccharides [2].
The structure, biosynthesis, and biological activity
of natural polysaccharides are intensively studied due
to their important role in various cellular processes. A
3
In this work, we report the synthesis and the results
of NMR and theoretical conformational analysis for the
series of n-propyl glycosides of di- and trisaccharides
special and increasing interest in fucoidans is con-
nected with their various physiological activities
(namely, anticoagulant, antitumor, antiinflammatory,
antiangiogenic, and antiviral; cf. references cited in [2,
3]). However, the information on the fucoidan structure
is yet fragmentary, because the structural characteriza-
tion of these compounds is complicated due to their
irregularity, heterogeneity, and the absence of methods
for their directed and controlled cleavage.
with the (1
2)-linked fucobioside moiety. These are
a nonsulfated (I) [2], 4-é-sulfated (II), and 4,4'-di-é-
sulfated (III) fucobiosides; as well as nonsulfated (IV)
[2], 4-é-sulfated (V), 4,4'-di-é-sulfated (VI), and
4,4',4''-tri-é-sulfated (VII) 2,3-branched trisaccharides
composed of α-L-fucopyranose residues.
We are systematically synthesizing the known and
presumable fucoidan fragments and are studying their
spectroscopic (NMR) and conformational characteris-
tics to reveal the key structural motifs and pharmaco-
phore groups determining their biological properties.
Among the fucoidan biological activities, we primarily
investigated the antiinflammatory (the ability to inhibit
L- and P-selectins) and antiangiogenic actions, and also
the inhibition of microbial adhesion. Previously, we
RESULTS AND DISCUSSION
Synthesis of Sulfated Di- and Trisaccharide Derivatives
(II), (III), and (V)–(VII)
The target sulfated disaccharide derivatives (II) and
(III) were synthesized from the common precursor, dis-
accharide (VIII) [2]. It was sulfated to monosulfate
(IX) in 85% yield by the treatment with Py · SO3 com-
plex. The sequential removal of protective groups
resulted in (II). The presence of sulfate group at C4 of
(II) was confirmed by the downfield shift of the H4 pro-
ton signal by 0.8 ppm as compared with that of H4 in
had studied the linear 4,4',4''-tri-é-sulfated (1
3)-
1
For Part V, see [1].
Corresponding author; phone: +7 (095) 135-8784; e-mail:
nen@ioc.ac.ru
Abbreviations: All, allyl; Bn, benzyl; Bz, benzoyl; DFT, density
functional theory; and Me SiOTf, trimethylsilyl triflate.
2
3
its nonsulfated analogue (I) (δ 3.84 ppm
see Table 1).
4.64 ppm,
3
1068-1620/04/3002-0137 © 2004 MAIK “Nauka /Interperiodica”

138
GERBST et al.
OPr
O
OPr
O
Me
O
Me
O
OH
O
OH
R1O
O
R1O
Me
OH
Me
O
O
Me
OR2
OH
OH
OH
OH
R3O
R2O
(IV) R1 = R2 = R3 = H
(I) R1 = R2 = H
(II) R1 = SO3Na, R2 = H
(III) R1 = R2 = SO3Na
(V) R1 = SO3Na, R2 = R3 = H
(VI) R1 = R2 = SO3Na, R3 = H
(VII) R1 = R2 = R3 = SO3Na
OAll
OAll
OAll
Me
Me
Me
O
O
O
OAll
OH
O
O
O
OBn
OR
Me
OBn
OH
Me
OBn
OR
Me
Me
O
O
O
O
O
OBn
OBn
OBn
O
Me
OBz
OBz
OH
OH
OBz
OR
Me
(VIII) R = H
(X)
(XI) R = H
(XIII)
(IX) R = SO3Na
(XII) R = SO3Na
OAll
O
OAll
O
Me
O
Me
O
OR1
OR2
Me
OBn
O
RO
OR
OBn
OBz
Me
O
O
Me
O
O
OBn
Me
OBn
OBz
OBz
OBz
OBz
OBz
OBz
BzO
(XVI) R1 = R2 = C(CH3)2
(XVII) R1 = R2 = H
(XIV) R = H
(XV) R = C(NH)CCl3
(XVIII) R = H
(XIX) R = SO3Na
OAll
OAll
Me
O
OAll
O
Me
O
Me
O
O
O
O
O
OH
OH
Me
OBn
O
Me
O
RO
Me
OBz
Me
O
O
Me
O
OBn
OBn
Me
OR
OBn
OBz
OH
(XXI)
OH
OH
OBz
BzO
(XXII) R = H
(XXIII) R = SO3Na
(XX)
OPr
OH
OAll
OAll
Me
O
Me
Me
O
O
O
O
O
O
OBn
O
OBn
O
O
O
OH
O
OR1
OBz
OH
O
RO
OH
Me
Me
O
Me
R2O
Me
OR
OBn
OBn
Me
O
Me
OH
OR1
OBz
OH
R2O
RO
(XXIV) R1 = R2 = H
(XXV) R1 = Bz, R2 = H
OH
OH
(XXVI) R = SO3Na
(XXVII)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 2 2004

THE SYNTHESIS AND NMR AND CONFORMATIONAL STUDIES … VI.
Table 1. Chemical shifts (δ, ppm) in the 1H NMR spectra* of (I)–(VII)
139
Compound
Residue
H1
H2
H3
H4
H5
H6
(I)
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-(1
5.10
5.04
5.10
5.00
5.07
5.02
5.17
5.08
5.13
5.18
5.10
5.28
5.20
5.13
5.28
5.21
5.13
5.31
3.85
3.76
3.87
3.75
3.85
3.77
4.04
3.78
3.83
4.13
3.78
3.82
4.12
3.85
3.77
4.11
3.85
3.86
3.96
3.95
4.05
3.91
4.05
4.01
4.06
3.72
3.74
4.24
3.70
3.75
4.25
3.82
3.79
4.25
3.85
3.93
3.84
3.81
4.64
3.80
4.62
4.57
4.09
3.79
3.79
4.85
3.80
3.77
4.87
4.60
3.80
4.87
4.63
4.60
4.09
4.25
4.20
4.27
4.18
4.36
4.08
4.15
4.15
4.17
4.15
4.22
4.18
4.28
4.22
4.19
4.28
4.35
1.22
1.21
1.26
1.19
1.23
1.22
1.24
1.24
1.24
1.30
1.25
1.25
1.30
1.30
1.25
1.30
1.31
1.31
2)
2)
2)
(II)
(III)
(IV)
2)
3)
(V)
2)
3)
(VI)
(VII)
2)
3)
2)
3)
* The spectra were registered at 30°C in D O using acetone (δ 2.25 ppm) as a standard. The signals of the propyl aglycon: OCH CH CH :
2
2
2
3
0.92 ppm; OCH CH CH : 1.62–1.64 ppm; and OCH CH CH : 3.49–3.65 and 3.62–3.84 ppm.
2
2
3
2
2
3
The disulfated disaccharide (III) was obtained from subsequent α-stereospecific 3-é-monofucosylation of
(VIII) by, first, the modification of the terminal fucose the resulting diol (XVII) with trichloroacetimidate
residue: its benzoyl groups were removed by sodium (XV) led to monohydroxy trisaccharide (XVIII) in
methylate in methanol, and the resulting triol (X) was 81% yield. The configuration of the newly created gly-
regioselectively 3'-é-benzoylated via the intermediate coside linkage was confirmed by the value of the J1'', 2''
1
stannylidene derivative [4] to diol (XI) in 66% yield
(calculated for the two steps). The disulfation of (XI)
and the subsequent deprotection of resulting disaccha-
ride (XII) led to the target (III). The presence in (III) of
sulfate groups at C4 and C4' was confirmed by the
downfield shift of the H4 and H4' signals in its 1H NMR
coupling constant (3.5 Hz) in the H NMR spectrum.
The sulfation of trisaccharide (XVIII) and subsequent
deprotection of the intermediate (XIX) resulted in the
target (V). The presence of sulfate group at C4 in (V)
was confirmed by the downfield shift of H4 signal
(4.09 ppm
4.85 ppm, Table 1).
spectrum (3.84 ppm
4.62 ppm and 3.81 ppm
The synthesis of trisaccharide (VI) containing sul-
fate groups at O4 of the reducing and at O2 of the ter-
minal residues was carried out starting from disaccha-
ride (XVI). The removal of benzoyl protective groups
from disaccharide (XVI), the regioselective mono-3'-
é-benzoylation of the intermediate diol (XX) via its
stannylidene derivative, and subsequent deacetonation
by the treatment with 90% aqueous CF3COOH resulted
in disaccharide (XXI) in 77% yield. The presence of
benzoyl group at O3' in (XXI) was confirmed by the
4.57 ppm, Table 1).
Trisaccharides (V)–(VII) were synthesized as fol-
lows. Acetonide (XIII) [2] was glycosylated with
trichloroacetimidate (XV), the choice of which as the
fucosyl donor was based on our previous study [5],
where we demonstrated the co-participation of benzoyl
substituents at O3 and O4, which enhances the yield of
α-isomer.
The interaction of acetonide (XIII) and trichloro-
acetimidate (XV) in the presence of Me3SiOTf resulted
in the α-stereospecific formation of disaccharide (XVI)
in 80% yield. The α-configuration of glycoside linkage
in (XVI) was confirmed by the value of J1', 2' coupling
1
downfield shift of the H3'signal in the H NMR spec-
trum by 1.59 ppm in comparison with the H3' chemical
shift in (XVI) (3.98 ppm
5.57 ppm, see Table 2).
The 3-é-fucosylation of 3,4,4'-triol (XXI) with
trichloroacetimidate (XV) proceeded regio- and ste-
constant (3.4 Hz) in the 1H NMR spectrum.
The removal of isopropylidene protective group reospecifically like that in the case of the trisaccharide
from disaccharide (XVI) by acidic hydrolysis and the (XVIII) synthesis. The substituted 2,3-branched trisac-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 2 2004

140
GERBST et al.
Table 2. Chemical shifts (δ, ppm) and coupling constants J1, 2 (Hz) in the 1H NMR spectra* of (VIII)–(XII) and (XV)–(XXVI)
Compound
Residue
J1.2
H1
H2
H3
H4
H5
H6
(VIII)
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc
3.4
3.3
3.4
3.4
3.3
3.4
3.5
3.4
3.4
3.4
3.4
8.1
3.4
3.4
3.4
3.4
3.4
3.4
3.5
3.4
3.5
3.4
3.4
3.4
3.4
3.4
3.4
3.4
3.4
3.5
3.4
3.4
3.5
3.4
3.4
3.4
3.4
3.4
3.3
3.4
3.4
5.10
5.16
5.01
5.32
5.26
5.22
5.08
5.10
5.03
5.14
6.69
5.99
5.01
5.23
5.10
5.23
5.26
5.34
5.17
5.28
5.53
5.89
4.98
5.16
5.04
5.10
5.21
5.22
5.13
5.85
5.40
5.28
5.10
5.13
5.04
5.21
5.23
5.03
5.15
5.32
5.63
4.09
4.16
4.15
4.10
3.87
4.30
4.14
4.08
4.08
4.11
4.31
4.19
3.93
4.20
3.96
4.21
4.29
4.23
4.25
4.33
4.20
4.23
3.75
3.68
3.87
4.15
4.28
4.15
4.21
4.25
4.16
4.25
4.08
3.68
3.74
4.21
4.13
4.19
4.18
4.14
4.16
4.16
5.88
4.05
5.70
4.14
4.03
4.03
5.62
3.98
5.56
5.81
5.51
4.45
5.85
4.15
5.84
4.34
5.85
5.86
4.43
6.02
5.91
4.18
3.98
4.07
5.57
4.33
5.60
5.75
4.46
5.76
6.07
4.05
3.87
3.89
4.30
5.50
5.55
4.36
5.64
5.85
3.93
5.52
4.85
5.28
3.66
4.05
3.91
3.93
4.82
4.59
4.77
5.65
4.15
5.74
3.90
5.73
3.88
5.84
5.91
4.94
5.85
5.90
3.71
4.14
3.85
4.12
3.88
4.28
5.88
4.93
4.95
5.85
3.81
3.65
3.65
3.83
4.24
4.32
4.80
4.89
4.95
4.01
4.50
4.10
4.35
4.35
4.11
3.99
4.33
4.04
4.36
4.56
4.14
4.23
4.70
4.08
4.64
4.02
4.65
4.82
4.03
1.34
0.93
1.35
0.75
1.36
1.20
1.33
1.01
1.33
0.97
1.27
1.35
1.40
1.21
1.35
1.25
1.39
1.35
1.30
1.40
1.22
1.12
1.25
1.19
1.30
1.30
1.20–1.41
2)
2)
2)
2)
2)
(IX)
(X)
(XI)
(XII)
(XV)
(XVI)
(XVII)
(XVIII)
β-L-Fuc
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OAll
α-L-Fuc-(1
α-L-Fuc-(1
2)
2)
2)
3)
(XIX)
2)
3)
4.83
4.12
4.22
4.05
4.40
3.99
4.46
4.76
4.05
4.55
4.86
3.85
4.15
4.24
3.96
4.42
4.62
3.85
4.85
4.62
(XX)
2)
2)
(XXI)
(XXII)
2)
3)
(XXIII)
(XXIV)
(XXV)
1.00–1.40
1.15–1.30
2)
3)
2)
3)
1.35
1.37
1.30
1.25
1.35
1.30
2)
3)
(XXVI)
2)
3)
* The spectra were registered at 30°C in CDCl for (VIII), (X), (XI), and (XIII)–(XVIII) and in CD OD for (IX), (XII), (XIX), (XXIII), and
3
3
(XXVI). Other signals: OCH CH=CH : 5.13–5.41 ppm; OCH CH=CH : 5.78–6.05 and 5.70–5.87 ppm; OCH CH=CH : 4.22–4.45 and
2
2
2
2
6 5
2
2
4.00–4.15 ppm; PhCH , 4.50–4.80 ppm; C H CH : 6.90–8.20 ppm; C H CO: 7.45–7.93 ppm.
2
6
5
2
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 2 2004

THE SYNTHESIS AND NMR AND CONFORMATIONAL STUDIES … VI.
141
2.54 Å
2.17 Å HO
HO
H
PrO
O
H
PrO
O
OH
OR
H
H
OH
OR
O
O
Me
2.73 Å O
H
2.34 ÅO
Me
H
OH
OR
OH
OR
Me
Me
(‡)
(b)
Fig. 1. The main conformer of compounds (I)–(III) according to (a) computations by molecular mechanics and (b) computations
using the DFT method.
charide (XXII) was obtained in 80% yield. The config- map, which takes a lot of time in the case of quantum
uration of the created glycoside linkage was confirmed mechanical computations, was impractical. In addition
by the value of J1'', 2'' (3.4 Hz) in the 1H NMR spectrum.
to the theoretical computations, conformations of oli-
gosaccharides (I)–(VII) were also studied using the
NOE experiments. A comparison of the experimental
NOE values and those calculated on the basis of the dis-
tribution of conformers theoretically predicted was
used to control the correctness of computations. The
NOE values were calculated as we described earlier [1].
The bissulfation of diol (XXII) (80% yield) and the
removal of protective groups in (XXIII) resulted in the
target trisaccharide (VI). The presence of sulfate
groups at C4 and C4' in trisaccharide (VI) was con-
firmed by the downfield shift of H4 and H4' signals in
the 1H NMR spectrum (4.09 ppm
and 3.79 ppm
4.87 ppm for H4
Unlike the oligofucosides studied previously [1, 4],
4.60 ppm for H4', Table 2).
the (1
2)-linked disaccharides (I)–(III) the quan-
The trisulfated trisaccharide (VII) was obtained
from trisaccharide (XVIII) by the following series of
reactions. It was debenzoylated, the resulting pentaol
(XXIV) was transformed into bisstannylidene deriva-
tive, and the subsequent treatment with 2.2 equiv of
benzoyl chloride gave 3',3''-di-é-benzoylated triol
(XXV) in 82% yield. The presence of benzoyl groups at
O3' and O3'' of trisaccharide (XXV) was proved by the
tum mechanical calculations using the MM3 force field
led to the results that markedly differed from the exper-
imental data (see Table 3). For example, we get too high
NOE values for H1'. This indicated that the molecular
mechanics erroneously predicts the predominance of
conformer with H1 and H1' drawn together (Fig. 1).
Disaccharides (I)–(III) contain 1,2-pseudobranch-
ing due to the vicinal arrangement of propyl aglycon
1
downfield shift of the H3' and H3'' signals in the H
and the (1
2)-linked fucose residue. Note that
NMR spectrum as compared with those for H3' and H3''
molecular mechanics correctly describes the conforma-
tional distribution around the glycoside bond bearing
the propyl aglycon. The computations demonstrated
that the éëç2 group of aglycon is preferentially in the
conformation shown in Fig. 2. A significant NOE
between H1 and one of the protons of the methylene
group (ç‡) and a markedly lower NOE between H1
and the second methylene proton (çb) should be
observed in this conformation. A NOE between çb and
H5 of the pyranose ring should also be observed in this
case, but it should be several times lower. All these
effects were experimentally registered, and, in the case
of disaccharides (I)–(III), the NOE ratios between
in trisaccharide (XXIV) (3.87 ppm
5.50 ppm for
H3 and 3.89 ppm
5.55 ppm for H3'', Table 2). The
trisulfation of triol (XXV) (89% yield) and the removal
of protective groups afforded the target trisaccharide
(VII). The presence of sulfate groups at C4 in all the
fucose residues of trisaccharide (VII) was confirmed by
the downfield shifts of the H4, H4', and H4'' signals in
1
the H NMR spectrum (4.09
4.87 ppm for H4,
3.79
H4'', Table 2).
4.63 ppm for H4', and 3.79 4.60 ppm for
Conformational Analysis of (I)–(VII)
The method of computations using the MM3 force
field of molecular mechanics was similar to that used in
the case of fucoidan fragments investigated previously
[1, 4]. The quantum mechanical computations were
carried out using the Priroda software [6] with a step of
10° for each coordinate as in the case of computations
using the MM3 force field, but the ranges for ϕ and ψ
values were 0° to 70° and –70° to +80°, respectively.
These ranges were chosen for scanning, because the
computations by molecular mechanics showed that the
portion of conformers lying beyond it is negligibly
small. Therefore, the scan of the whole conformational
Et
Ha
Hb
O
H
H
O
O R
Me
OH
OH
Fig. 2. The dominating conformation of the propyl aglycon
in (1 2)-linked disaccharides.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 2 2004

142
GERBST et al.
Table 3. The values of NOE for (I)–(VII) and 3,4-branched trifucoside (XXVII) determined experimentally and computed
(in parentheses) from the conformation distribution using the MM3 force field
Compound
Bond
ç2'
ç1
ç2
100
H3
H4
H6
(I)
120(108)
114(108)
145(108)
146(150)
100(100)
164(150)
138(145)
150(150)
150(140)
151(150)
123(140)
(150)
89.5(140)
88(135)
(II)
(III)
(IV)
100
65(140)
100
(1
(1
(1
(1
(1
(1
(1
(1
(1
(1
(1
(1
(1
(1
(1
(1
2)
3)
2)
3)
2)
3)
2)
3)
2)
3)
2)
3)
2)
3)
3)
4)
109(100)
100(100)
172(45 + 110)
(V)
133(120)
100(115)
123(120)
(120)
100(100)
100(100)
100(100)
(100)
100(100)
100(100)
100(100)
(100)
143(155)
125(135)
130(150)
(320)
(VI)
(VII)a
(VII)b
(VII)c
(VII)d
(XXVII)
(105)
(145)
(120)
(100)
(105)
(100)
(350)
(150)
(128)
(100)
(105)
(100)
(350)
80(95)
115(102)
70(105)
20(0)
100(100)
100(100)
40(55)
a
b
c
d
Computed data for dianion with a nondissociated sulfate group in the bisglycosylated unit.
Trianion with all sulfate groups dissociated.
Dianion with one nondissociated sulfate group in the fucose residue at O2.
Dianion with one nondissociated sulfate group in the fucose residue at O3.
Ha/H1 and Hb/H1 and between Hb/H1 and Hb/H5
The computation of trisaccharide (XXVII) confor-
were found to be 2 : 1 and 1 : (2 to 1) : 4, respectively. mations by the MM3 method led to the results that sat-
isfactorily agree with the experimental data (Table 3).
These results allowed us to presume that the dis-
One can see from Table 3 that the presence of fucose
agreement between the experimental and the computed
residue at O4 results in an increase in the relative NOE
NOE values may be due to the incorrect description of
at H3. By analogy, this should cause an increase in the
the conformer distribution around the (1
2)-linked
NOE at H2 in the case of the (1
actually observed in the experiment, but was not
reflected in the computations. We think that molecular
2) linkage; this was
by molecular mechanics. We further investigated this
problem by calculating also for the NOE values for
3,4-branched trifucoside (XXVII) [2]. Like disaccha-
rides (I)–(III), this compound is characterized by the
vicinal axial–equatorial disubstitution, and the stere-
mechanics may incorrectly describe the (1
2)-gly-
coside linkage in the presence of propyl aglycon, which
causes lesser steric hindrances than the fucose residue.
Therefore, for the conformational analysis of (I)–(III),
we used the DFT method [7], which has a higher theo-
retical rank.
ochemical situation for the (1
side unit is similar to that for the (1
in disaccharides (I)–(III).
3)-linked fucobio-
2)-linked unit
The NOE values computed using the DFT method
agreed with the experimental values (Table 4), which
means that the DFT method correctly describes the
Table 4. The values of NOE for (1
2)-fucobiosides
(I)–(III) determined experimentally and computed (in parenthe-
ses) from the conformation distribution using the DFT method
conformation of the (1
the NOE values demonstrated that the introduction of
sulfate group in position O4 of the (1 3)-linked
2) linkage. An analysis of
Compound
H2'
H1
H2
(I)
(II)
(III)
120 (105)
114 (105)
145 (128)
89.5 (75)
88 (69)
65 (60)
100
100
100
difucosides does not cause such severe conformational
changes, as it does in the case of the previously studied
(1
2)-linked difucosides [4]. Obviously, this is due
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 2 2004

THE SYNTHESIS AND NMR AND CONFORMATIONAL STUDIES … VI.
ψ, deg
143
180
(a)
(b)
90
0
–90
–180
–180
–90
0
90
180 –180
ϕ, deg
–90
0
90
180
Fig. 3. Conformational maps for (a) disaccharide Fuc-α-(1 3)-Fuc-α-OPr [4] and (b) the (1
branched trisaccharide (IV) obtained using the MM3 force field.
3)-α-linked fragment of 2,3-
to the spatial remoteness of the sulfate group at O4 by the {H4–H1'} NOE values, which exceeded the
from the (1
2) linkage.
NOE value at H3 by a factor of 1.3–1.5.
Previously [4], we studied a series of (1
3)-
Interestingly, an increase in the experimental and
the computed NOE values at H2' in comparison with
the interunit NOE is observed when passing from non-
sulfated (1
fated derivative (III). Such a change was not observed
in the analysis of the corresponding (1 3)-disaccha-
rides [4]. We believe that this could indicate an increase
in the valent angle at the glycoside oxygen atom in
(III). Such a changed angle is really observed in com-
putations; its value is approximately 2°.
linked fucobiosides and observed a marked change in
conformation due to 4-é-sulfation. However, a com-
parison of data for nonsulfated trisaccharide (IV) with
those for its sulfated derivatives (V)–(VII) did not
reveal any significant conformational differences. This
indicates that, in the case of 2,3-branched trisaccha-
2)-fucobioside (I) to its 4,4'-di-é-sul-
rides under study, the (1
2)-fucobioside fragment is
the main structural element determining the general
conformational state, whereas the influence of 4-é-sul-
fation is less significant.
The (1
2)-linked fucobioside unit can occur in
The computation of conformations for trisulfated
trisaccharide (VII) led to the situation we previously
observed during the study of 4,4',4''-trisulfated linear
fucoidans not only as a part of linear chains, but also as
a 2,3-branched fragment [8, 9]. To investigate the pos-
sible effect of the fucose substituent at O3 on the (1
2)-unit conformation, we studied the branched trisac-
charides (IV)–(VII). In this case, the results of confor-
mational computations using MM3 and DFT methods
agreed with the experimental NOE values (Table 3). An
insignificant change in the conformation of the (1
2)-fucobioside fragment was observed when passing
(1
3)-fucotrioside [1]: the agreement of theoretical
and experimental NOE values was reached only when
using the structure containing one nondissociated sul-
fate group of three present sulfate moieties. In this case
(Table 3), the best agreement was obtained for the form
containing the uncharged sulfate group in the bisglyco-
sylated unit.
from (1
2)-linked disaccharides (I)–(III) to the cor-
However, one cannot argue that this result unambig-
uously proves that the solution of (VII) actually con-
tains a partially ionized form, which should be labile in
aqueous medium because of autolysis. There is no
information other than the results of NOE computa-
responding trisaccharides (IV)–(VII). In trisaccharides,
H1 and H1' were in a closer proximity, which was con-
firmed by some increase in NOE at H1 of the glycosy-
lated residue.
The conformation of the (1
3)-fucobioside frag- tions that points out to such feasibility. In the main con-
ment in trisaccharides (IV)–(VII) is determined by the former of (VII), sulfate groups are not spatially drawn
presence of neighboring glycoside residue at O2, which together (Fig. 4), and, initially, it is not obvious that the
restricts conformational mobility, especially around the ionization of two of them should impede ionization of
C3–O3 bond (rotational angle ψ). This is obvious from the third group. The spatial hindrances for the counteri-
the analysis of conformational maps given in Fig. 3. ons in the triionized structure are also hardly expected.
The conformational changes mentioned above results The results of studies of trisaccharide (VII) and its lin-
in the approach of H1' and H4. This is also confirmed ear isomer [1] show that the conformational analysis of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 2 2004

144
GERBST et al.
Analysis of 13C NMR Spectra of (I)–(VII)
8.94 Å
Me
OPr
O
O
We also studied the special features of the NMR
spectra of oligosaccharides (I)–(VII) and their relation
to their conformational characteristics. The 1H and 13C
NMR spectral data for (I)–(VII) are given in Tables 1
and 5, whereas in Table 6, the values of sulfation effect
in 13C NMR spectra of (II), (III), and (V)–(VII) are
presented; they were calculated according to the equa-
tion: ∆δCi = δCi (Ä) – δCi (B), where A and B are the
sulfated and nonsulfated derivative, respectively.
OSO3
HO
O
O
8.62 Å
O
OH
Me
OSO3
HO
Me
The sulfation effects for oligosaccharides corre-
sponding to fucoidan fragments observed in NMR
spectra were studied previously [1, 4] within the frames
of the Grant and Cheney conception [10]. We also used
a similar approach for (I)–(VII).
OSO3
OH
8.95 Å
Fig. 4. The main conformer of branched trisaccharide (VII)
and the distances between sulfate groups.
The presence of sulfo group at O4 of the glycosy-
lated residue in difucosides (II) and (III), which is spa-
tially distant from the (1
2)-linkage, has a low
oligoionic oligosaccharide derivatives are associated
with possible mistakes even when using the methods of
such high level as DFT, because of the compulsory sim-
plification of these complex compounds at the preset of
computation model. Revealing the reasons for the inad-
equate description of conformations of multiply
charged carbohydrate molecules, trisaccharide (VII)
being among them, using the methods of molecular
mechanics requires further investigation.
effect on the conformation of this bond. Therefore, the
sulfation effects in both glycosylated and glycosylating
residue are determined only by the electron-acceptor
properties of sulfate group. The effect of difference in
the interproton distances because of different confor-
mations (the Grant–Cheney conception) is absent in
this case. This manifests itself in that the sulfation
effects for the glycosylated residues in (II) and (III) are
comparable with the sulfation effect for the glycosylat-
ing residue in (III) (Table 6).
Table 5. Chemical shifts (δ, ppm) in 13C NMR spectra* of (I)–(VII)
Compound
Residue
C1
C2
C3
C4
C5
C6
(I)
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-(1
96.6
97.1
96.3
97.1
96.6
97.4
96.2
96.6
95.4
96.3
96.8
95.6
96.3
96.8
95.8
96.4
96.9
95.9
73.3
69.2
73.5
68.9
74.0
69.3
70.6
68.95
69.1
71.0
69.1
69.2
71.3
69.2
69.2
71.5
69.2
69.4
69.2
70.6
68.4
70.3
68.5
69.6
73.1
71.1
71.0
71.4
71.2
71.4
71.5
70.1
71.3
71.8
70.0
70.2
73.1
73.0
81.4
72.7
81.9
81.8
68.6
72.9
72.9
78.0
72.9
72.9
78.1
81.4
72.9
78.2
81.4
81.5
67.7
68.2
66.6
67.8
66.9
67.5
67.2
68.3
68.4
67.2
68.1
68.4
67.2
67.9
68.2
67.2
67.9
67.6
16.5
16.4
16.5
16.1
16.9
16.9
16.5
16.5
16.5
17.1
16.6
16.5
17.1
17.1
16.6
17.1
17.1
17.1
2)
2)
2)
(II)
(III)
(IV)
2)
3)
(V)
2)
3)
(VI)
(VII)
2)
3)
2)
3)
* The spectra were registered at 30°C in D O using acetone (δ 31.5 ppm) as an internal standard. Signals of the propyl aglycon:
2
OCH CH CH , 11.1 ppm; OCH CH CH , 23.2–23.3 ppm; and OCH CH CH , 71.3 ppm.
2
2
3
2
2
3
2
2
3
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THE SYNTHESIS AND NMR AND CONFORMATIONAL STUDIES … VI.
145
Table 6. The values of sulfation effects (∆δ, ppm) in 13C NMR spectra of (II), (III), and (V)–(VII)
Compound
Residue
C1
C2
C3
C4
C5
C6
(II)
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-(1
α-L-Fuc-OPr
α-L-Fuc-(1
α-L-Fuc-(1
0.2
0.5
0
0.6
0.2
0.7
0.1
0.4
0.2
0.1
0.7
0.3
0.1
0.9
0.3
0.3
–0.4
0.2
8.8
0.1
8.8
8.8
9.4
0
–0.7
0.1
0.5
0.2
0.4
0.5
0.6
0.1
0
2)
2)
(III)
(V)
–0.7
–1.0
–1.7
0.1
–0.8
–0.7
0
0.3
0.1
0.2
0.2
0.1
0.2
0.4
0.2
0.3
0.5
2)
3)
–0.2
0
0.4
0
(VI)
–1.6
–1
9.5
8.5
0
0
0.6
0.6
0.1
0.6
0.6
0.6
2)
3)
–0.4
–0.2
0
0.3
(VII)
–1.3
–1.1
–0.8
9.6
8.5
8.6
2)
3)
–0.4
–0.8
A marked increase in the sulfation effects on C4 of determining the conformation of both (1
2)- and
the central residues as compared with the influence on (1
3)-linkage in the series of vicinally branched
C4 of glycosylating residues in sulfated trisaccharides fucotriosides, whereas the presence of sulfate group at
(V)–(VII) is due to the change in the conformation of O4 was established to influence it to a markedly lesser
(1
3)-linkage as compared with that in nonsulfated extent.
trisaccharide (IV) (Table 6). According to the experi-
mental and computed NOE values (Table 3), sulfated
trisaccharides (V)–(VII) are characterized by a
decrease in NOE at H4 and an increase in NOE at H3
of the central residue as a response to the excitation of
EXPERIMENTAL
The procedures for purification of solvents and
reagents were reported in [11]. 1H and 13C NMR spec-
tra of oligosaccharides were registered on a Bruker
DRX500 spectrometer in D2O containing 0.05% ace-
H' of the (1
3)-linked residue. This correlates with
an increase in the average distance between H1' and H4.
tone as a standard (1H, 2.225 ppm; 13C, 31.45 ppm). The
2D experiments with the included gradient pulse, such
as gCOSY, gNOESY, gHSQC, and also TOCSY 2D
experiment, were used for the assignment of reso-
nances in the 1D spectra. Experimental NOE values
were obtained using gNOESY 2D technique on a
Bruker DRX500 spectrometer in D2O (99.98% D,
Merck) at 303 K with mixing time of 500 ms and relax-
ation time of 5 s. The values of optical rotation were
measured on a Jasco DIP-360 digital polarimeter at 18–
25°ë. For TLC, silica gel 60 (Merck) precoated plates
were used. Spots were visualized by spraying with 10
vol% solution of orthophosphoric acid in ethanol and
subsequent heating at ~150°ë. Column chromatogra-
phy was carried out on 0.063–0.2 mm silica gel 60
(Fluka). Gel chromatography of unprotected sulfated
compounds (II), (III), and (V)–(VII) was carried out
on a Sephadex G-10 (2 × 20 cm) column using water as
an eluent.
Unlike (1 3)-linked disaccharides [4] and linear
trisaccharides [1], 2,3-branched trisaccharides (V)–
(VII) showed no marked positive effect of 4-é-sulfa-
tion of the central residue on the chemical shifts of C1'
atoms in the (1
3)-linked residues. This indicates
insignificant changes in the conformation of the (1
3)-glycoside bond in comparison with its change upon
sulfation of (1
3)-linked difucosides [4].
As expected, the sulfation effects in 2-é-glycosylat-
ing residues of trisaccharides (V)–(VII) are comparable
with those in (1
(Table 5).
2)-linked disaccharides (II)−(III)
CONCLUSIONS
During this study, we synthesized and analyzed con-
formations of a series of fucooligosaccharides contain-
ing α-(1
2)-glycoside linkage and 2,3-diequatorial
branching. The coincidence of computed and experi-
General sulfation procedure. A Py · SO3 complex
(80 mg, 0.5 mmol) was added to a solution of a nonsul-
fated derivative (0.1 mmol) in DMF (1 ml), and the
mixture was stirred for 1 h. Then, NaHCO3 (150 mg)
mental NOE values for the (1
2)-linked fucobio-
sides can only be achieved with the use of the nonem-
pirical DFT method. 4-O-Sulfation was shown to exert
a little effect on the conformation of the (1
2)-gly-
coside linkage. The presence of fucose residue in the and KU-2 (Na+) cation exchanger were added, and the
neighboring position was found to be the main factor mixture was stirred for 1 h. The solution was filtered
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 2 2004

146
GERBST et al.
from NaHCO3 and the cation exchanger and concen- vacuum, and the product of glycosylation was isolated
from the residue by chromatography.
trated to a volume of 1 ml. The sulfated derivative was
isolated by column chromatography on silica gel.
Allyl
2-O-(2-O-benzyl-3,4-di-O-benzoyl-a-L-
fucopyranosyl)-3,4-O-isopropylidene-a-L-fucopyr-
anoside (XVI) was obtained by glycosylation of ace-
tonide (XIII) [2] (58 mg, 0.23 mmol) with trichloroace-
timidate (XV) (140 mg, 0.23 mmol); yield 123 mg
(78%), Rf 0.73 (2 : 1 toluene–ethyl acetate), [α]D –247°
(c 2, ethyl acetate).
Allyl
2-O-(2-O-benzyl-3,4-di-O-benzoyl-a-L-
fucopyranosyl)-3-é-benzyl-4-é-sulfo-a-L-fucopyr-
anoside sodium salt (IX) was obtained by sulfation of
(VIII) [2] (83 mg, 0.11 mmol); yield 80 mg (85%), Rf
0.46 (5 : 1 dichloromethane–methanol), [α]D –227°
(c 2, methanol).
Allyl
2-O-(2-O-benzyl-3,4-di-O-benzoyl-a-L-
Allyl 2-O-(2-O-benzyl-a-L-fucopyranosyl)-3-O-
benzyl-a-L-fucopyranoside (X). A 1 M solution of
sodium methylate in methanol (0.1 ml) was added to a
solution of disaccharide (VIII) (114 mg, 0.15 mmol) in
methanol (2 ml), and the mixture was kept for 3 h at
40°ë. The solution was neutralyzed with KU-2 (H+
form), filtered from the cation exchanger, and concen-
trated. Column chromatography of the residue resulted
in triol (X) (70 mg, 88%), Rf 0.14 (1 : 1 toluene–ethyl
acetate), [α]D –159° (c 1, methanol). Found, %: C
65.61, H 6.93. Calc. for C29H38O9 (M 530.62): C 65.66,
H 7.17.
fucopyranosyl)-a-L-fucopyranoside (XVII). Disac-
charide (XVI) (68 mg, 0.1 mmol) was dissolved in a
mixture of dichloromethane (1 ml) and 90% aqueous
CF3COOH (0.5 ml), kept for 30 min, concentrated, and
coevaporated with toluene (5 × 5 ml). The residue was
chromatographed to isolate diol (XVII) (56 mg, 87%);
Rf 0.1 (2 : 1 toluene–ethyl acetate); [α]D –233° (c 2,
ethyl acetate).
Allyl 2,3-di-O-(2-O-benzyl-3,4-di-O-benzoyl-a-
L-fucopyranosyl)-a-L-fucopyranoside (XVIII) was
obtained by glycosylation of diol (XVII) (115 mg,
0.18 mmol) with trichloroacetimidate (XV) (109 mg,
0.18 mmol); yield 157 mg (80%); Rf 0.54 (2 : 1 tolu-
ene–ethyl acetate); [α]D –219° (c 2, ethyl acetate).
Allyl 2,3-di-O-(2-O-benzyl-3,4-di-O-benzoyl-a-L-
fucopyranosyl)-4-O-sulfo-a-L-fucopyranoside sodium
salt (XIX) was obtained by sulfation of trisaccharide
(XVIII) (160 mg, 0.15 mmol); yield 138 mg (90%), Rf
Allyl 2-O-(2-O-benzyl-3-O-benzoyl-a-L-fucopyr-
anosyl)-3-é-benzyl-a-L-fucopyranoside (XI).
A
mixture of disaccharide (X) (64 mg, 0.12 mmol) and
Bu2SnO (33 mg, 0.13 mmol) in anhydrous toluene
(2 ml) was refluxed until complete dissolution. The
solution was concentrated to a volume of 0.5 ml, treated
with benzoyl chloride (0.02 ml, 0.14 mmol), and the
mixture was kept for 1 h and then concentrated. The 0.93 (5 : 1 dichloromethane–methanol); [α]D –230°
residue was chromatographed to give benzoate (XI)
(56 mg, 75%), Rf 0.58 (1 : 2 toluene–ethyl acetate),
[α]D –191° (c 1, ethyl acetate).
Allyl 2-O-(2-O-benzyl-3-O-benzoyl-4-O-sulfo-a-
L-fucopyranosyl)-3-é-benzyl-4-é-sulfo-a-L-fucopy-
ranoside disodium salt (XII) was obtained by sulfa-
tion of diol (XI) (82 mg, 0.13 mmol) with Py · SO3 com- (c 1, methanol).
plex (206 mg, 1.3 mmol); yield 95 mg (87%), Rf 0.21
(10 : 3 dichloromethane–methanol), [α]D –185° (c 1, anosyl)-a-L-fucopyranoside (XXI) was obtained by
methanol).
(c 2, methanol).
Allyl 2-O-(2-O-benzyl-a-L-fucopyranosyl)-3,4-
O-isopropylidene-a-L-fucopyranoside (XX) was
obtained by debenzoylation of disaccharide (XVI)
(41 mg, 0.06 mmol) as described for (X); yield 25 mg
(88%), Rf 0.17 (1 : 1 toluene–ethyl acetate); [α]D –184°
Allyl 2-O-(2-O-benzyl-3-O-benzoyl-a-L-fucopyr-
monobenzoylation of diol (XX) (35 mg, 0.06 mmol) as
described for (XI) and subsequent removal of the iso-
propylidene protective group as described for (XVII);
yield 30 mg (77%); Rf 0.17 (2 : 3 toluene–ethyl acetate);
[α]D –194° (c 1, methanol).
2-O-Benzyl-3,4-di-O-benzoyl-a-L-fucopyranosyl
trichloroacetimidate (XV). Hemiacetal (XIV) [2]
(462 mg, 1 mmol) was dissolved in dichloromethane
(15 ml) under argon, CCl3CN (1 ml) was added, the
mixture was cooled to –30°ë, 1,8-diazabicy-
Allyl 2-O-(2-O-benzyl-3-O-benzoyl-a-L-fucopyra-
clo[5.4.0]undec-7-ene (0.06 ml) was added, and the nosyl)-3-O-(2-O-benzyl-3,4-di-O-benzoyl-a-L-fucopy-
reaction mixture was kept for 1 h. Column chromatog- ranosyl)-a-L-fucopyranoside (XXII) was obtained by
raphy on silica gel passivated by triethylamine resulted glycosylation of triol (XXI) (22 mg, 0.04 mmol) with
in imidate (XV) as a mixture of anomers; yield 588 mg trichloroacetimidate (XV) (242 mg, 0.04 mmol); yield
(97%), Rf 0.54 (β-anomer) and 0.73 (α-anomer) in 32 mg (80%), Rf 0.56 (1 : 1 toluene–ethyl acetate), [α]D
1 : 1 : 0.01 toluene–ethyl acetate–triethylamine.
–167° (c 1, methanol).
General glycosylation procedure. Trichloroace-
Allyl 2-O-(2-O-benzyl-3-O-benzoyl-4-O-sulfo-a-
timidate (XV) (1 mmol) was mixed with a fucosyl L-fucopyranosyl)-3-O-(2-O-benzyl-3,4-di-O-benzoyl-
acceptor (1 mmol) under argon. The mixture was dis- a-L-fucopyranosyl)-4-O-sulfo-a-L-fucopyranoside
solved in dichloromethane (22 ml), cooled to –30°ë, disodium salt (XXIII) was obtained by sulfation of
mixed with 0.1 M solution of Me3SiOTf in dichlo- diol (XXII) (44 mg, 0.05 mmol); yield 53 mg (89%),
Rf 0.21 (5 : 1 dichloromethane–methanol), [α]D –153°
romethane (50 µl), and kept for 30 min. Triethylamine
(1 ml) was added, the mixture was concentrated in a (c 1, methanol).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 2 2004

THE SYNTHESIS AND NMR AND CONFORMATIONAL STUDIES … VI.
147
Allyl 2,3-di-O-(2-O-benzyl-a-L-fucopyranosyl)-
Propyl 2,3-di-O-(4-O-sulfo-a-L-fucopyranosyl)-
a-L-fucopyranoside (XXIV) was obtained by deben- 4-é-sulfo-a-L-fucopyranoside trisodium salt (VII)
zoylation of trisaccharide (XVIII) (50 mg, 0.05 mmol) was obtained from (XXVI) (71 mg, 0.06 mmol); yield
as described for (X); yield 26 mg (90%), Rf 0.38 (10 : 1 37 mg (76%); [α]D –147° (c 0.63, H2O).
ethyl acetate–methanol); [α]D –145° (c 1, methanol).
Allyl
2,3-di-O-(2-O-benzyl-3-O-benzoyl-a-L-
ACKNOWLEDGMENTS
fucopyranosyl)-a-L-fucopyranoside (XXV) was
obtained by benzoylation of trisaccharide (XXIV)
(30 mg, 0.04 mmol) as described for (XI) [Bu2SnO
(25 mg, 0.1 mmol) and benzoyl chloride (0.03 ml,
0.2 mmol)]; yield 32 mg (82%); Rf 0.49 (1 : 1 toluene–
ethyl acetate), [α]D –168° (c 1, ethyl acetate).
The work was supported by the Russian Foundation
for Basic Research (project nos. 01-03-3059a, 01-04-
48401, 02-03-06589-MAC, and 02-03-06588-MAC),
by VI Estimation Competition (grant no. 129 (no. 15 in
the Division 7: General and Technical Chemistry), the
Foundation forAssistance to Domestic Science, and the
International Foundation in the Field of Exact Sciences.
Allyl
2,3-di-O-(2-O-benzyl-3-O-benzoyl-4-O-
sulfo-a-L-fucopyranosyl)-4-é-sulfo-a-L-fucopyra-
noside trisodium salt (XXVI) was obtained by trisul-
fation of triol (XXV) (30 mg, 0.03 mmol); yield 36 mg
(89%); Rf 0.33 (3 : 1 dichloromethane–methanol); [α]D
–160° (c 1, methanol).
REFERENCES
1. Gerbst, A.G., Ustuzhanina, N.E., Grachev, A.A., Kha-
tuntseva, E.A., Tsvetkov, D.E., Shashkov, A.S.,
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and Nifantiev, N.E., J. Carbohydr. Chem., 2003, vol. 22,
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General procedure for the deprotection and
transformation of aglycon. A 10% Pd/C catalyst
(10 mg) was added to a solution of a protected oli-
gosaccharide (0.06 mmol) in methanol (3 ml), the mix-
ture was degassed, and the flask was filled with hydro-
gen. The reaction mixture was stirred for 1 h, separated
from Pd/C by filtration through a Celite layer, and con-
centrated in a vacuum. The dry residue was dissolved in
distilled water (1 ml), 0.5 M aqueous NaOH (0.8 ml)
was added, and the mixture was kept for 3 h. The depro-
tected oligosaccharide was isolated from the reaction
mixture by gel filtration on Sephadex G-10.
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Propyl 2-O-(a-L-fucopyranosyl)-4-O-sulfo-a-L-
fucopyranoside sodium salt (II) was obtained from
(IX) (50 mg, 0.06 mmol); yield 22 mg (80%); [α]D
−140° (c 1, H2O).
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Propyl 2-O-(4-O-sulfo-a-L-fucopyranosyl)-4-é-
sulfo-a-L-fucopyranoside disodium salt (III) was
obtained from (XII) (50 mg, 0.058 mmol); yield 27 mg
(80%), [α]D –144° (c 1, H2O).
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Propyl 2,3-di-O-(a-L-fucopyranosyl)-4-é-sulfo-
a-L-fucopyranoside sodium salt (V) was obtained
from (XIX) (60 mg, 0.05 mmol); yield 23 mg (78%);
[α]D –117° (c 0.77, H2O).
9. Patankar, M.S., Oehninger, S., Barnett, T., Will-
iams, R.L., and Clark, G.F., J. Biol. Chem., 1993,
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Propyl 2-O-(4-O-sulfo-a-L-fucopyranosyl)-3-é-
(a-L-fucopyranosyl)-4-é-sulfo-a-L-fucopyranoside
disodium salt (VI) was obtained from (XXIII) (95 mg,
0.08 mmol); yield 41 mg (74%); [α]D –140° (c 0.7,
H2O).
10. Grant, D.M. and Cheney, B.V., J. Am. Chem. Soc., 1967,
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 2 2004