Synthesis, NMR, and conformational studies of fucoidan fragments. V.[1] linear 4,4′,4″-tri-O-sulfated and parent non-sulfated (1→3)-fucotrioside fragments

Article abstract of DOI:10.1081/CAR-120020481

Propyl 4,4′,4″-tri-O-sulfated and non-sulfated (1 → 3)-α-L-fucotriosides which are related to fragments of natural fucoidans have been synthesized. Their spectral and conformational properties have been investigated by ¹H and ¹³C NMR

Full text of DOI:10.1081/CAR-120020481

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Synthesis, NMR and Conformational Studies of Fucoidan
Fragments. V. Linear 4,4′,4″‐Tri‐O‐Sulfated and Parent
Non‐sulfated (1→3)‐Fucotrioside Fragments
Alexey G. Gerbst a , Nadezhda E. Ustuzhanina a , Alexey A. Grachev a , Elena A.
Khatuntseva a , Dmitry E. Tsvetkov a , Alexander S. Shashkov a , Anatoly I. Usov a , Marina E.
Preobrazhenskaya b , Natalya A. Ushakova b & Nikolay E. Nifantiev a
a N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky
Prospect 47, 119991, Moscow, B‐334, Russia
b V.N. Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences,
Moscow, Russia
Published online: 16 Aug 2006.
To cite this article: Alexey G. Gerbst , Nadezhda E. Ustuzhanina , Alexey A. Grachev , Elena A. Khatuntseva , Dmitry E.
Tsvetkov , Alexander S. Shashkov , Anatoly I. Usov , Marina E. Preobrazhenskaya , Natalya A. Ushakova & Nikolay E. Nifantiev
(2003) Synthesis, NMR and Conformational Studies of Fucoidan Fragments. V. Linear 4,4′,4″‐Tri‐O‐Sulfated and Parent
Non‐sulfated (1→3)‐Fucotrioside Fragments, Journal of Carbohydrate Chemistry, 22:2, 109-122, DOI: 10.1081/CAR-120020481
To link to this article: http://dx.doi.org/10.1081/CAR-120020481
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 22, No. 2, pp. 109–122, 2003
Synthesis, NMR, and Conformational Studies of Fucoidan
Fragments. V.[1] Linear 4,4’,4@-Tri-O-Sulfated and Parent
Non-sulfated (1!3)-Fucotrioside Fragments
Alexey G. Gerbst,1 Nadezhda E. Ustuzhanina,1 Alexey A. Grachev,1
Elena A. Khatuntseva,1 Dmitry E. Tsvetkov,1 Alexander S. Shashkov,1
Anatoly I. Usov,1 Marina E. Preobrazhenskaya,2 Natalya A. Ushakova,2
and Nikolay E. Nifantiev1,
*
1N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
Moscow, Russia
2V.N. Orekhovich Institute of Biomedical Chemistry, Russian Academy of
Medical Sciences, Moscow, Russia
ABSTRACT
Propyl 4,4’,4@-tri-O-sulfated and non-sulfated (1 ! 3)-a-L-fucotriosides which are
related to fragments of natural fucoidans have been synthesized. Their spectral and
conformational properties have been investigated by 1H and 13C NMR, NOE and
molecular modeling. Molecular mechanics calculations of the tri-O-sulfated
compound as a trianion did not give agreement with the experimental NOE values,
while the model with the non-dissociated sulfo group on the non-reducing end
worked successfully. (1 ! 3)-Fucobioside fragments in both trisaccharides inves-
tigated were shown to have the same range of conformations as in previously de-
scribed propyl (1 ! 3)-a-L-fucobiosides, but with the increase of the relative
*
Correspondence: Nikolay E. Nifantiev, N.D. Zelinsky Institute of Organic Chemistry, Russian
Academy of Sciences, Leninsky Prospect 47, 119991 Moscow B-334, Russia; Fax: + 7-095-
1358784; E-mail: nen@ioc.ac.ru.
109
DOI: 10.1081/CAR-120020481
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

110
Gerbst et al.
population of the conformer with the spatial proximity of H-1’ and H-4 in the case of
non-sulfated fucotrioside.
Key Words: Fucoidan fragments; Synthesis; NMR; Conformational analysis; NOE.
INTRODUCTION
Fucoidans, a group of highly sulfated polysaccharides of brown seaweeds, have
received increasing interest as readily available biopolymers having many promising
biological activities. Complete structural characterization of fucoidans can not generally
be performed due to their irregularity and heterogeneity. In the framework of the
investigation of fucoidan structure and assessment of pharmacophores in fucoidan
chains, we perform the systematic synthesis of their supposed fragments for the study
of their NMR and conformational characteristics and various types of biological
activities (see the citations in Ref. [2]). Recently we described the synthesis and NMR
studies of propyl 3-O-a-L-fucopyranosyl-a-L-fucopyranoside,[1,2] its 4-O-sulfate and
4,4’-di-O-sulfate.[1] In this communication we report the synthesis, NMR and confor-
mational analyses of propyl glycosides of linear (1 ! 3)-linked a-L-fucotrioside (1) and
its 4,4’,4@-tri-O-sulfate (2). The presence of 1 !3-linked a-L-fucopyranose sequences
was long ago suggested as the main structural feature of many fucoidans[3] and unam-
biguously confirmed by several investigators recently.[4–6]
RESULTS AND DISCUSSION
Synthesis of compounds 1 and 2. The target linear fucotriosides 1 and 2 were
prepared by use of the readily accessible partially protected disaccharide 3[1] as glycosyl
acceptor. Its regioselective and stereospecific fucosylation by 3,4-di-O-benzoyl-2-O-
benzylfucosyl bromide[2] in the presence of Hg(CN)2 and HgBr2 gave the trisaccharide
derivative 4 in 65% yield. The a-anomeric configuration of the unit at the non-reducing
end in 4 was confirmed by a characteristic value of J1,2 = 3.0 Hz in the 1H NMR
spectrum. The location of the newly introduced fucosyl residue at O-3 followed from
characteristic chemical shifts of C-3 in the 13C NMR spectrum (Table 1).

Fucoidan Fragments. V.
Table 1. 13C NMR chemical shiftsa for compound 4.
111
Residue
C-1
C-2
C-3
C-4
C-5
C-6
! 3)a-L-Fuc-OAll
! 3)-a-L-Fuc-(1 !
a-L-Fuc-(1 !
95.8
94.0
94.3
74.5
75.1
73.2
74.8
75.5
71.1
68.2
68.2
72.3
64.9
65.7
65.5
16.2
16.0
15.8
aIn ppm, recorded at 30 °C in CDCl3. Signals of allyl aglycon: OCH2CH = CH2 d 117.7;
OCH2CH = CH2 d 134.0; OCH2CH = CH2 d 68.2.
O-Debenzoylation of 4 with methanolic sodium methoxide gave tetraol 5. Its
regioselective benzoylation via a stannylidene intermediate gave the 3@-O-benzoylated
derivative 6 in 62% yield. A downfield chemical shift of the H-3@ signal to d = 5.54
ppm (Table 2) evidenced the location of the benzoate group at C-3@.
Treatment of triol 6 with sulfur trioxide-pyridine complex in DMF gave sulfated
derivative 7 in 80% yield. Characteristic downfield chemical shifts of the H-4 signals
(Table 2) indicated the presence of sulfate groups in 7 at C-4, C-4’ and C-4@.
Catalytic hydrogenolysis and subsequent saponification of products 4 and 7
gave the corresponding target trisaccharides 1 and 2 which were purified by gel
filtration on a Sephadex G-10 column. The presence of O-sulfonato groups in 2
was confirmed by low-field chemical shifts of the respective H-4 and C-4 signals
(Tables 3–5).
1
NMR analysis of compounds 1 and 2. Tables 3–5 show the H NMR and 13C
1
NMR chemical shifts of compounds 1 and 2. Assignments of the H NMR spectra
1
(Tables 3, 4) of these compounds were made using a combination of H–1H COSY and

112
Gerbst et al.

Fucoidan Fragments. V.
Table 3. 1H NMR chemical shiftsa of oligosaccharides 1 and 2.
113
Compound
Residue
H-1
H-2
H-3
H-4
H-5
H-6
1
! 3)a-L-Fuc-OPr
! 3)-a-L-Fuc-(1 !
a-L-Fuc-(1 !
4.92
5.10
5.06
4.92
5.12
5.14
3.93
3.93
3.81
3.95
3.87
3.76
3.96
4.01
3.97
4.03
4.05
4.01
4.03
4.02
3.83
4.75
4.77
4.61
4.08
4.28
4.32
4.19
4.43
4.48
1.23
1.23
1.23
1.25
1.25
1.25
2
a-L-Fuc-OPr
! 3)-a-L-Fuc-(1 !
a-L-Fuc-(1 !
aIn ppm, recorded at 40 °C in D2O with acetone as an internal standard. Signals of aglycon:
OCH2CH2CH3 d 0.92; OCH2CH2CH3 d 1.62–1.64; OCH2CH2CH3 d 3.49–3.65 and 3.62–3.84.
2D TOCSY experiments. Assignments of the 13C NMR spectra (Table 5) were made
1
using 2D H-13C HSQC and HMQC correlation spectroscopy.
The location of sulfates at C-4 in 2 was confirmed by the characteristic low-field
position of the H-4 and C-4 signals in the spectra of 2. The O-sulfation effects observed
in 13C NMR spectra of compound 2 are shown in Table 6. These effects (Dd) are the
differences in the chemical shifts of the respective signals in spectra of the O-sulfated
compound and its non-sulfated parent. The sulfation effect on the C-4 of the terminal
residue at the non-reducing end is ca. 2 ppm smaller than those in the other two
residues. At the same time, C-3 atoms in these two residues lack a b-effect (slight
negative change of the chemical shift[7]) present in the terminal residue. Also sub-
stantial positive sulfation effects are observed on C-1 in the terminal and middle
residues, while for the reducing end Dd13C is 0. Similar tendencies were observed
previously in the case of fucobiosides 8 and 9.[1]
The similarity in the 13C spectra of compounds 1, 2 and 8, 9 suggests their
conformational similarity. To confirm this we studied the trisaccharides using the
approach suggested before[1] for (1 !3)-fucobiosides 8 and 9 by means of molecular
mechanics calculations and NOE experiments.
Conformational analysis of compounds 1 and 2. In a previous work[1] we ac-
complished the experimental conformational analysis of fucosides via a steady-state
NOE technique. This method cannot be applied to compounds 1 and 2, because the
1
Table 4. Coupling constants in H NMR spectra of oligosaccharides 1 and 2.
Compound
Residue
J1,2
J2,3
J3,4
J4,5
J5,6
1
! 3)a-L-Fuc-OPr
! 3)-a-L-Fuc-(1 !
a-L-Fuc-(1 !
2.6
3.8
3.8
3.9
3.7
3.6
n.d.a
n.d.a
10.7
10.3
10.4
10.5
< 2 Hz
< 2 Hz
3.0
< 2 Hz
< 2 Hz
< 2 Hz
< 2 Hz
< 2 Hz
< 2 Hz
6.5
6.5
6.5
6.4
6.5
6.5
2
! 3)a-L-Fuc-OPr
! 3)-a-L-Fuc-(1 !
a-L-Fuc-(1 !
2.4
2.3
2.5
aNot determined due to the overlap of the multiplets.

114
Gerbst et al.
Table 5. 13C NMR chemical shiftsa for oligosaccharides 1 and 2.
Compound
Residue
C-1
C-2
C-3
C-4
C-5
C-6
1
! 3)a-L-Fuc-OPr
! 3)-a-L-Fuc-(1 !
a-L-Fuc-(1 !
99.6
96.5
97.1
99.6
99.5
98.9
67.7
67.8
69.2
67.7
68.3
69.8
75.9
76.4
70.9
77.3
76.8
70.2
69.6
69.9
73.3
80.4
80.4
82.5
67.6
68.0
68.3
67.2
67.7
67.7
16.7
16.7
16.7
17.0
17.0
17.0
2
! 3)a-L-Fuc-OPr
! 3)-a-L-Fuc-(1 !
a-L-Fuc-(1 !
aIn ppm, recorded at 40 °C in D2O with acetone as an internal standard. Signals of propyl
aglycon: OCH2CH2CH3 d 11.1; OCH2CH2CH3 d 23.2–23.3; OCH2CH2CH3 d 71.3.
resonance lines of the two protons of interest, H-1’ and H-1@, are separated by less than
15 Hz, which makes impossible their selective pre-irradiation. In the current study the
2D-NOESY method followed by quantitative integration of cross-peaks was used. The
resulting numbers correspond to transient NOE values. At the employed mixing time
of 500 ms the build-up of NOE is in the linear range and NOE values can be
estimated[8,9] as
1
f /
r6
A theoretical conformational analysis was performed using the MM3 force field.[10]
Conformational maps of the fucotriosides were built via a procedure similar to the one
previously described.[1] Glycosidic linkages were treated independently: first, one
linkage was restrained and the other allowed to rotate freely, then vice-versa. Thus, two
conformational maps were produced for each fucotrioside. For minima found on these
maps we analyzed the state of the glycosidic linkage that was not restrained during the
construction of the current map. This yielded four conformations for each fucotrioside,
which were checked further against identity: that is, if j and c angle values for two
conformations differed by less than 10° and their energies by less than 0.2 kcal/mol, we
considered these conformations the same. Conformational maps of trisaccharides 1 and
2 are shown in Figure 1.
One of the approaches to the conformational analysis of higher oligosaccharides
was to use pre-defined glycosidic linkages[11] in the same conformation as in a disac-
charide fragment. Though we did not apply such constraints to the conformation of
(1 !3)-linkages in the studied compounds, our calculations showed that its con-
formational behavior in general resembles that of (1 ! 3)-linkage in fucobiosides. For
Table 6. O-Sulfation effects (Dd) in 13C NMR spectrum of O-sulfated derivative 2.
Residue
DdC-1
DdC-2
DdC-3
DdC-4
DdC-5
DdC-6
! 3)a-L-Fuc-OPr
! 3)-a-L-Fuc-(1 !
a-L-Fuc-(1 !
0.0
3.0
1.8
0.0
0.5
0.6
1.4
0.4
10.8
10.5
8.8
ꢀ 0.4
ꢀ 0.3
ꢀ 0.6
0.3
0.3
0.3
ꢀ 0.7

Fucoidan Fragments. V.
115
Figure 1. Conformational maps for the inter-unit linkages in trisaccharides 1 (A,B) and 2 (C,D).
each linkage there exist two main conformers. The first is characterized by the spatial
proximity of protons H-1’ and H-4 (positive c), while in the second the proton H-1’ is
closer to H-3 (negative c). We also encountered some noteworthy details that were not
present in the disaccharides, and they are discussed below.
Figure 2. Conformational maps of disaccharides 8 and 9.

116
Gerbst et al.
Table 7. Experimental relative integral values of cross-peaks in 2D NOESY spectra of
compounds 1 and 8 and computed relative transient NOE values (in parenthesis).
H-4/H-1’
Linkage
H-2’/H-1’
H-3/H-1’
NOEa
NOEb
1
a-L-Fuc-(1 ! 3)-a-L-Fuc-OPr
a-L-Fuc-(1 ! 3)-a-L-Fuc
214c (120 + 100)
119 (110)
125 (102)
143 (155)
143 (160)
109 (92)
(250)
(235)
(180)
100 (100)
100 (100)
8
aTheoretical NOEs calculated over all conformations lying within 10% of the global energy
minimum.
bTheoretical NOEs calculated over 3 conformations shown on Figure 3.
cMeasured for the sum of H-3/H-1’ and H-2’/H-1’ cross-peaks.
Compounds 1, 8 and 9. For non-sulfated trisaccharide 1 three different conformers
were observed. Their schematic representations and proportion in the total ensemble are
given in Figure 2. The dominant conformation is that with spatial proximity of H-1’/H-
4 and H-1@/H-4’ in both linkages. It is the same as in fucobioside 8 (Figure 2), but the
value of its statistical weight is greater than 80%, while the weight of the conformer
with such geometry of the inter-unit linkage in the fucobioside was about 60%. Rota-
tion about either inter-unit linkage in order to shorten the H-1’—H-3 distance results in
the increase of the energy and consequently in the decrease of the population. Con-
Figure 3. Conformers of the non-sulfated trisaccharide 1 with their statistical weights and
schematic representation of one of the conformers of the related fucobioside 8 conformer.

Fucoidan Fragments. V.
117
formations in which both H-1 protons are close to H-3 are populated scarcely more
than 1% and are not shown.
Values obtained from 2D-NOESY experiments for compounds 1 and 8 (Table 7)
confirmed the tendency predicted from the calculations. Differences in ratio of NOE
values of H-4 and H-3 protons between compounds 8 and 1 reveal that the average
H-4/H’-1 distance in the fucotrioside is shorter than in fucobioside 8. The same can
be concluded from the conformational changes.
Comparison of the theoretical NOE values calculated from the weights shown
above to the experimental data gave only qualitative agreement (Table 7): computed
NOE values on H-4 were about twice greater than expected both for the disaccharide
and trisaccharide. This observation means that the representation of a saccharide as an
ensemble of only few conformers may be a rather coarse approximation.
It is worth noting that other researchers[12] also admitted that there are situations
when NOEs computed directly from the map minima fail to reproduce experimental
results. One way to avoid this is to perform molecular dynamics simulations, which
give an average conformation but no information about local minima. To keep illus-
trativity provided by the conformational map, we employed the procedure described in
the previous work,[1] taking into calculation of transient NOE all the conformations
with energies lying within 10% of the global minimum. Thus computed values are
shown in Table 7.
Compound 2. The method described above for the parent trifucoside did not
provide correspondence to the experimental 2D-NOESY data. Surprisingly, our calcu-
lations showed no significant changes in conformation of the glycosidic linkage upon
introduction of sulfates. At the same time according to the experimental NOESY data
the conformation of the trisaccharide should change in the same manner as for the
difucoside (Tables 7 and 8), that is, the weight of the conformation with spatial prox-
imity of H-1’ and H-3 for both linkages should increase.
We supposed that such trisulfated molecule might not be fully dissociated in
solution. As there are three sulfonato-groups and no presumptions could be made which
of them is more likely to be non-dissociated, three computations were carried out, each
time leaving one of the sulfo groups protonated. After that NOESY values over all
conformers lying within 10% of the energy of the global minimum were calculated for
each hypothetical compound. Resulting values (Tables 8, 9) showed good correlation
with the experiment only when the proton was left on the sulfate group of the reducing
Table 8. Experimental relative integral values of cross-peaks in 2D NOESY spectra of
compounds 2 and 9 and computed transient NOE values (in parenthesis).
H-4/H-1’
Linkage
H-2’/H-1’
H-3/H-1’
NOEa
NOEb
2
a-L-Fuc-(1 ! 3)-a-L-Fuc-OPr
a-L-Fuc-(1 ! 3)-a-L-Fuc
80 (90)
70 (95)
90 (82)
100 (100)
100 (100)
100 (100)
57.5 (35)
57.5 (40)
49 (42)
(85)
(95)
(70)
9
aTheoretical NOEs calculated over all conformations lying within 10% of the global energy
minimum.
bTheoretical NOEs calculated over 3 conformations shown on Figure 4.

118
Gerbst et al.
Table 9. Theoretical transient H4/H1’ NOE values for different anions of compound 2.
H-4/H-1’
Fragment
Trianion
dianion-1a
dianion-2b
a-L-Fuc-(1 ! 3)-a-L-Fuc-OPr
a-L-Fuc-(1 ! 3)-a-L-Fuc
148
140
155
150
145
140
adianion with the sulfate in the non-reducing end.
bdianion with the sulfate in the middle ring.
end. The resulting conformations and their statistical weights are presented in Figure 4.
This compound also had three main conformations, but in the most stable one both
H-1’—H-3 and H-1@—H-3’ distances are short.
Analysis of 13C chemical shifts in spectra of fucotriosides 1 and 2. In the case
of fucobiosides[1] it was possible to rationalize differences in sulfation effects (Table 6)
in terms of the Grant-Cheney theory.[13] The same could be applied in part to the
studied trisaccharides. Thus the lack of the b-effect on C-3 in the reducing and the
middle ring is due to the decrease of the average distance between H-1’ and H-3 upon
4-O-sulfation. A positive sulfation effect on C-1 of the middle and non-reducing ring
appears due to the the increase of the H-1’—H-4 distance.
In this study we also had an opportunity to compare trifucosides 1 and 2 to the
corresponding disaccharide fragments 8 and 9. In Table 10 the values of deviations
Figure 4. Conformers of tri-O-sulfated trisaccharide 2, their statistical weights and schematic
representation of related fucobioside 9 conformer.

Fucoidan Fragments. V.
119
Table 10. Deviations from additivity in compounds 1 and 2, computed relative to the fucobiosides.
Compound
Residue
DdC-1
DdC-2
DdC-3
DdC-4
DdC-5
DdC-6
1
! 3)a-L-Fuc-OPr
! 3)-a-L-Fuc-(1 !
a-L-Fuc-(1 !
0
ꢀ 0.1
0
ꢀ 0.1
0.4
0
0.1
0.4
0
0
0
0
0.4
0
0
0.6
0.1
0.8
0.2
ꢀ 0.2
ꢀ 0.1
0.5
0
2
! 3)a-L-Fuc-OPr
! 3)-a-L-Fuc-(1 !
a-L-Fuc-(1 !
0.7
0.2
0.0
0.4
0.4
0.4
0.1
0.6
0.1
0.0
0.0
0.0
0.1
from additivity are presented. They are calculated as differences between chemical
shifts of a trisaccharide and the corresponding disaccharide fragment (8 or 9).
For trisaccharide 2 both NOE data and the theoretical calculations show a simi-
larity to disulfated fucobioside 9. According to the Grant and Cheney concept[13] DdC
values should be 0. In fact, they are slightly positive for some atoms. More inte-
restingly, the same tendency takes place in compound 1, where DdC are also positive
or close to 0. This evidence is contradictory to that provided by NOE and calculations,
that show significant decrease of the H-1’—H-4 distance. This observation shows that
the Grant and Cheny theory may not correlate with experimental NOE data.
CONCLUSIONS
Two linear fucotrioside fucoidan fragments have been synthesized and investigated
1
by H, 13C and NOE NMR spectroscopy and molecular modeling. For the non-sulfated
saccharide combined molecular mechanics modeling and 2D NOESY experiments have
shown that the statistical weight of the dominant conformation with spatial proximity of
H-1’ and H-4 protons is increased if compared with that of the previously studied
fucobioside. It was shown that molecular modeling results for the tri-O-sulfated tri-
saccharide do not correspond to the experimental NOESY data when the molecule is
considered as a tri-anion, while theoretical regard of the sulfate group in the reducing
end in the non-dissociated state restores the agreement with the experiment. It was
found that NOE data and Dd13C values predicted from the Grant-Cheney concept do
not always correlate.
EXPERIMENTAL
General methods. TLC was performed on silica gel 60 F254 (Merck) with
EtOAc– toluene (A, 1:1, B, 1:2, C, 1:5), CH2Cl2– MeOH (D, 4:1); spots were detected
by charring with H3PO4. Column chromatography was performed on Silica Gel 0.063–
0.2 mm (Fluka) by gradient elution. Gel chromatography was performed on a Sephadex
G-10 column (2 ꢁ 20 cm) by elution with water at a flow rate of 1 mL/min, and a
Sephadex LH-20 column (2 ꢁ 40 cm) by elution with methanol at a flow rate of 1
mL/min. Optical rotations were determined with a Jasco DIP-360 digital polarimeter at
26-30°C. All solvents used for syntheses were purified according to conventional
procedures.[14] NMR spectra for substituted compounds 4–7 were recorded on Bruker
1
spectrometers WM-250 and AM-300 at 303 K. H and 13C NMR spectra for oligo-

120
Gerbst et al.
saccharides 1 and 2 were recorded in D2O on a Bruker spectrometer DRX-500 with
0.05% acetone as reference (1H 2.225 ppm; 13C 31.45 ppm). Gradient enhanced 2D
gCOSY, gNOESY and gHSQC experiments as well as TOCSY experiments were used
for resonance assignment. Experimental NOEs were measured using a 2D-NOESY
technique with pulse field gradient (PFG) on a Bruker DRX-500 instrument in D2O
(99.98% D, Merck) solutions at 303 K, mixing time 500 ms, relaxation delay 5 s. The
following PFG parameters were used: homospile/gradient pulse of 1 ms, delay for
homospile/gradient recovery of 1 ms. Computations were performed using TINKER
software package with the implemented MM3 force field. The dielectric constant e was
set to 81. All sulfate groups were treated as anions, unless stated otherwise. Parameters
for their modelling were based on published work.[15] No solvent molecules were
considered in the calculation. The starting structures were produced by geometry opti-
mization with MM3. In each point of a conformational map the same starting geometry
was used, and the dihedral angles were restrained with force constant of 10 kcal/deg2
before the optimization.
!
Allyl 3,4-Di-O-benzoyl-2-O-benzyl- -L-fucopyranosyl-(1 3)-2-O-benzyl- -L-
!
fucopyranosyl-(1 3)-2-O-benzyl- -L-fucopyranoside (4). A mixture of triol 3
(111 mg, 0.21 mmol), Hg(CN)2 (58 mg, 0.23 mmol), HgBr2 (10 mg), and molecular
˚
sieves 4 A (500 mg) in CH2Cl2 (3.5 mL) was stirred for 1 h at 20 °C under Ar. Using a
syringe, a solution of 3,4-di-O-benzoyl-2-O-benzyl-a-L-fucosyl bromide (prepared from
3,4-di-O-benzoyl-2-O-benzyl-L-fucoside,2 146 mg, 0.32 mmol) was added portionwise
during 1 h. The mixture was stirred for 24 h, then filtered through Celite, diluted with
CH2Cl2, washed with satd aq KBr and NaHCO3 solutions and concentrated. Adsorption
chromatography on silica of the residue followed by gel chromatography on a Sepha-
dex LH-20 column gave the trisaccharide 4 (133 mg, 65%): [a]D ꢀ 253° (c 2, MeOH);
1
RF 0.57 (solvent B). The H and 13C NMR data for 4 are presented in Tables 1 and 2.
Anal. Calcd for C56H62O15 (975.11): C, 68.98%; H, 6.41%. Found: C, 68.91%;
H, 6.55%.
!
Allyl 2-O-Benzyl- -L-fucopyranosyl-(1 3)-2-O-benzyl- -L-fucopyranosyl-
!
(1 3)-2-O-benzyl- -L-fucopyranoside (5). To a solution of 4 (97 mg, 0.10 mmol)
in CH2Cl2 (1 mL) a solution of 1.2 M MeONa in MeOH (0.1 mL) was added. The
mixture was kept for 1 h at rt and then neutralized with KU-2 (H + ) resin, filtered, and
concentrated to dryness. Column chromatography of the residue gave compound 5 (68
1
mg, 90%): [a]D ꢀ 115° (c 0.67, MeOH); RF 0.17 (solvent A). The H NMR data for 5
are presented in Table 1.
Anal. Calcd for C42H54O13(766.89): C, 65.78%; H, 7.10%. Found: C, 65.63%;
H, 6.99%.
!
Allyl 3-O-Benzoyl-2-O-benzyl- -L-fucopyranosyl-(1 3)-2-O-benzyl- -L-fuco-
!
pyranosyl-(1 3)-2-O-benzyl- -L-fucopyranoside (6). A mixture of compound 5
(20 mg, 0.026 mmol), Bu2SnO (7 mg, 0.03 mmol) and toluene (1.7 mL) was refluxed
until complete dissolution and then concentrated to a volume of 0.7 mL. BzCl (4 mL,
0.034 mmol) was added, the solution was kept at rt for 1.5 h and concentrated in vacuo.
Column chromatography of the residue gave amorphous 6 (14 mg, 62%): [a]D ꢀ 159°
1
(c 1, EtOAc); RF 0.79 (solvent C). The H NMR data for 6 are presented in Table 1.
Anal. Calcd for C49H58O14 (871.00): C, 67.57%; H, 6.71%. Found: C, 67.45%;
H, 6.69%.

Fucoidan Fragments. V.
121
Trisodium salt of Allyl 3-O-Benzoyl-2-O-benzyl-4-O-sulfonato- -L-fucopyrano-
!
!
syl-(1 3)-2-O-benzyl-4-O-sulfonato- -L-fucopyranosyl-(1 3)-2-O-benzyl-4-O-
sulfonato- -L-fucopyranoside (7). A solution of 6 (25 mg, 0.03 mmol) in DMF
(0.5 mL) was treated with SO3ꢂPy complex (57 mg, 0.36 mmol) for 1 h at rt, then
quenched with NaHCO3 (30 mg) and stirred for 1 h. The solid was filtered off 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 concentrated.
Column chromatography of the residue gave amorphous 7 (28 mg, 80%): [a]D ꢀ 143°
1
(c 1, MeOH); RF 0.27 (solvent D). The H NMR data for 7 are presented in Table 1.
Anal. Calcd for C49H55Na3O23S3(1177.13): C, 50.00%; H, 4.71%. Found: C,
50.13%; H, 4.87%.
!
!
Propyl -L-Fucopyranosyl-(1 3)- -L-fucopyranosyl-(1 3)- -L-fucopyranoside
(1). A solution of 4 (43 mg, 0.047 mmol) in MeOH (6 mL) was subjected to catalytic
hydrogenolysis with 10% Pd-C at 20°C and atm. pressure for 2 h. The mixture was
filtered through Celite, and the solvent was evaporated in vacuo. The residue was
dissolved in water (1 mL), 0.1 M NaOH (0.5 mL) was added. The mixture was kept for
1 h at rt and then was subjected to gel chromatography on a Sephadex G-10 column in
water to give amorphous 1 (18 mg, 75%): [a]D ꢀ 248° (c 0.33, H2O). The H and 13C
1
NMR data for 1 are presented in Tables 3–5.
Anal. Calcd for C21H38O13 (498.53): C, 50.60%; H, 7.68%. Found: C, 50.72%;
H, 7.88%.
!
Trisodium salt of Propyl 4-O-sulfonato- -L-fucopyranosyl-(1 3)-4-O-sulfo-
!
nato- -L-fucopyranosyl-(1 3)-4-O-sulfonato- -L-fucopyranoside (2). Debenzyla-
tion and debenzoylation of 7 (28 mg, 0.024 mmol) via the same procedure as de-
1
scribed for 1 gave amorphous 2 (15 mg, 78%): [a]D ꢀ 150° (c 0.5, H2O). The H and
13C NMR data for 2 are presented in Tables 3–5.
Anal. Calcd for C21H35Na3O22S3 (804.66): C, 31.35%; H, 4.38%. Found: C,
31.23%; H, 4.48%.
ACKNOWLEDGMENTS
This work was supported by GlycoSense AG (Jena, Germany), the Russian
Foundation for Basic Research (grants 01-03-33059a, 01-04-48401a, 02-03-06588 and
02-03-06589) and Russian Academy of Sciences (grant 129-6-1999 for young re-
searchers). We are grateful to Natalya S. Zlotina (Higher Chemical College, Moscow)
for technical assistance. We thank Professor J. W. Ponder (Department of Biochemistry
and Molecular Biophysics, School of Medicine, Washington University, St. Louis,
Missouri, USA) for providing us with TINKER software.
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Received March 25, 2002
Accepted December 26, 2002