Synthesis, NMR, and conformational studies of fucoidan fragments. IV.[1] 4-Mono- and 4,4′-disulfated (1→3)-α-l-fucobioside and 4-sulfated fucoside fragments

Article abstract of DOI:10.1081/CAR-120013500

Propyl 4-O-sulfonato- and 4,4′-di-O-sulfonato-3-O-α-L-fucopyranosyl-α-L- fucopyranosides, which are related to fragments of brown algal fucoidans, have been synthesized. Their spectral (¹H and ¹³C NMR, NOE) and conformational propert

Full text of DOI:10.1081/CAR-120013500

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
SYNTHESIS, NMR, AND CONFORMATIONAL
STUDIES OF FUCOIDAN FRAGMENTS 4: 4-MONO-
AND 4,4′-DISULFATED (1→3)-α-l-FUCOBIOSIDE AND
4-SULFATED FUCOSIDE FRAGMENTS
Alexey G. Gerbst , Nadezhda E. Ustuzhanina , Alexey A. Grachev , Natalya S.
Zlotina , Elena A. Khatuntseva , Dmitry E. Tsvetkov , Alexander S. Shashkov ,
Anatoly I. Usov & Nikolay E. Nifantiev
To cite this article: Alexey G. Gerbst , Nadezhda E. Ustuzhanina , Alexey A. Grachev , Natalya S.
Zlotina , Elena A. Khatuntseva , Dmitry E. Tsvetkov , Alexander S. Shashkov , Anatoly I. Usov &
Nikolay E. Nifantiev (2002) SYNTHESIS, NMR, AND CONFORMATIONAL STUDIES OF FUCOIDAN
FRAGMENTS 4: 4-MONO- AND 4,4′-DISULFATED (1→3)-α-l-FUCOBIOSIDE AND 4-SULFATED
FUCOSIDE FRAGMENTS, Journal of Carbohydrate Chemistry, 21:4, 313-324, DOI: 10.1081/
CAR-120013500
To link to this article: http://dx.doi.org/10.1081/CAR-120013500
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 21, No. 4, pp. 313–324, 2002
SYNTHESIS, NMR, AND CONFORMATIONAL STUDIES
OF FUCOIDAN FRAGMENTS. IV.[1] 4-MONO- AND
!
4,4’-DISULFATED (1 3)-a-L-FUCOBIOSIDE AND
4-SULFATED FUCOSIDE FRAGMENTS
Alexey G. Gerbst,1 Nadezhda E. Ustuzhanina,1 Alexey A. Grachev,2
Natalya S. Zlotina,2 Elena A. Khatuntseva,1 Dmitry E. Tsvetkov,1
Alexander S. Shashkov,1 Anatoly I. Usov,1 and Nikolay E. Nifantiev1,
*
1N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of
Sciences, Leninsky Prospect 47, 119991 Moscow B-334, Russia
2Higher Chemical College, Russian Academy of Sciences,
Moscow, Russian Federation
ABSTRACT
Propyl 4-O-sulfonato- and 4,4’-di-O-sulfonato-3-O-a-L-fucopyranosyl-a-L-
fucopyranosides, which are related to fragments of brown algal fucoidans,
have been synthesized. Their spectral (1H and 13C NMR, NOE) and con-
formational properties have been studied in combination with molecular
modeling and compared with the respective non-sulfated propyl fucobioside.
Correlations between chemical shifts and conformational properties of these
compounds were investigated.
INTRODUCTION
Fucoidans, a unique class of sulfated polysaccharides of brown seaweeds, consist
mainly of L-fucose with some galactose, xylose, mannose, and uronic acid[2] as
carbohydrate components. These biopolymers have been known for about a century,
but their chemical structure, and especially their structural diversity in dependence on
*
Corresponding author. Fax: + 7-095-1358784; E-mail: nen@ioc.ac.ru
313
DOI: 10.1081/CAR-120013500
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2002 by Marcel Dekker, Inc.

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314
GERBST ET AL.
algal species, is poorly understood. Recent investigations have shown that (1 ! 3)-
linked a-L-fucopyranose 4-sulfate residue may be regarded as one of the principal
structural units.
Although fucoidans have been extensively studied over the period of several
decades due to their diverse biological activities, especially anticoagulant,[3] antiviral[4]
and antiinflammatory,[5] the relationships between structure and biological activity are
not clearly established.
The study presented here was performed as a part of the project aimed at the
investigation of fucoidan structure and assessment of pharmacophores in their chains.
Recently, we described[6] the synthesis and NMR studies of 2,3- and 3,4-branched a-L-
fucotriosides which relate to non-sulfated vicinally branched fucoidan fragments. In this
communication we report on the synthesis, NMR and conformational studies of propyl
3-O-a-L-fucopyranosyl-a-L-fucopyranoside 3,[6] its 4-O-sulfonato- and 4,4’-di-O-sulfo-
nato derivatives 1 and 2. We also investigated the 4-sulfated monosaccharide 4 along
with the non-sulfated analog 5.[6]
RESULTS AND DISCUSSION
Synthesis of compounds 1, 2 and 4. The preparation of target sulfated fuco-
biosides 1 and 2 was performed using the readily accessible disaccharide 6[1] as the
starting compound. Its treatment with sulfur trioxide–pyridine complex in DMF gave
sulfated derivative 7 in 85% yield. The presence of a sulfate group at C-4 in 2 was
confirmed by a downfield chemical shift of the H-4 signal to 5.28 ppm (Table 1) in its
1H NMR spectrum.
O-Debenzoylation of 6 with methanolic sodium methoxide gave triol 8. Its
regioselective benzoylation with benzoyl chloride via the stannylidene procedure gave 3-
O-benzoate 9 in 77% yield. A downfield chemical shift of the H-3 signal in the 1H NMR
spectrum of compound 9 (Table 1) indicated the location of a benzoate group at C-3.
Treatment of compound 9 with sulfur trioxide–pyridine complex in DMF gave
sulfated derivative 10 in 95% yield. Similarly, monohydroxy fucoside 11[6] was

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FUCOIDAN FRAGMENTS. IV
Table 1. 1H NMR Shiftsa for Protected Compounds 6–12
315
Compound
Residue
H-1
H-2
H-3
H-4
H-5
H-6
6[6]
a-L-Fuc-OAll
(1 ! 3)-a-L-Fuc
a-L-Fuc-OAll
(1 ! 3)-a-L-Fuc
a-L-Fuc-OAll
(1 ! 3)-a-L-Fuc
a-L-Fuc-OAll
(1 ! 3)-a-L-Fuc
a-L-Fuc-OAll
(1 ! 3)-a-L-Fuc
a-L-Fuc-OAll
a-L-Fuc-OAll
5.10
5.06
5.03
5.41
4.85
4.82
4.89
4.89
4.96
5.50
4.78
4.73
4.03
3.96
4.37
4.08
3.79
3.70
3.86
4.11
4.01
4.13
3.48
3.70
4.17
4.14
4.49
5.56
4.00
3.94
4.07
5.54
4.28
5.67
3.82
3.76
3.91
5.47
5.28
4.94
3.65
3.65
3.68
3.97
4.89
4.57
3.90
4.73
4.03
4.35
1.34
1.01
1.55
0.75
1.29
1.10
1.29
1.08
1.30
1.15
4.78
1.25
7
8
3.91
4.15
3.93
4.31
3.96
4.52
1.21
3.94
9
10
11[6]
12
aIn ppm, compounds 6, 8, 9, 11 and 12 were recorded in CDCl3; compounds 7 and 10 were recorded
in CD3OD. Other signals: OCH2CH CH2 d 5.13–5.41; OCH2CH CH2 d 5.78–6.05 and 5.70–
5.87; OCH2CH CH2 d 4.22–4.45 and 4.00–4.15; PhCH2 d 4.50–4.80; C6H5CH2 d 6,90–8,20;
C6H5CO d 7.45–7.93.
sulfated to give 12. Catalytic hydrogenolysis and subsequent saponification of
compounds 7, 10 and 12 gave target disaccharides 1 and 2 and fucoside 4 which
were purified by gel filtration on a Sephadex G-10 column. The presence of the sulfate
group at C-4 in compounds 1, 2 and 4 was confirmed by downfield chemical shifts
1
(Table 2) of the H-4 signals in the corresponding H NMR spectra.

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316
GERBST ET AL.
Table 2. 1H NMR Shiftsa and Coupling Constants (Hz) for Oligosaccharides 1–5
No.
Residue
H-1 H-2 H-3 H-4 H-5 H-6 J1,2 J2,3 J3,4 J4,5 J5,6
1
a-L-Fuc-OPr
4.99 3.98 4.07 4.81 4.23 1.34 3.8 10.3 3.4 < 2 6.5
a-L-Fuc(1 ! 3) 5.17 3.80 3.96 3.87 4.37 1.26 4.0 10.3 3.5 < 2 6.7
a-L-Fuc-OPr 4.92 3.92 4.03 4.76 4.18 1.28 3.9 10.4 2.8 < 2 6.5
a-L-Fuc(1 ! 3) 5.13 3.78 4.00 4.60 4.44 1.26 3.9 10.4 2.9 < 2 6.5
3[6] a-L-Fuc-OPr 4.91 3.91 3.91 4.02 4.08 1.27 < 3 ndb ndb ndb 6.7
2
a-L-Fuc(1 ! 3) 5.07 3.81 3.95 3.82 4.28 1.21 3.9 10.4 3.4 < 2 6.7
4
a-L-Fuc-OPr
4.90 3.81 3.97 4.60 4.21 1.26 3.9 10.3 3.4 < 2 6.7
4.88 3.78 3.86 3.80 4.10 1.22 3.8 10.2 3.5 < 2 6.5
5[6] a-L-Fuc-OPr
aIn ppm, recorded at 40°C in D2O with 0.05% acetone as internal standard. Signals of propyl
aglycon: OCH2CH2CH3 d 0.92; OCH2CH2CH3 d 1.62–1.64; OCH2CH2CH3 d 3.49–3.84.
bCoupling constants not determined due to the overlapping of multiplets.
1
NMR analysis of compounds 1–5. Tables 2 and 3 show the H NMR and 13C
NMR chemical shifts for compounds 1, 2 and 4 and parent non-sulfated 3 and 5.
1
Assignment of the H NMR spectra (Table 2) of these compounds was made using a
combination of 1H–1H COSY and 2D TOCSY experiments. 13C NMR spectral
1
assignments (Table 3) were made using 2D H–13C HMQC correlation spectroscopy.
To evaluate the influence of sulfate on 13C NMR spectra of compounds 1, 2 and
4 we calculated the sulfation effects (Dd), which are the differences in chemical shifts
of the respective signals in spectra of sulfated and non-sulfated compounds. As was
expected, we observed substantial sulfation effects in 13C NMR spectra (Table 4) on
C-4 and C-3 atoms of the glycosylated unit and C-1’ atom of the glycosylating unit.
The values of DdC-4 for saccharides 1 and 2 are ca. 2 ppm larger than that for
monosaccharide 4. Sulfation effects in 4 are similar to those observed in the terminal
residue of disaccharide 2 except for DdC-1’ value, which both in 1 and 2 are
considerably positive. The sulfation effect on C-3 is negative in the monosaccharide
which is obviously due to the so called b-effect, i.e., upfield shift of the b-carbon atom
upon the introduction of an electronegative subtituent.[7] It is noteworthy that it
Table 3. 13C NMR Shiftsa for Oligosaccharides 1–5
Compound
Residue
C-1
C-2
C-3
C-4
C-5
C-6
1
a-L-FucOPr
99.6
99.1
99.5
98.7
99.5
96.4
99.1
99.5
68.0
69.7
67.8
69.7
67.6
69.2
69.3
69.2
76.8
71.2
76.6
70.2
75.8
70.7
69.9
70.9
80.1
73.2
80.0
81.9
69.3
73.1
81.5
73.1
67.2
68.3
67.1
67.6
67.5
68.1
66.8
67.7
16.6
17.1
17.0
17.0
16.5
16.5
16.6
16.5
a-L-Fuc(1 ! 3)
2
a-L-FucOPr
a-L-Fuc(1 ! 3)
a-L-FucOPr
3[6]
a-L-Fuc(1 ! 3)
4
5[6]
a-L-FucOPr
a-L-FucOPr
aIn ppm, recorded at 40°C in D2O with 0.05% acetone as internal standard. Signals of propyl
aglycon: OCH2CH2CH3 d 11.1; OCH2CH2CH3 d 23.2–23.3; OCH2CH2CH3 d 71.3.

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FUCOIDAN FRAGMENTS. IV
317
Table 4. Sulfation Effects (Dd) in 13C NMR Spectra of Compounds 1, 2 and 4
Compound
Residue
DdC-1
DdC-2
DdC-3
DdC-4
DdC-5
DdC-6
1
a-L-FucOPr
0.1
2.7
0.4
0.5
0.2
0.5
0.1
1.0
0.5
10.8
0.1
À 0.3
0.2
0.1
0.6
0.5
0.5
0.1
a-L-Fuc(1 ! 3)
2
4
a-L-FucOPr
0.0
2.3
0.8
10.7
8.8
À 0.4
À 0.5
À 0.9
a-L-Fuc(1 ! 3)
a-L-FucOPr
À 0.5
À 1.0
À 0.4
8.4
becomes slightly positive in disaccharides 1 and 2, thus revealing that some other
influence overcomes the b-effect. In order to explain the observed DdC we investigated
conformations of these compounds.
Conformational analysis of compounds 1–3. The common approach to
investigate the conformational properties of oligosaccharides consists of building up
a conformational map, i.e., plotting either potential energy or relative population based
on it against j and c angles around the glycoside linkage. Potential energy can be
estimated according to one of the well established protocols using various molecular
mechanics force fields.[8] Early investigators used algorithms of partial geometry
optimisation leaving the carbohydrate ring rigid.[9] However, in recent years con-
formational analysis with full geometry optimisation has become the usual
procedure.[10] The reliability of the obtained results is tested by comparison of an
experimental physical property with the one calculated from the conformational map.
Conformational modeling with the use of an MM3 force field[11] was shown to give
better coincidence between calculated and experimental characteristics, particularly
NOE values. The MM3 force field was also successfully used in conformational
analysis of sulfated galactobiosides[12] and therefore was employed in conformational
study of fucosides 1–3.
To confirm the adequacy of the molecular modeling results we used the
comparative analysis of theoretical and experimental NOE values for compounds 1–3
measured after the pre-irradiation of their H-1’ proton (Figure 1). Depending on the
Pre-irradiation
Interunit NOEs
H-1
Pr
O
H-3
OH
O
H-5
HO
OR2
O
Me
O
OH
H-4
Me
1
OR
Figure 1. A schematic representation of the inter-unit NOE interactions observed for compound
1–3 (R1, R2 H or SO3 À ).

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318
GERBST ET AL.
Table 5. Relative (%) Experimental and Calculated NOE Values for Trisaccharides 1–3
(Saturation of H-1’ of the Fucose Unit at ‘‘Non-reducing’’ End; Irradiation Time of 7.2 s)
Compound
Proton
Experimental NOE
Calculated NOE
1
H-2
H-3
0
100
À 0.3
100
49
H-4
H-5
41.5
À 13.3
0
À 11.9
À 0.3
100
2
3
H-2
H-3
100
H-4
H-5
49.5
À 10.2
100a
88.8
À 12.9
41.5
À 12.0
100a
85
H-2 + H-3
H-4
H-5
À 14.1
aGiven for the sum of H-2 and H-3 due to the overlap of their signals in H NMR spectrum of 3
1
(Table 2).
NMR technique used the NOE values can be measured in either steady-state or
transient mode.[13] The values of the steady-state NOE are a function of all the inter-
proton distances, and thus are very conformation dependent. The sensitivity of steady-
state technique is higher.[13] The disadvantage of this method is that a resonance line
should be irradiated selectively and during the longer time needed for the system to
reach the new equilibrium state. Thus, the steady-state technique can be useful only for
1
molecules with well resolved resonance lines in their H NMR spectrum. As the H-1’
signal in H NMR spectra of compounds 1–3 has separate location from other lines, we
employed this technique to obtain steady-state NOE values (Table 5).
1
To calculate the theoretical steady-state NOE value for a single conformation we
employed the iterative Noggle and Shirmer[14] Eq. 1. The obtained NOE values for
conformers were further averaged over the multiple minima on the conformational map
according to a Boltzman distribution. In every minimum the computed NOE was
multiplied by Boltzman factor e À E/RT, and the sum of all the obtained values was then
divided by the sum of their Boltzman factors.
X
1
2
1
2
fik
¼
Á ðrikÞÀ6
À
Á
ðrkdÞÀ6 Á fdi
ð1Þ
d
The conformational maps for disaccharides 1–3 were built via a grid search
procedure using 10° steps both in j and c directions. These compounds were
characterized by the presence of two main minima: broad minimum A located around
point j =40°, c =40°, and narrow minimum B located near the point j =20°,
c = À 50° (Figure 2). Minimum A corresponds to the conformation with spatial
proximity of the anomeric proton H-1’ of the glycosylating residue and proton H-4 of
the glycosylated fucose unit (Figure 3A). In conformation B, H-1’ is close to H-3
(Figure 3B). The conformational map of compound 2 had also a minimum C (j =40°,
c = À 60°).

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FUCOIDAN FRAGMENTS. IV
319
180
180
90
0
(ϕ=40°,
ψ=40°)
A
90
( ϕ=40°,
ψ =40°)
A
0
ψ
ψ
B
B
( ϕ=30°,
ψ=-50°)
C
-90
-90
( ϕ=30°,
ψ=-40° )
(ϕ=40°,
ψ=-60°)
-180
-180
-180
0
-90
0
90
180
-180
-90
90
180
ϕ
ϕ
1
2
180
(ϕ=40°,
ψ=30°)
A
90
0
ψ
B
-90
(ϕ=20°,
ψ=-50°)
-180
-180
0
-90
90
180
ϕ
3
Figure 2. Conformational maps of compounds 1–3. Population levels are presented every 10%.
In the case of non-sulfated compound 3, the population ratio for local minima A
and B is 1.6:1 as calculated according to Boltzman distribution. For 4-O-sulfated
compound 1 the ratio of population of minimum A to the sum of populations of
minima B and C is 1:1.4, showing the tendency for H-1’ to stay in proximity with H-3
rather than H-4. This can be explained by the presence of the sulfate moiety at C-4.
Figure 3. Inter-proton interactions in conformations A and B in compounds 1–3 (R H or
SO3 À ).

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320
GERBST ET AL.
NOE values for compounds 1 and 3 were calculated based on the above statistical
ratios. These values are consistent with the experimental data (Table 5).
The conformational map of disaccharide 1 (Figure 2) contained a number of
almost equally populated conformations lying between conformations A and B.
Therefore we could not correctly select any of them to estimate an NOE. To overcome
this problem we varied energy limits (percent from the global minimum energy) within
which lay the points to include in the calculation of an NOE. It was thus empirically
determined that satisfactory coincidence with the experimental data can be obtained by
setting this limit to 10% of the energy of the minimum. It was further found that NOEs
for compounds 2 and 3 computed in the same manner with the same limit also
coincided with the experimental values (Table 5).
According to Table 5 both experimental NOE and calculated relative NOE on
H-4 in compounds 1 and 2 are nearly twofold lower than in compound 3. This leads
to the suggestion that mono-4-O-sulfation of the disaccharide increases the weight of
the conformation in which protons H-1’ and H-3 are spatially close. This result is
similar to that obtained by Stortz and Cerezo[12] for 4-O-sulfated galactobioside.
Experimental NOE values for compounds 1 and 2 did not reflect considerable
differences in conformations, but theoretical calculations showed that compound 1 was
probably more flexible than 2.
Our calculations did not indicate any substantial change in the conformation of
sugar rings. It is also in accordance with the fact that coupling constants in the studied
disaccharides do not change significantly (Table 2).
Using these results we tried to explain the difference in sulfation effects (DdC,
Table 4) for glycosylating and glycosylated units in 13C NMR spectra of disaccharides 1
and 2. We regarded sulfate groups in disaccharides as anions and assumed their free
rotation. These assumptions allowed us to neglect possible anisotropy of the sulfates. Both
disaccharides were characterized by the considerable weight of the conformation with
close H-1’ and H-3 protons (Figure 3B, Table 6). In terms of the Grant and Cheney
conception[15] the interaction between these protons causes a downfield shift of C-3 and
C-1’ signals. The significance of this effect is demonstrated by the fact that the chemical
shift of C-3 becomes positive in the disaccharides 1 and 2 while the b-effect makes it
negative in monosaccharide 4 and in the glycosylating residue of disulfated disaccharide
2. The downfield shift of C-1’ of over 2 ppm is also observed for disaccharides 1 and 2.
Interaction between H-1’ and H-4 causes a strong upfield shift of C-4 in confor-
mations of type A (Figure 3A), and a slight downfield shift of the same signal in con-
formation B (Figure 3B). Due to the increase of the statistical weight of the conformation
˚
Table 6. H-1’–H-3 and H-1’–H-4 Distances (A) in Conformations A and B for Trisaccharides
1–3
H-1’–H-3
H-1’–H-4
Compound
A
B
A
B
1
2
3
2.56
2.55
2.56
2.27
2.26
2.21
2.21
2.21
2.22
3.92
3.43
3.72

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FUCOIDAN FRAGMENTS. IV
321
of type B in compounds 1 and 2, the sulfation effect on atom C-4 becomes about 2 ppm
larger than in monosaccharide 4 or in the glycosylating residue in compound 2.
CONCLUSIONS
Several 4-O-sulfated (1 !3)-linked fucobiosides were synthesized and investi-
1
gated by H and 13C NMR spectroscopy. Conformational analysis of these compounds
and of parent non-sulfated fucobioside using combined molecular mechanics and
steady-state NOE experiments showed the interaction between protons located around
inter-residue linkage (H-1’, H-3 and H-4). The correlation between these interactions
and 13C NMR chemical shift values has been investigated. This information will be of
use in the analysis of fucoidan NMR spectra.
EXPERIMENTAL
General methods. TLC was performed on Silica Gel 60 F254 (Merck) with
EtOAc–toluene (A, 1:2), CH2Cl2–MeOH (B, 5:1, C, 10:1); spots were detected by
charring with H3PO4. Column chromatography of non-sulfated compounds was per-
formed on Silica Gel 0.063–0.2 mm (Fluka) by gradient elution with toluene–EtOAc.
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 MeOH 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.[16]
NMR spectra for substituted compounds 7–10 and 12 were recorded on Bruker
spectrometers WM-250 and AMX-300 at 303 K. 1H and 13C NMR spectra for
oligosaccharides 1, 2 and 4 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 NOE were measured by differential spectro-
scopy on a Bruker DRX-500 instrument in D2O (99.98% D, Merck) solutions at 303 K.
Computations were performed using the TINKER program with the implemented
MM3 force field. The dielectric constant e was set to 81. All sulfate groups were treated as
anions. Parameters for their modeling were based on published work[17] with slight
modifications. No solvent molecules were considered in the calculation. The starting
structures were produced by geometry optimization with MM + (HyperChem[18]). For
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.
Sodium Allyl 3-O-(3,4-Di-O-benzoyl-2-O-benzyl-a-L-fucopyranosyl)-2-O-ben-
zyl-4-O-sulfonato-a-L-fucopyranoside (7). A solution of 6 (100 mg, 0.14 mmol) in
DMF (1 mL) was treated with SO3ÁPy complex (176 mg, 0.9 mmol) for 1h at rt, then
quenched with NaHCO3 (100 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-

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GERBST ET AL.
exchange resin for 20 min, the resin was filtered off, and the filtrate was concentrated.
Gel chromatography on a Sephadex LH-20 column in MeOH afforded amorphous 7 (95
1
mg, 85%): [a]D À 220° (c 2, MeOH); RF 0.36 (solvent C). The H NMR data for 7 are
presented in Table 1.
Anal. Calcd for C43H45NaO14S (840.87): C, 61.42%; H, 5.39%. Found: C,
61.33%; H, 5.36%.
Allyl 3-O-(2-O-Benzyl-a-L-fucopyranosyl)-2-O-benzyl-a-L-fucopyranoside (8).
A solution of 7 (248 mg, 0.33 mmol) in 2 mL of 0.1M MeONa in MeOH 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 triol 8 (151 mg, 85%): [a]D À 142° (c 2,
1
EtOAc); RF 0.14 (solvent A). The H NMR data for 8 are presented in Table 1.
Anal. Calcd for C29H38O9 (530.62): C, 65.66%; H, 7.17%. Found: C, 65.65%; H,
6.95%.
Allyl 3-O-(3-O-Benzoyl-2-O-benzyl-a-L-fucopyranosyl)-2-O-benzyl-a-L-fuco-
pyranoside (9). A mixture of triol 8 (64 mg, 0.12 mmol), Bu2SnO (33 mg, 0.13
mmol) and toluene (1.7 mL) was refluxed until complete dissolution and then
concentrated to the volume of 0.7 mL. BzCl (0.02 mL, 0.17 mmol) was added, the
solution was kept at rt for 3 h and concentrated in vacuo. Column chromatography of
the residue gave amorphous 9 (59 mg, 77%): [a]D À 166° (c 2, EtOAc); RF 0.39
1
(solvent A). The H NMR data for 9 are presented in Table 1.
Anal. Calcd for C36H42O10 (634.72): C, 68.12%; H, 6.67%. Found: C, 68.20%; H,
6.69%.
Sodium Allyl 3-O-(Sodium 4-O-Sulfonato-3-O-benzoyl-2-O-benzyl-a-L-fuco-
pyranosyl)-2-O-benzyl-4-O-sulfonato-a-L-fucopyranoside (10). Sulfation of 9 (43
mg, 0.07 mmol) by SO3ÁPy complex (0.7 mmol) as described for 7, gave 10 (58 mg,
1
95%): [a]D À 151° (c 2.5, MeOH); RF 0.29 (solvent B). The H NMR data for 10 are
presented in Table 1.
Anal. Calcd for C36H40Na2O16S2 (838.80): C, 51.55%; H, 4.81%. Found: C,
51.63%; H, 4.89%.
Sodium Allyl 2,3-Di-O-benzyl-4-O-sulfonato-a-L-fucopyranoside (12). Sulfa-
tion of 11[6] (35 mg, 0.09 mmol) by SO3ÁPy complex (72 mg, 0.45 mmol) as described
1
for 7, gave 12 (41 mg, 93%). The H NMR data for 12 are presented in Table 1.
Anal. Calcd for C23H27NaO8S (486.51): C, 56.78%; H, 5.59%. Found: C,
56.62%; H, 5.66%.
Sodium Propyl 3-O-(a-L-Fucopyranosyl)-4-O-sulfonato-a-L-fucopyranoside
(1). A solution of 7 (46.1 mg, 0.06 mmol) in MeOH (3 mL) was subjected to
catalytic hydrogenolysis with 10% Pd–C at 20°C and atm. pressure for 2 h. The

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FUCOIDAN FRAGMENTS. IV
323
mixture was filtered through Celite, and the solvent was evaporated in vacuo. The
residue was dissolved in water (1 mL), 0.1M NaOH (0.5 mL) was added. The mixture
was kept for 1 h at rt and then was subjected to gel filtration on a Sephadex G-10
column in water to give amorphous 1 (20 mg, 75%): [a]D À 103° (c 0.675, H2O). The
1H and 13C NMR data for 1 are presented in Tables 2 and 3.
Anal. Calcd for C15H27NaO12S (454.42): C, 39.65%; H, 5.99%. Found: C,
39.53%; H, 5.88%.
Sodium Propyl 3-O-(Sodium-4-O-sulfonato-a-L-fucopyranosyl)-4-O-sulfonato-
a-L-fucopyranoside (2). Debenzylation and debenzoylation of 10 (56 mg, 0.067
mmol), as described for 1, yielded amorphous 2 (28 mg, 75%), [a]D À 185° (c 1, H2O).
1
The H and 13C NMR data for 2 are presented in Tables 2 and 3.
Anal. Calcd for C15H26Na2O15S2 (556.46): C, 32.38%; H, 4.71%. Found: C,
32.43%; H, 4.89%.
Sodium Propyl 4-O-Sulfonato-a-L-fucopyranoside (4). Debenzylation of 12
(40 mg, 0.08 mmol), as described for 1, yielded amorphous 4 (18 mg, 75%):
1
[a]D À 74° (c 0.725, H2O). The H and 13C NMR data for 4 are presented in Tables 2
and 3.
Anal. Calcd for C9H17NaO8S (308.28): C, 35.07%; H, 5.56%. Found: C, 35.13%;
H, 5.76%.
ACKNOWLEDGMENTS
This work was supported by GlycoSense AG (Jena, Germany), the Russian
Foundation for Basic Research (grant 01-03-33059a) and Russian Academy of Sciences
(grant 129-6-1999 for young researchers). 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.
REFERENCES
1. Gerbst, A.G.; Ustuzhanina, N.E.; Grachev, A.A.; Khatuntseva, E.A.; Tsvetkov, D.E.;
Whitfield, D.M.; Berces, A.; Nifantiev, N.E. Synthesis, NMR and conformational
studies of fucoidan fragments. III. Effect of benzoyl group at O-3 on stereoselectivity
of glycosylation by 3-O- and 3,4-di-O-benzoylated 2-O-benzyl fucosyl bromides. J.
Carbohydr. Chem. 2001, 20 (9), 821–831.
2. Painter, T.J. Algal Polysaccharides. In The Polysaccharides; Aspinall, G.O., Ed.;
Academic Press: New York, 1983; Vol. 2, 195–285.
3. Nagumo, T.; Nishino, T. Fucan Sulfates and Their Anticoagulant Activities. In
Polysaccharides in Medicinal Applications; Dumitriu, S., Ed.; Marcel Dekker: New
York, 1996; 545–574.
4. Schaeffer, D.J.; Krylov, V.S. Anti-HIV activity of extracts and compounds from
algae and cyanobacteria. Ecotoxicol. Environ. Saf. 2000, 45 (3), 208–227.

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©2002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.
324
GERBST ET AL.
5. Preobrazhenskaya, M.E.; Berman, A.E.; Mikhailov, V.I.; Ushakova, N.A.; Mazurov,
A.V.; Semenov, A.V.; Usov, A.I.; Nifant’ev, N.E.; Bovin, N.V. Fucoidan inhibits
leucocyte recruitment in a model peritoneal inflammation in rat and blocks in-
teraction of P-selectin with its carbohydrate ligand. Biochem. Mol. Biol. Int. 1997,
43 (2), 443–451.
6. Khatuntseva, E.A.; Ustuzhanina, N.E.; Zatonskii, G.V.; Shashkov, A.S.; Usov, A.I.;
Nifant’ev, N.E. Synthesis, NMR and conformational studies of fucoidan fragments
1: Desulfated 2, 3- and 3, 4-branched trisaccharide fragments and constituting
disaccharides. J. Carbohydr. Chem. 2000, 19 (9), 1151–1173.
7. Bock, K.; Pedersen, C. Carbon-13 Nuclear Magnetic Resonance Spectroscopy of
Monosaccharides. In Advances in Carbohydrate Chemistry and Biochemistry;
Horton, D., Tipson, R.S., Eds.; Academic Press: New York, 1983; Vol. 41, 27–66.
8. Perez, S.; Imberty, A.; Engelsen, S.B.; Gruza, J.; Mazeau, K.; Barbero, J.J.; Poveda,
A.; Espinosa, J.F.; Van Eyck, B.; Johnson, G.; French, A.; Kouwijzer, M.L.;
Grootenhuis, P.; Bernardi, A.; Raimondi, L.; Senderowitz, H.; Durier, V.; Vergoten,
G.; Rasmussen, K. A comparison and chemometrics analysis of several molecular
mechanics force fields and parameters sets for carbohydrates. Carbohydr. Res.
1998, 314, 141–155.
9. Zsiska, M.; Meyer, B. Influence of sulfate and carboxylate groups on the con-
formation of chondroitin sulfate related disaccharides. Carbohydr. Res. 1993, 243,
225–258.
10. Stortz, C.A. Disaccharide conformational maps: How adiabatic is an adiabatic map?
Carbohydr. Res. 1999, 322 (1–2), 77–86.
11. Allinger, N.L.; Yuh, Y.H.; Lii, J.-H. Molecular mechanics. The MM3 force field for
hydrocarbons. J. Am. Chem. Soc. 1989, 111 (23), 8551–8566.
12. Stortz, C.A.; Cerezo, A.S. Conformational analysis of sulfated a-(1 ! 3)-linked
D-galactobioses using the MM3 force-field. J. Carbohydr. Chem. 1998, 17 (9),
1405–1420.
13. Claridge, T.D.W. High-Resolution NMR Techniques in Organic Chemistry; Per-
amon: Oxford, 2000; 384 pp.
14. Shirmer, R.E.; Noggle, J.H.; Davis, J.P.; Hart, P.A. Determination of molecular
geometry by quantative application of the nuclear overhauser effect. J. Am. Chem.
Soc. 1970, 92 (11), 3266–3273.
15. Grant, D.M.; Cheney, B.V. Carbon-13 magnetic resonance. VII. Steric perturbation
of the carbon-13 chemical shift. J. Am. Chem. Soc. 1967, 89 (21), 5315–5319.
16. Sherman, A.A.; Yudina, O.N.; Shashkov, A.S.; Menshov, V.M.; Nifantiev, N.E.
Synthesis of Neu5Ac-and Neu5Gc-a-(2! 6’)-lactosamine 3-aminopropyl glyco-
sides. Carbohydr. Res. 2001, 330 (4), 445–458.
17. Lamba, D.; Glover, S.; Mackie, W.; Rahid, A.; Scheldrick, B.; Perez, S. Insights
into stereochemical features of sulfated carbohydrates. Glycobiology 1994, 4, 151–
163.
18. HyperChemTM, Hypercube, Inc.: 1115 NW 4th Street, Gainesville, Florida 32601,
USA.
Received May 29, 2001
Accepted February 18, 2002