Synthesis of the oligosaccharides related to branching sites of fucosylated chondroitin sulfates from sea cucumbers

Article abstract of DOI:10.3390/md13020770

Natural anionic polysaccharides fucosylated chondroitin sulfates (FCS) from sea cucumbers attract great attention nowadays due to their ability to influence various biological processes, such as blood coagulation, thrombosis, angiogenesis, inflammation, b

Full text of DOI:10.3390/md13020770

Mar. Drugs 2015, 13, 770-787; doi:10.3390/md13020770
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Synthesis of the Oligosaccharides Related to Branching Sites of
Fucosylated Chondroitin Sulfates from Sea Cucumbers
Nadezhda E. Ustyuzhanina 1,*, Polina A. Fomitskaya 2, Alexey G. Gerbst 1,
Andrey S. Dmitrenok 1 and Nikolay E. Nifantiev 1,*
1
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47,
119991 Moscow B-334, Russia; E-Mails: alger@ioc.ac.ru (A.G.G.); dmt@ioc.ac.ru (A.S.D.)
2
M.V. Lomonosov Moscow State University, Leninskie gory, 119991 Moscow, Russia;
E-Mail: fomitskaya.p@gmail.com
* Authors to whom correspondence should be addressed; E-Mails: nen@ioc.ac.ru (N.E.N.);
ustnad@gmail.com (N.E.U.); Tel./Fax: +7-499-137-8784.
Academic Editor: Yoshihide Usami
Received: 1 December 2014 / Accepted: 22 January 2015 / Published: 2 February 2015
Abstract: Natural anionic polysaccharides fucosylated chondroitin sulfates (FCS) from sea
cucumbers attract great attention nowadays due to their ability to influence various
biological processes, such as blood coagulation, thrombosis, angiogenesis, inflammation,
bacterial and viral adhesion. To determine pharmacophore fragments in FCS we have
started systematic synthesis of oligosaccharides with well-defined structure related to
various fragments of these polysaccharides. In this communication, the synthesis of
non-sulfated and selectively O-sulfated di- and trisaccharides structurally related to
branching sites of FCS is described. The target compounds are built up of propyl
β-D-glucuronic acid residue bearing at O-3 α-L-fucosyl or α-L-fucosyl-(1→3)-α-L-fucosyl
substituents. O-Sulfation pattern in the fucose units of the synthetic targets was selected
according to the known to date holothurian FCS structures. Stereospecific α-glycoside
bond formation was achieved using 2-O-benzyl-3,4-di-O-chloroacetyl-α-L-fucosyl
trichloroacetimidate as a donor. Stereochemical outcome of the glycosylation was
explained by the remote participation of the chloroacetyl groups with the formation of the
stabilized glycosyl cations, which could be attacked by the glycosyl acceptor only from the
α-side. The experimental results were in good agreement with the SCF/MP2 calculated
energies of such participation. The synthesized oligosaccharides are regarded as model
compounds for the determination of a structure-activity relationship in FCS.

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Keywords: fucosylated chondroitin sulfate; oligosaccharides; synthesis; glycosylation;
stereoselectivity; remote participation; sulfation
1. Introduction
Different types of natural anionic polysaccharides attract increasing attention nowadays due to their
biological activity of different types that makes possible their use as pharmacological regulators of
several diseases related to biological processes, such as blood coagulation, thrombosis, angiogenesis,
inflammation, bacterial and viral adhesion and some others. The most famous biopolymer of the
discussed type is glycosaminoglycan heparin, which was found to be a leader on anticoagulant and
antithrombotic market for decades [1,2]. Besides, this biopolymer was shown to attenuate metastasis
and inflammation in a way of inhibition of P- and L-selectins binding to their cellular ligands [3,4].
Due to several side effects of heparin treatment, new biologically active compounds are intensively
searched for and are under development as potential alternative drugs. Among them are fucosylated
chondroitin sulfates (FCS) isolated from different sea cucumber species. These polysaccharides
demonstrated a wide spectrum of biological activities including anticoagulant, antithrombotic, antitumor,
immunostimulatory, anti-hyperglycemia, antiangiogenic, antibacterial, antiviral and some others [5–10].
A number of studied to date FCSs are glycosaminoglycans built up of alternating
→4)-linked β-D-glucuronic acid and →3)-linked N-acetyl β-D-galactosamine residues in a backbone.
Unlike mammalian chondroitin sulfates, these polysaccharides bear side chains containing O-sulfated
fucosyl residues attached to O-3 of glucuronic acid units (Figure 1) [5,11]. It should be noted that the
presence of side chains is essential for biological properties of FCS [12]. The structures of
fucosyl-branches vary accordingly to the type of sea cucumber species and determine in many respects
the level and the character of its biological activity [5,11]. Moreover, the degree of O-sulfation and
fucose content in FCS also depend on the geographic range and season of harvesting [13,14]. The known
structures of non-sulfated and selectively O-sulfated α-L-fucosyl and α-L-fucosyl-(1→3)-α-L-fucosyl
fragments which were discovered as the side chains in FCS from sea cucumbers [5,11,15] are shown
in Figure 1.
Figure 1. Branched fragments of fucosylated chondroitin sulfates from sea cucumbers.

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In 2013 Tamura et al. reported synthesis of the trisaccharide β-D-GalNAc(4,6-diS)(1→4)
[α-L-Fuc(2,4-diS)(1→3)]-β-D-GlcA related to branching site of FCS [16]. But to determine
pharmacophore fragments of FCS, a series of compounds with well-defined structure are required.
We have started the systematic synthesis of oligosaccharides related to various fragments of these
types of natural polysaccharides. In this communication, the first synthesis of non-sulfated and
selectively O-sulfated di- and trisaccharides 1–8 related to the known structures of branching sites of
FCS is described (Figure 2). The target compounds are built up of the propyl β-D-glucuronic acid
residue bearing at O-3 the α-L-fucosyl or α-L-fucosyl-(1→3)-α-L-fucosyl substituents. The sulfation
pattern of the fucosyl units was selected according to the data of holothurian FCS structures described
in literature (see above for citations).
Figure 2. The target compounds 1–8 related to branching sites of fucosylated chondroitin
sulfates (FCS).
2. Results and Discussion
Two monosaccharides 9 and 10 (Figure 3) bearing free hydroxyl group at C-3 were studied as
glycosyl acceptors for assembling of target compounds 1–8. These compounds were prepared from
2,4-di-O-acylated derivatives of 3,6-lactone of allyl glucuronide as described previously [17].
Since the target compounds contain α-L-fucosyl residues, an efficient method for α-L-fucosylation
should be applied in their synthesis. Earlier we have shown that the presence of acyl groups at O-3 and
O-4 of the fucosyl donor was essential for α-glycoside formation [18–20]. Thus, the use of
2-O-benzyl-3-O-acetyl-4-O-benzoyl-L-fucosyl trichloroacetimidate 13 gave the only α-isomeric
glycosylation product [20]. The stereochemical result of the reaction was explained by the remote
participation of acyl groups with the formation of the stabilized glycosyl cation (similar to cations II
and III in Figure 4 below), which could be attacked by an acceptor only from the α-side. This
approach was quite different then what was used by Tamura et al., where fucosyl fluoride with
non-participating allyl and benzyl groups was applied [16].
Figure 3. Monosaccharide building blocks applied for the synthesis of FCS related oligosaccharides.

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Figure 4. Expected non-stabilized (I) and stabilized (II), (III) fucosyl cations in
glycosylation reactions of fucosyl donor 12. 3D structures of cations were obtained by
SCF/MP2 method.
An additional requirement to the structure of the fucosyl donor is the presence of selectively
removable protective groups at O-3 and O-4, which are necessary for the preparation of the partially
O-sulfated oligosaccharides 3–5 and 8. It is known that chloroacetyl group (CA) could be selectively
removed in the presence of benzoyl and acetyl groups. Thus, we investigated first whether the CA
group could stabilize glycosyl cation in the same way as had been observed for other acyls and could
thus direct the glycosylation towards the formation of the α-isomer. Following our synthetic strategy,
2-O-benzyl-3,4-di-O-chloroacetyl trichloroacetimidate 12 was regarded as a donor of choice (Figure 3).
We conducted theoretical calculations to investigate a possibility of the discussed stereocontrolling
effect of remote CA groups. At the primary level, as very coarse estimate, we performed molecular
mechanics calculations following the technique described in [21] using the evaluation version of
HyperChem 1.0 for Linux (HyperChem is the trademark of Hypercube, Inc. (Gainesville, FL, USA) [22]).
The MM+ force field (HyperChem version of MM2 [23]) was employed with the electrostatic term
using charge-charge interactions. Atomic charges were obtained from single point calculations using
semiempirical PM3 approximation [24,25]. Geometry optimizations of a non-stabilized and stabilized
forms of the oxocarbenium ion (Figure 4) were conducted and the corresponding “stabilization
energies” were calculated as energy differences in pairs E(I)—E(II) and E(I)—E(III). At this very low
level of theory, it was found that the CA group (especially that at O-3) might have the ability to
stabilize efficiently the oxocarbenium ion. Stabilization energies were computed as 11.1 and 6.2
kcal/mol for the CA groups at O-3 and O-4, respectively. Calculated values were only slightly lower
than those found for the stabilizing groups in donor 13 (Table 1).
Then the study was carried out at the ab initio level with the account for electron correlation.
Geometries of all the cations were optimized using the SCF/MP2 approximation with the 6-31+G*

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basis set as provided along with the NWChem 6.3 software package [26]. This calculation resulted in
an increase in the difference in stabilization energies between 12 and 13 (Table 1), but the stabilization
energies for the CA groups at O-3 and O-4 of 12 still remained rather high, suggesting their ability to
interact with the cationic center.
Table 1. Stabilization energy of the fucosyl cations.
Stabilization Energy
Calculated at MM+ Level,
Kcal/Mole
Stabilization
The Stabilizing
Group
Donor
Energy Calculated at
SCF/MP2 Level, Kcal/Mole
3-O-Ac
4-O-Bz
11.6
9.7
15.5
15.5
10.4
8.6
3-O-C(O)CH2Cl
4-O-C(O)CH2Cl
11.1
6.2
Compound 12 was prepared from diol 11 [27] by per-O-chloroacetylation followed by deallylation
and subsequent trichloroacetimidation in a yield of 78% over three steps. The efficiency of this donor
was then studied in direct experiments.
An attempt to involve the 2,4-di-O-benzoylated glucuronyl acceptor 9 into glycosylation with donor
12 in the presence of TMSOTf failed. The main product of the reaction was the respective
N-glycosylated trichloroacetamide, while compound 9 was recovered unchanged. On the contrary, the
2,4-di-O-acetylated glucuronyl acceptor 10 under the same conditions reacted rapidly with the
formation of the desired α-linked disaccharide 14 exclusively in a yield of 92% (Scheme 1). The
difference in the reactivity of the acceptors 9 and 10 could be explained by the different steric
availability of the hydroxyl group at C-3. Bulk benzoyl groups at O-2 and O-4 in 9 hindered the access
of glycosyl donor to hydroxyl group. It should be noted that the stereochemical result of the
glycosylation was still in good agreement with the theoretical prediction.
Selective removal of CA-groups in 14 using thiourea in the presence of collidine gave diol 15 in a
yield of 94% (Scheme 1). This compound was used as the precursor of all target disaccharides 1–5. Thus,
hydrogenolysis of 15 followed by saponification gave the non-sulfated propyl glycoside 1.
Per-O-sulfation of 15 with subsequent hydrogenolysis and saponification led to the 3′,4′-di-O-sulfated
disaccharide 3. Acetylation of 15 followed by hydrogenolysis, O-sulfation and saponification gave the
2′-O-sulfated disaccharide 2.
To prepare compounds 4 and 5, selective 3′-O-benzoylation of diol 15 via a stannylidene
intermediate was performed with the formation of disaccharide 16 in a yield of 80%. Further
O-sulfation of 16 and deprotection of the sulfated derivative gave target disaccharide 4.
Hydrogenolysis of 15 followed by O-sulfation and saponification gave the 2′,4′-di-O-sulfated
disaccharide 5.

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Scheme 1. Synthesis of the disaccharides 1–5. Reagents and conditions: (i) TMSOTf,
CH2Cl2, −30 °C, 92%; (ii) (NH2)2CS, collidine, MeOH, 94%; (iii) Bu2SnO, BzCl (1.1 eq),
80%; (iv) (a) H2, Pd(OH)2/C; (b) LiOH, H2O, THF; (c) NaOH, H2O, 79%; (v) (a) AcCl,
Py; (b) H2, Pd(OH)2/C; (c) SO3·Py, DMF; (d) Amberlite IR-120 (Na+), NaHCO3;
(e) LiOH, H2O, THF; (f) NaOH, H2O, 64%; (vi) (a) SO3·Py, DMF; (b) Amberlite IR-120
(Na+), NaHCO3; (c) H2, Pd(OH)2/C; (d) LiOH, H2O, THF; (e) NaOH, H2O, 62%;
(vii) (a) H2, Pd(OH)2/C; (b) SO3·Py, DMF; (c) Amberlite IR-120 (Na+); NaHCO3;
(d) LiOH, H2O, THF; (e) NaOH, H2O, 52%.
The synthesis of trisaccharides 6–8 was performed using [2 + 1] strategy for the assembling of the
carbohydrate chain. Allyl glucuronide 10 was used as an acceptor, and difucoside 18 was chosen as a
donor. Compound 18 was prepared by stereo- and regioselective glycosylation of diol 11 with
monosaccharide 12 followed by 4-O-chloroacetylation, deallylation and trichloacetimidation steps
(Scheme 2). Finally, the anomeric mixture of trichloroacetimidates 18 was obtained in a yield of 66%.
Scheme 2. Preparation of the difucoside donor 18. Reagents and conditions:
(i) (a) TMSOTf, CH2Cl2, −30 °C; (b) (ClСH2CO)2O, Py, 78%; (ii) (a) PdCl2, MeOH;
(b) CCl3CN, Cs2CO3, 84%.
Coupling of thus obtained compounds 18 and 10 in the presence of TMSOTf gave exclusively
trisaccharide 19 in a yield of 90% (Scheme 3). Removal of chloroacetyl groups in 19 gave
corresponding triol 20, which was further transformed into target trisaccharides 6–8 using the
procedure sequences applied for the synthesis of the disaccharides 1–3, respectively.

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Scheme 3. Synthesis of the trisaccharides 6–8. Reagents and conditions: (i) TMSOTf,
CH2Cl2, −30 °C, 90%; (ii) (NH2)2CS, collidine, MeOH, 92%; (iii) (a) H2, Pd(OH)2/C;
(b) LiOH, H2O, THF; (c) NaOH, H2O, 82%; (iv) (a) AcCl, Py, (b) H2, Pd(OH)2/C;
(c) SO3·Py, DMF; (d) Amberlite IR-120 (Na+), NaHCO3; (e) LiOH, H2O, THF; (f) NaOH,
H2O, 54%; (v) (a) SO3·Py, DMF; (b) Amberlite IR-120 (Na+), NaHCO3; (c) H2,
Pd(OH)2/C; (d) LiOH, H2O, THF; (e) NaOH, H2O, 62%.
1
All the target compounds were characterized with H and 13C NMR spectroscopy (Tables 2 and 3).
The presence of the O-sulfate group was confirmed by the downfield shift of the signal of the neighbor
proton and carbon atoms in the 1H and 13C NMR spectra. Introduction of the sulfate group at O-2 also
influenced the shift of the signals of anomeric proton and carbon atoms.
Table 2. 13C data for the target compounds 1–8.
Compound
1
Residue
C1
C2
C3
C4
C5
C6
→3)-β-D-GlcA
α-L-Fuc-(1→3
→3)-β-D-GlcA
α-L-Fuc-(1→3
→3)-β-D-GlcA
α-L-Fuc-(1→3
→3)-β-D-GlcA
α-L-Fuc-(1→3
→3)-β-D-GlcA
α-L-Fuc-(1→3
→3)-β-D-GlcA
→3)-α-L-Fuc-(1→3
α-L-Fuc-(1→3
→3)-β-D-GlcA
→3)-α-L-Fuc-(1→3
α-L-Fuc-(1→3
→3)-β-D-GlcA
→3)-α-L-Fuc-(1→3
α-L-Fuc-(1→3
103.2
100.7
103.0
98.4
74.8
69.6
74.7
76.8
74.6
67.8
73.4
68.6
74.7
76.6
74.6
70.7
69.3
75.2
74.7
76.5
73.4
68.6
67.8
83.5
70.8
83.2
68.7
83.5
76.6
82.2
68.6
83.2
67.8
83.6
73.2
68.2
83.2
74.3
68.6
82.2
74.0
76.5
71.7
73.2
71.6
73.3
71.6
80.4
70.5
81.0
71.5
82.1
71.7
69.5
75.9
71.6
70.0
73.4
70.5
81.2
80.4
78.0
68.1
77.9
68.0
78.1
67.8
76.8
66.4
77.9
67.4
78.0
68.1
67.9
78.0
68.2
67.6
76.8
66.4
67.8
176.9
16.5
2
3
4
5
6
176.8
16.5
103.5
100.5
102.1
99.1
176.8
17.0
176.8
17.0
103.0
98.3
176.8
16.9
103.1
100.7
96.5
176.8
16.5
16.5
7
8
103.1
98.5
176.9
16.5
95.5
16.5
102.1
99.1
176.8
16.9
100.5
17.0

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Table 3. 1H NMR data for the target compounds 1–8.
Compound
Residue
H1
H2
H3
H4
H5
H6
→3)-β-D-GlcA
α-L-Fuc-(1→3
→3)-β-D-GlcA
α-L-Fuc-(1→3
→3)-β-D-GlcA
α-L-Fuc-(1→3
→3)-β-D-GlcA
α-L-Fuc-(1→3
→3)-β-D-GlcA
α-L-Fuc-(1→3
→3)-β-D-GlcA
4.48 3.51 3.60
5.27 3.79 3.92
4.49 3.55 3.65
5.57 4.48 4.05
4.48 3.55 3.67
5.36 4.00 4.65
4.47 3.50 3.62
5.30 3.83 4.02
4.47 3.55 3.63
5.60 4.47 4.17
4.49 3.55 3.63
3.63 3.74
-
1
2
3
4
5
3.82 4.37 1.20
3.65 3.74
3.90 4.44 1.21
3.63 3.74
4.90 4.52 1.27
3.62 3.73
4.62 4.50 1.23
3.66 3.73
4.69 4.55 1.27
3.65 3.74
-
-
-
-
-
6
7
8
→3)-α-L-Fuc-(1→3 5.31 3.98 3.84
4.04 4.31 1.21
3.95 4.37 1.21
α-L-Fuc-(1→3
→3)-β-D-GlcA
5.09 3.82 3.95
4.49 3.63 3.65
3.67 3.74
-
→3)-α-L-Fuc-(1→3 5.60 4.57 4.07
4.10 4.42 1.24
3.91 4.49 1.26
α-L-Fuc-(1→3
→3)-β-D-GlcA
5.34 4.45 4.12
4.46 3.55 3.63
3.65 3.73
-
→3)-α-L-Fuc-(1→3 5.32 4.00 4.08
α-L-Fuc-(1→3 5.20 3.92 4.62
4.76 4.46 1.24
4.90 4.46 1.27
The synthesized oligosaccharides are regarded as model compounds for the investigation of a
structure-activity relationship in FCS. The results of conformational analysis and biological studies of
the compounds 1–8 will be published elsewhere. Synthesis and interdisciplinary studies of larger
oligosaccharides related to structural fragments of FCS are in progress at this laboratory.
3. Experimental Section
3.1. Materials
Dimethylformamide (DMF, ≥99.5%) and pyridine (≥99.5%) were purchased from Sigma-Aldrich
(Saint Louis, MO, USA). Dichloromethane (CH2Cl2) was successively distilled from diethanolamine,
P2O5, and CaH2 under argon. Analytical thin-layer chromatography (TLC) was performed on Silica
Gel 60 F254 aluminum sheets (Merck, Darmstadt, Germany), and visualization was accomplished using
UV light or by charring at ~150 C with 10% (v/v) H3PO4 in ethanol. Liquid column chromatography
was performed on Silica Gel 60, 40–63 µm (Merck, Darmstadt, Germany). Gel chromatography was
performed on the Sephadex G-15 column (260 cm) by elution with water at a flow rate of 1 mL/min.
3.2. Characterization of Compounds
1H and 13C NMR spectra were recorded on Bruker DRX500 and AV600 spectrometers at 303 K in
CDCl3 and D2O. Chemical shifts were reported in ppm referenced to the residual CHCl3 peak (δ 7.27)
1
13
for substituted compounds and to acetone peak (0.05% as internal standard, H δ 2.22 and C δ 30.9)
1
13
for the oligosaccharides 1–8. Signal assignment in H and C NMR spectra were made using COSY,

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1
TOCSY, ROESY and H-13C HSQC techniques. High-resolution mass spectra were acquired by
electrospray ionization on a Bruker Daltonics micrOTOF II instrument [28]. Optical rotation values
were measured using a JASCO DIP-360 polarimeter at the ambient temperature in solvents specified.
3.3. Chemical Synthesis
All glycosylation reactions were carried out under dry argon. Molecular sieves for glycosylation
reactions were activated prior to application at 180 °C in vacuum of an oil pump during 2 h.
3.3.1. 2-O-Benzyl-3,4-di-O-chloroacetyl-L-fucopyranosyl Trichloroacetimidate (12)
To a solution of the monosaccharide 11 (800 mg, 2.72 mmol) in CH2Cl2 (20 mL), Py (2 mL) and
(ClCH2C(O))2O (1 g, 5.9 mmol) were added. The reaction mixture was kept at room temperature (rt)
for 1 h, then diluted with EtOAc (50 mL) and washed with HCl (0.1 M) (20 mL) and distilled water
(2 × 30 mL). Organic layer was separated and concentrated in vacuo. Chromatography of the residue
on a silica gel column gave the totally protected monosaccharide as an amorphous solid. The product
was dissolved in MeOH (30 mL), and PdCl2 (127 mg, 0.8 mmol) was added. The mixture was stirred for
3 h at rt, then it was filtered through a celite pad and the filtrate was concentrated in vacuo. Flash column
chromatography of the residue on silica gel gave the respective semiacetales as an amorphous solid,
which was then dissolved in CH2Cl2 (15 mL), and CCl3CN (300 µL, 3.0 mmol) together with Cs2CO3
(50 mg, 0.15 mmol) were added. The reaction mixture was stirred for 1 h at rt, then it was filtered
through a celite pad and the filtrate was concentrated in vacuo. Column chromatography of the residue
on a silica gel inactivated by Et3N (0.1%) gave the compound 12 as a mixture of α and β isomers in a
ratio of 1:1 (1.16 g, 2.12 mmol, 78%). HRMS (ESI) C19H20Cl5NO7 [M + Na]+ calc.: 571.9580,
found: 571.9575.
α-Isomer: Rf 0.7 (Toluene–EtOAc 3:1); 1H NMR (600 MHz, CDCl3): δ 1.20 (d, J5,6 6.5 Hz, 3H, H-6),
3.99 (d, Jgem 2.0 Hz, 2H, ClCH2CO), 4.05 (dd, J1,2 3.5 Hz, J2,3 10.4 Hz, 1H, H-2), 4.15 (s, 2H,
ClCH2CO), 4.42 (q, 1H, H-5), 4.65 (d, Jgem 12.0 Hz, 1H, PhCHH), 4.74 (d, Jgem 12.0 Hz, 1H, PhCHH),
5.45 (d, J3,4 3.1 Hz, 1H, H-4), 5.50 (dd, 1H, H-3), 6.56 (d, J1,2 3.5 Hz, 1H, H-1), 7.20–7.40 (m, 5H,
Ar), 8.68 (s, 1H, NH).
1
β-Isomer: Rf 0.6 (Toluene–EtOAc 3:1); H NMR (600 MHz, CDCl3): δ 1.29 (d, J5,6 6.5 Hz, 3H,
H-6), 3.83 (d, Jgem 11.7 Hz, 1H, ClCHHCO), 3.90 (d, Jgem 11.7 Hz, 1H, ClCHHCO), 3.95 (dd,
J1,2 8.1 Hz, J2,3 10.0 Hz, 1H, H-2), 4.03 (q, 1H, H-5), 4.19 (s, 2H, ClCH2CO), 4.68 (d, Jgem 11.4 Hz,
1H, PhCHH), 4.92 (d, Jgem 11.4 Hz, 1H, PhCHH), 5.18 (dd, 1H, J3,4 3.4 Hz, H-3), 5.34 (d, 1H, H-4),
5.86 (d, J1,2 8.1 Hz, 1H, H-1), 7.20–7.40 (m, 5H, Ar), 8.78 (s, 1H, NH).
3.3.2. Allyl 2-O-benzyl-3,4-di-O-chloroacetyl-α-L-fucopyranosyl-(1→3)-(methyl 2,4-di-O-acetyl-β-D-
glucopyranosyl Uronate) (14)
A solution of the monosaccharide 12 (274 mg, 0.5 mmol) and the monosaccharide 10 (166 mg,
0.5 mmol) in CH2Cl2 (5 mL) was stirred at rt under argon atmosphere with molecular sieves 4 Å
(500 mg) for 1 h. The mixture was cooled to −30 °С and TMSOTf of (3 µL) was added. The mixture
was stirred for 15 min at −30 °С, then Et3N (0.05 mL) was added. The mixture was filtered through a

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celite pad, and the filtrate was concentrated in vacuo. Column chromatography of the residue on silica
gel gave the disaccharide 14 as an amorphous solid (330 mg, 0.46 mmol, 92%): Rf 0.4 (Toluene–EtOAc
1
3:1), [α]D = −63° (c 1, EtOAc); H NMR (600 MHz, CDCl3): δ 1.08 (d, J5,6 6.6 Hz, 3H, H-6),
1.85 (s, 3H, COCH3), 2.08 (s, 3H, COCH3), 3.78 (m, 5H, C(O)OCH3, H-2Fuc, H-3GlcA), 3.93 (m, 3H,
H-5GlcA, ClCH2CO), 4.10 (s, 2H, ClCH2CO), 4.12 (m, 1H, CHHCH=CH2), 4.23 (q, 1H, H-5Fuc),
4.37 (m, 1H, CHHCH=CH2), 4.58 (d, J1,2 7.7 Hz, 1H, H-1GlcA), 4.62 (s, 2H, PhCH2), 4.93 (d,
J1,2 3.3 Hz, 1H, H-1Fuc), 5.10 (t, J2,3 7.5 Hz, 1H, H-2GlcA), 5.20 (m, 3H, H-4GlcA, CH2CH=CH2),
5.30 (m, 2H, H-3Fuc, H-4Fuc), 5.85 (m, 1H, CH2CH=CH2), 7.20–7.40 (m, 5H, Ar). HRMS (ESI)
C31H38Cl2O15 [M + Na]+ calc.: 743.1479, found: 743.1480.
3.3.3. Allyl 2-O-benzyl-α-L-fucopyranosyl-(1→3)-(methyl 2,4-di-O-acetyl-β-D-glucopyranosyl
Uronate) (15)
To a solution of the disaccharide 14 (300 mg, 0.42 mmol) in MeOH (5 mL) thiourea (96 mg,
1.26 mmol) and collidine (50 µL) were added. The mixture was kept at 40 °С for 2 h, then it was
diluted with EtOAc (20 mL) and washed with HCl (0.1 M) (10 mL) and distilled water (2 × 15 mL).
Organic layer was separated and concentrated in vacuo. Chromatography of the residue on a silica gel
column gave the diol 15 as an amorphous solid (222 mg, 0.39 mmol, 94%): Rf 0.2 (Toluene–EtOAc
1:1), [α]D = −87° (c 1, EtOAc); 1H NMR (600 MHz, CDCl3): δ 1.09 (d, J5,6 6.6 Hz, 3H, H-6), 1.82 (s,
3H, COCH3), 1.98 (s, 3H, COCH3), 3.23 (s, 2H, 2OH), 3.54 (dd, J1,2 3.5 Hz, J2,3 10.0 Hz, 1H, H-2Fuc),
3.60 (d, J3,4 3.3 Hz, 1H, H-4Fuc), 3.66 (m, 4H, C(O)OCH3, H-3GlcA), 3.74 (dd, 1H, H-3Fuc), 3.83 (d,
J4,5 9.8 Hz, 1H, H-5GlcA), 3.91 (q, 1H, H-5Fuc), 4.04 (m, 1H, CHHCH=CH2), 4.28 (m, 1H,
CHHCH=CH2), 4.49 (d, J1,2 7.9 Hz, 1H, H-1GlcA), 4.55 (d, Jgem 11.9 Hz, 1H, PhCHH), 4.60 (d,
Jgem 11.9 Hz, 1H, PhCHH), 4.77 (d, J1,2 3.5 Hz, 1H, H-1Fuc), 4.98 (dd, J2,3 9.1 Hz, J3,4 7.9 Hz, 1H,
H-2GlcA), 5.03 (t, J3,4 = J4,5 9.3 Hz, 1H, H-4GlcA), 5.14 (m, 1H, CH2CH=CHH), 5.21 (m, 1H,
CH2CH=CHH), 5.76 (m, 1H, CH2CH=CH2), 7.20-7.40 (m, 5H, Ar). HRMS (ESI) C27H36O13
[M + Na]+ calc.: 591.2054, found: 591.2050.
3.3.4. Allyl 2-O-benzyl-3-O-benzoyl-α-L-fucopyranosyl-(1→3)-(methyl 2,4-di-O-acetyl-β-D-
glucopyranosyl Uronate) (16)
A mixture of the compound 15 (210 mg, 0.37 mmol) and Bu2SnO (102 mg, 0.41 mmol) in toluene
(5 mL) was refluxed with aseotropic removal of water to a volume of 3 mL, treated with benzoyl
chloride (50 µL, 0.41 mmol), and the mixture was kept for 1 h. Then MeOH (5 mL) was added, and
the mixture was concentrated. Column chromatography of the residue on silica gel afforded the
1
compound 16 (200 mg, 0.30 mmol, 80%): Rf 0.5 (Toluene–EtOAc 1:1), [α]D = −72° (c 1, EtOAc); H
NMR (600 MHz, CDCl3): δ 1.20 (d, J5,6 6.4 Hz, 3H, H-6), 1.86 (s, 3H, COCH3), 2.08 (s, 3H, COCH3),
3.78 (s, 3H, C(O)OCH3), 3.82 (t, J2,3 8.6 Hz, J3,4 9.0 Hz, 1H, H-3GlcA), 3.94 (d, J4,5 9.6 Hz, 1H,
H-5GlcA), 4.02 (m, 2H, H-2Fuc, H-4Fuc), 4.13 (m, 1H, CHHCH=CH2), 4.22 (q, 1H, H-5Fuc), 4.39 (m, 1H,
CHHCH=CH2), 4.60 (d, J1,2 7.6 Hz, 1H, H-1GlcA), 4.68 (dd, 2H, PhCH2), 5.00 (s, 1H, H-1Fuc), 5.16
(t, 1H, H-2GlcA), 5.22 (m, 2H, H-3Fuc, H-4GlcA), 5.30 (d, 1H, CH2CH=CHH), 5.40 (m, 1H,
CH2CH=CHH), 5.88 (m, 1H, CH2CH=CH2), 7.20–8.00 (m, 10H, Ar). HRMS (ESI) C34H40O14
[M + Na]+ calc.: 695.2316, found: 695.2321.

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3.3.5. Allyl 2-O-benzyl-3,4-di-O-chloroacetyl-α-L-fucopyranosyl-(1→3)-2-O-benzyl-4-O-cloroacetyl-
α-L-fucopyranoside (17)
A solution of the monosaccharide 12 (300 mg, 0.55 mmol) and the monosaccharide 11 (162 mg,
0.55 mmol) in CH2Cl2 (5 mL) was stirred at rt under argon atmosphere with molecular sieves 4 Å
(500 mg) for 1 h. The mixture was cooled to −30 °С and TMSOTf of (5 µL) was added. The mixture
was stirred for 15 min −30 °С, then Et3N (0.05 mL) was added. The mixture was filtered through a
celite pad, and the filtrate was concentrated in vacuo. Column chromatography of the residue on a
silica gel gave the disaccharide, which was dissolved in CH2Cl2 (5 mL), and Py (0.5 mL) together with
(ClCH2C(O))2O (150 mg, 0.9 mmol) were added. The reaction mixture was kept at rt for 1 h, then
diluted with EtOAc (20 mL) and washed with HCl (0.1 M) (10 mL) and distilled water (2 × 20 mL).
Organic layer was separated and concentrated in vacuo. Chromatography of the residue on a silica gel
column gave the disaccharide 17 as an amorphous solid (326 mg, 0.43 mmol, 78%): Rf 0.6
(Toluene–EtOAc 4:1), [α]D = −170° (c 1, EtOAc); 1H NMR (600 MHz, CDCl3): δ 0.70 (d, J5,6 6.4 Hz,
3H, H-6), 1.15 (d, J5,6 6.4 Hz, 3H, H-6), 3.80 (m, 2H, H-2Fuc1, H-2Fuc2), 3.92–4.30 (m, 10H, H-3Fuc1
,
H-5Fuc1, 3xClCH2CO, CH2CH=CH2), 4.50 (q, 1H, H-5Fuc2), 4.52–4.80 (m, 4H, 2x PhCH2), 5.00 (d, 1H,
H-1Fuc1), 5.10 (d, 1H, H-1Fuc2), 5.35 (m, 5H, H-3Fuc2, H-4Fuc1, H-4Fuc2, CH2CH=CH2), 5.92 (m, 1H,
CH2CH=CH2), 7.20–7.42 (m, 10H, Ar). HRMS (ESI) C35H41Cl3O12 [M + Na]+ calc.: 781.1561,
found: 781.1557.
3.3.6. 2-O-Benzyl-3,4-di-O-chloroacetyl-α-L-fucopyranosyl-(1→3)-2-O-benzyl-4-O-cloroacetyl-L-
fucopyranosyl Trichloroacetimidate (18)
To a solution of the disaccharide 17 (230 mg, 0.30 mmol) in MeOH (5 mL) PdCl2 (22 mg,
0.12 mmol) was added. The mixture was stirred for 3 h at rt, then it was filtered through a celite pad
and the filtrate was concentrated in vacuo. Flash column chromatography of the residue on a silica gel
gave the respective semiacetales as an amorphous solid, which was then dissolved in CH2Cl2 (3 mL),
and CCl3CN (50 µL, 0.49 mmol) together with Cs2CO3 (10 mg, 0.03 mmol) were added. The reaction
mixture was stirred for 1 h at rt, then it was filtered through a celite pad and the filtrate was
concentrated in vacuo. Column chromatography of the residue on a silica gel inactivated by Et3N
(0.1%) gave the compound 18 as a mixture of α and β isomers in a ratio of 1:1 (215 mg, 0.25 mmol,
84%). HRMS (ESI) C34H37Cl6NO12 [M + Na]+ calc.: 884.0345, found: 884.0350.
1
α-Isomer: Rf 0.7 (Toluene–EtOAc 4:1); H NMR (600 MHz, CDCl3): δ 0.62 (d, J5,6 6.5 Hz, 3H,
H-6Fuc2), 1.2 (d, J5,6 6.5 Hz, 3H, H-6Fuc1), 3.80 (dd, J1,2 3.4 Hz, J2,3 10.5 Hz, 1H, H-2Fuc2), 3.84 (d,
Jgem 15.0 Hz, 1H, ClCHHCO), 3.90 (d, Jgem 15.0 Hz, 1H, ClCHHCO), 3.94 (d, Jgem 15.0 Hz, 1H,
ClCHHCO), 4.04 (m, 4H, H-2Fuc1, ClCH2CO, ClCHHCO), 4.33 (m, H-3, H-3Fuc1, H-5Fuc1, H-5Fuc2),
4.50 (d, Jgem 12.2 Hz, 1H, PhCHH), 4.58 (d, Jgem 10.1 Hz, 1H, PhCHH), 4.72 (d, Jgem 12.2 Hz, 1H,
PhCHH), 4.80 (d, Jgem 10.1 Hz, 1H, PhCHH), 4.90 (d, J3,4 2.7 Hz, 1H, H-4Fuc2), 5.10 (d, J1,2 3.4 Hz,
1H, H-1Fuc2), 5.39 (dd, 1H, H-3Fuc2), 5.49 (d, 1H, H-4Fuc1), 6.68 (d, J1,2 3.4 Hz, 1H, H-1Fuc1),
7.20–7.40 (m, 10H, Ar), 8.62 (s, 1H, NH).
1
β-Isomer: Rf 0.3 (Toluene–EtOAc 4:1); H NMR (600 MHz, CDCl3): δ 0.60 (d, J5,6 6.5 Hz, 3H,
H-6Fuc2), 1.29 (d, J5,6 6.5 Hz, 3H, H-6Fuc1), 3.78 (dd, J1,2 3.5 Hz, J2,3 9.5 Hz, 1H, H-2Fuc2), 3.81 (d,

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Jgem 15.0 Hz, 1H, ClCHHCO), 3.92 (m, 4H, H-2Fuc1, H-3Fuc1, H-5Fuc1, ClCHHCO), 4.01 (m, 3H,
ClCH2CO, ClCHHCO), 4.10 (d, Jgem 15.2 Hz, 1H, ClCHHCO), 4.15 (q, 1H, H-5Fuc2), 4.50 (d,
Jgem 12.2 Hz, 1H, PhCHH), 4.58 (d, Jgem 10.1 Hz, 1H, PhCHH), 4.72 (d, Jgem 12.2 Hz, 1H, PhCHH),
4.82 (d, J3,4 2.9 Hz, 1H, H-4Fuc2), 5.05 (d, J1,2 3.5 Hz, 1H, H-1Fuc2), 5.08 (d, Jgem 10.1 Hz, 1H, PhCHH),
5.37 (dd, 1H, H-3Fuc2), 5.40 (d, 1H, H-4Fuc1), 5.82 (d, J1,2 7.8 Hz, 1H, H-1Fuc1), 7.20–7.40 (m, 10H, Ar),
8.75 (s, 1H, NH).
3.3.7. Allyl 2-O-benzyl-3,4-di-O-chloroacetyl-α-L-fucopyranosyl-(1→3)-2-O-benzyl-4-O-
chloroacetyl-α-L-fucopyranosyl-(1→3)-(methyl 2,4-di-O-acetyl-β-D-glucopyranosyl Uronate) (19)
A solution of the disaccharide 18 (200 mg, 0.23 mmol) and the monosaccharide 10 (76 mg,
0.23 mmol) in CH2Cl2 (2 mL) was stirred at rt under argon atmosphere with molecular sieves 4 Å
(200 mg) for 1 h. The mixture was cooled to −30 °С and TMSOTf of (2 µL) was added. The mixture
was stirred for 15 min at −30 °С, then Et3N (0.05 mL) was added. The mixture was filtered through a
celite pad, and the filtrate was concentrated in vacuo. Column chromatography of the residue on a
silica gel gave the trisaccharide 19 as an amorphous solid (214 mg, 0.20 mmol, 90%): Rf 0.4
(Toluene–EtOAc 4:1), [α]D = −152° (c 1, EtOAc); 1H NMR (600 MHz, CDCl3): δ 1.06 (d, J5,6 6.5 Hz,
6H, H-6Fuc1, H-6Fuc2), 1.76 (s, 3H, COCH3), 2.06 (s, 3H, COCH3), 3.76 (m, 4H, C(O)OCH3, H-2Fuc1),
3.83 (m, 2H, H-3GlcA, H-2Fuc2), 3.94 (m, 3H, H-5GlcA, ClCH2CO), 4.12 (m, 5H, H-3Fuc1, H-5Fuc1
,
CHHCH=CH2, ClCH2CO), 4.16 (s, 2H, ClCH2CO), 4.30 (q, 1H, H-5Fuc2), 4.38 (m, 1H,
CHHCH=CH2), 4.55 (d, Jgem 12.1 Hz, 1H, PhCHH), 4.58 (d, J1,2 7.7 Hz, 1H, H-1GlcA), 4.64 (d,
Jgem 11.2 Hz, 1H, PhCHH), 4.69 (d, Jgem 11.2 Hz, 1H, PhCHH), 4.73 (d, Jgem 12.1 Hz, 1H, PhCHH),
4.94 (d, J1,2 3.5 Hz, 1H, H-1Fuc1), 5.12 (m, 3H, H-2GlcA, H-4GlcA, H-1Fuc2), 5.21 (m, 2H, H-4Fuc2
,
CH2CH=CHH), 5.30 (m, 1H, CH2CH=CHH), 5.37 (d, J3,4 3.3 Hz, 1H, H-4Fuc1), 5.53 (dd, J2,3 10.5 Hz,
J3,4 3.2 Hz, 1H, H-3Fuc2), 5.85 (m, 1H, CH2CH=CH2), 7.29–7.44 (m, 10H, Ar). HRMS (ESI)
C46H55Cl3O20 [M + Na]+ calc.: 1055.2250, found: 1055.2246.
3.3.8. Allyl 2-O-benzyl-α-L-fucopyranosyl-(1→3)-2-O-benzyl-α-L-fucopyranosyl-(1→3)-(methyl
2,4-di-O-acetyl-β-D-glucopyranosyl Uronate) (20)
To a solution of the trisaccharide 19 (200 mg, 0.19 mmol) in MeOH (3 mL) thiourea (60 mg,
0.78 mmol) and collidine (100 µL) were added. The mixture was kept at 40 °С for 2 h, then it was
diluted with EtOAc (20 mL) and washed with HCl (0.1 M) (10 mL) and distilled water (2 × 20 mL).
Organic layer was separated and concentrated in vacuo. Chromatography of the residue on a silica gel
column gave the triol 20 as an amorphous solid (140 mg, 0.17 mmol, 92%): Rf 0.1 (TolueneEtOAc,
1
1:1), [α]D = −168 (c 1, EtOAc); H NMR (600 MHz, CDCl3): δ 1.18 (d, J5,6 6.5 Hz, 3H, H-6Fuc2),
1.21 (d, J5,6 6.5 Hz, 3H, H-6Fuc1), 1.89 (s, 3H, COCH3), 2.05 (s, 3H, COCH3), 3.11 (s, 3H, 3OH), 3.64
(d, J3,4 2.2 Hz, 1H, H-4Fuc2), 3.73 (m, 4H, H-3GlcA, H-2Fuc1, H-2Fuc2, H-4Fuc1), 3.75 (s, 3H, C(O)OCH3),
3.89 (m, 2H, H-5GlcA, H-3Fuc2), 3.98 (m, 2H, H-3Fuc1, H-5Fuc1), 4.13 (m, 2H, H-5Fuc2, CHHCH=CH2),
4.38 (m, 1H, CHHCH=CH2), 4.58 (m, 3H, H-1GlcA, PhCH2), 4.72 (s, 2H, PhCH2), 4.86 (d, J1,2 3.4 Hz,
1H, H-1Fuc2), 4.89 (d, J1,2 3.6 Hz, 1H, H-1Fuc1), 5.10 (t, J2,3 = J3,4 8.5 Hz, 1H, H-2GlcA), 5.14 (t,
J3,4 = J4,5 8.5 Hz, 1H, H-4GlcA), 5.22 (m, 1H, CH2CH=CHH), 5.30 (m, 1H, CH2CH=CHH), 5.87

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(m, 1H, CH2CH=CH2), 7.20–7.40 (m, 10H, Ar). HRMS (ESI) C40H52O17 [M + Na]+ calc.: 827.3102,
found: 827.3105.
3.3.9. Propyl α-L-fucopyranosyl-(1→3)-β-D-glucopyranosyl Uronic Acid Sodium Salt (1)
To a solution of the disaccharide 15 (57 mg, 0.10 mmol) in MeOH (1.5 mL) and EtOAc (1.5 mL)
Pd(OH)2/C (15 mg) was added. The mixture was stirred for 1 h at rt under hydrogen atmosphere, then
it was filtered through a celite pad, and the filtrate was concentrated in vacuo. Column chromatography
of the residue on a silica gel (Toluene–EtOAc) gave an amorphous solid, which was dissolved in THF
(1.0 mL), and 0.1N(aq) LiOH (0.5 mL) was added. The mixture was kept for 1 h at rt and then 0.1N(aq)
NaOH (0.5 mL) was added, and the solution was kept at 40 °C for 1 h. After the mixture was filtered
through the Whatman paper filter, the filtrate was concentrated in vacuo to a volume of 1 mL. Column
chromatography of the residue on the Sephadex G-15 gel in water gave the disaccharide 1 (32 mg,
0.08 mmol, 79%). [α]D = −44° (c 0.5, H2O). HRMS (ESI) C15H25NaO11 [M − Na]− calc.: 381.1397,
found: 381.1402.
3.3.10. Propyl 2-O-sulfonato-α-L-fucopyranosyl-(1→3)-β-D-glucopyranosyl Uronic Acid Disodium
Salt (2)
To a solution of the disaccharide 15 (57 mg, 0.10 mmol) in CH2Cl2 (1 mL) Py (0.1 mL) and AcCl
(0.1 mL) were added. The mixture was kept for 2 h at rt, then it was diluted with EtOAc (20 mL) and
washed with HCl (0.1 M) (10 mL) and distilled water (2 × 15 mL). Organic layer was separated and
concentrated in vacuo. Chromatography of the residue on a silica gel column (Toluene–EtOAc) gave
the totally protected disaccharide, which was dissolved in a 1:1 mixture MeOH–EtOAc (3 mL) and
Pd(OH)2/C (15 mg) was added. The mixture was stirred for 1 h at rt under hydrogen atmosphere, then
it was filtered through a celite pad, and the filtrate was concentrated in vacuo. Flash chromatography
of the residue on a silica gel column (Toluene–EtOAc) gave an amorphous solid, which was dissolved
in DMF (1 mL) and PySO3 (80 mg, 0.5 mmol) was added. The reaction mixture was kept for 1 h at rt
and then quenched with NaHCO3 (200 mg). The resin Amberlite IR-120 (Na+) and MeOH (1 mL) were
added, and the mixture was stirred for 1 h. Then the resin was filtered off, and the filtrate was
concentrated in vacuo to a volume of 1 mL. Chromatography of the residue on a silica gel column
(CH2Cl2–MeOH) gave the sulfated disaccharide, which was dissolved in THF (1.0 mL), and 0.1N(aq)
LiOH (0.5 mL) was added. The mixture was kept for 1 h at rt and then 0.1N(aq) NaOH (0.5 mL) was
added, and the solution was kept at 40 °C for 1 h. After the mixture was filtered through the Whatman
paper filter, the filtrate was concentrated in vacuo to a volume of 1 mL. Column chromatography of
the residue on the Sephadex G-15 gel in water gave the disaccharide 2 (33 mg, 0.065 mmol, 64%).
[α]D = −38° (c 0.6, H2O). HRMS (ESI) C15H24Na2O14S [M − Na]− calc.: 483.0784, found: 483.0790.
3.3.11. Propyl 3,4-di-O-sulfonato-α-L-fucopyranosyl-(1→3)-β-D-glucopyranosyl Uronic Acid
Trisodium Salt (3)
To a solution of the disaccharide 15 (57 mg, 0.10 mmol) in DMF (1 mL) PySO3 (80 mg, 0.5 mmol)
was added. The reaction mixture was kept for 1 h at rt and then quenched with NaHCO3 (200 mg). The

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resin Amberlite IR-120 (Na+) and MeOH (1 mL) were added, and the mixture was stirred for 1 h. Then
the resin was filtered off, and the filtrate was concentrated in vacuo to a volume of 1 mL.
Chromatography of the residue on a silica gel column (CH2Cl2–MeOH) gave the sulfated disaccharide,
which was dissolved in THF (1.0 mL) and H2O (50 µL), and Pd(OH)2/C (15 mg) was added. The
mixture was stirred for 30 min at rt under hydrogen atmosphere, then it was filtered through a celite
pad. To the filtrate 0.1N(aq) LiOH (0.5 mL) was added. The mixture was kept for 1 h at rt and then
0.1N(aq) NaOH (0.5 mL) was added, and the solution was kept at 40 C for 1 h. Then the mixture was
filtered through the Whatman paper filter, and the filtrate was concentrated in vacuo to a volume of
1 mL. Column chromatography of the residue on the Sephadex G-15 gel in water gave the disaccharide
3 (37 mg, 0.062 mmol, 62%). [α]D = −30° (c 0.7, H2O). HRMS (ESI) C15H23Na3O17S2 [M − Na]− calc.:
585.0172, found: 585.0178.
3.3.12. Propyl 4-O-sulfonato-α-L-fucopyranosyl-(1→3)-β-D-glucopyranosyl Uronic
Acid Disodium Salt (4)
To a solution of the disaccharide 16 (55 mg, 0.08 mmol) in DMF (1 mL) PySO3 (65 mg, 0.4 mmol)
was added. The reaction mixture was kept for 1 h at rt and then quenched with NaHCO3 (200 mg).
The resin Amberlite IR-120 (Na+) and MeOH (1 mL) were added, and the mixture was stirred for 1 h.
Then the resin was filtered off, and the filtrate was concentrated in vacuo to a volume of 1 mL.
Chromatography of the residue on a silica gel column (CH2Cl2–MeOH) gave the sulfated disaccharide,
which was dissolved in THF (1.0 mL) and H2O (50 µL), and Pd(OH)2/C (15 mg) was added. The
mixture was stirred for 30 min at rt under hydrogen atmosphere, then it was filtered through a celite
pad. To the filtrate 0.1N(aq) LiOH (0.5 mL) was added. The mixture was kept for 1 h at rt and then
0.1N(aq) NaOH (0.5 mL) was added, and the solution was kept at 40 C for 1 h. Then the mixture was
filtered through the Whatman paper filter, and the filtrate was concentrated in vacuo to a volume of
1 mL. Column chromatography of the residue on the Sephadex G-15 gel in water gave the disaccharide
4 (24 mg, 0.048 mmol, 62%). [α]D = −32° (c 0.6, H2O). HRMS (ESI) C15H24Na2O14S [M − Na]− calc.:
483.0784, found: 483.0790.
3.3.13. Propyl 2,4-di-O-sulfonato-α-L-fucopyranosyl-(1→3)-β-D-glucopyranosyl Uronic Acid
Trisodium Salt (5)
To a solution of the disaccharide 16 (47 mg, 0.07 mmol) in a 1:1 mixture MeOH–EtOAc (3 mL)
Pd(OH)2/C (15 mg) was added. The mixture was stirred for 1 h at rt under hydrogen atmosphere, then
it was filtered through a celite pad, and the filtrate was concentrated in vacuo. Flash chromatography
of the residue on a silica gel column (Toluene–EtOAc) gave an amorphous solid, which was dissolved
in DMF (1 mL) and PySO3 (110 mg, 0.7 mmol) was added. The reaction mixture was kept for 1 h at rt
and then quenched with NaHCO3 (200 mg). The resin Amberlite IR-120 (Na+) and MeOH (1 mL)
were added, and the mixture was stirred for 1 h. Then the resin was filtered off, and the filtrate was
concentrated in vacuo to a volume of 1 mL. Chromatography of the residue on a silica gel column
(CH2Cl2–MeOH) gave the sulfated disaccharide, which was dissolved in THF (1.0 mL), and 0.1N(aq)
LiOH (0.5 mL) was added. The mixture was kept for 1 h at rt and then 0.1N(aq) NaOH (0.5 mL) was
added, and the solution was kept at 40 C for 1 h. Then the mixture was filtered through the Whatman

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paper filter, and the filtrate was concentrated in vacuo to a volume of 1 mL. Column chromatography
of the residue on the Sephadex G-15 gel in water gave the disaccharide 5 (25 mg, 0.04 mmol, 58%).
[α]D = −28° (c 0.5, H2O). HRMS (ESI) C15H23Na3O17S2 [M − Na]− calc.: 585.0172, found: 585.0176.
3.3.14. Propyl α-L-fucopyranosyl-(1→3)-α-L-fucopyranosyl-(1→3)-β-D-glucopyranosyl Uronic Acid
Sodium Salt (6)
Treatment of the trisaccharide 20 (40 mg, 0.05 mmol) as described for the preparation of the
compound 1 gave the trisaccharide 6 (19 mg, 0.035 mmol, 69%). [α]D = −68° (c 0.5, H2O). HRMS
(ESI) C21H35NaO15 [M − Na]− calc.: 527.1976, found: 527.1981.
3.3.15. Propyl 2-O-sulfonato-α-L-fucopyranosyl-(1→3)-2-O-sulfonato-α-L-fucopyranosyl-(1→3)-β-D-
glucopyranosyl Uronic Acid Trisodium Salt (7)
Treatment of the trisaccharide 20 (40 mg, 0.05 mmol) as described for the preparation of the
compound 2 gave the trisaccharide 7 (20 mg, 0.027 mmol, 54%). [α]D = −48° (c 0.6, H2O). HRMS
(ESI) C21H33Na3O21S2 [M − Na]− calc.: 731.0731, found: 731.0736.
3.3.16. Propyl 3,4-di-O-sulfonato-α-L-fucopyranosyl-(1→3)-4-O-sulfonato-α-L-fucopyranosyl-(1→3)-
β-D-glucopyranosyl Uronic Acid Tetrasodium Salt (8)
Treatment of the trisaccharide 20 (45 mg, 0.056 mmol) as described for the preparation of the
compound 3 gave the trisaccharide 8 (30 mg, 0.035 mmol, 62%). [α]D = −42° (c 0.6, H2O). HRMS
(ESI) C21H32Na4O24S3 [M − Na]− calc.: 833.0139, found: 833.0143.
3.4. Calculations
Starting structures for geometry optimizations were built as follows: for the forms without
stabilization, torsional angles H3-C3-O3-CO were set to −40°, C3-O3-CO-O to 0°; and H4-C4-O4-CO
to +40°, C4-O4-CO-O to 0°. For the forms with supposed participation of the carbonyl oxygen at O3,
torsion H3-C3-O3-CO were set to 180°, and for the supposed participation from O4, torsion
H4-C4-O4-CO were set to 180°, putting the corresponding carbonyl oxygen into the proximity of the
cationic center.
MM+ optimizations were carried out as described in the text until the RMS gradient attained
0.01 kcal/mol·Å.
Ab initio calculations used the SCF/MP2 approach with frozen core orbitals: 1s were frozen for all
carbons and oxygens, 1s, 2s and 2p were frozen for chlorines. Optimizations were carried out until the
RMS gradient attained the value of 0.0001. After that, vibrational analysis was performed to check that
no negative frequencies were present.
4. Conclusions
The efficient synthesis of a series of non-sulfated and selectively O-sulfated di- and trisaccharides
related to branching sites of fucosylated chondroitin sulfates from holothurias has been performed. The
synthesized compounds are built up of the propyl β-D-glucuronic acid residue bearing at O-3

Mar. Drugs 2015, 13
785
α-L-fucosyl or α-L-fucosyl-(1→3)-α-L-fucosyl substituents. The sulfation pattern in the fucosyl units
was selected according to the known to date holothurian FCS structures. Stereospecific formation
of the α-glycoside bond was achieved using 2-O-benzyl-3,4-di-O-chloroacetyl-α-L-fucosyl
trichloroacetimidate as a donor. Stereochemical outcome of the glycosylation was explained by the
possible remote participation of the chloroacetyl groups at O-3 and O-4 with the intermediate
formation of the stabilized glycosyl cations, which are hindered from the β-side, and thus could only be
attacked by the glycosyl acceptor from the α-side. The experimental results in glycosylation reactions
were in good agreement with the SCF/MP2 calculated energies of such participation. Obtained NMR
characteristics of synthesized oligosaccharides can be used as model in further analyses of natural FCSs.
Acknowledgments
The work was supported by the Russian Scientific Foundation (Grant 14-13-01325). The authors
are grateful to A.O. Chizhov for recording of high-resolution mass spectra.
Author Contributions
NEN and NEU conceived the study. NEU synthesized and characterized compounds 9–12, 16–19
and 6–8. PAF synthesized and characterized compounds 14–15 and 1–5. AGG performed calculations
of the values of the energy of stabilization of fucosyl cations. ASD recorded NMR spectra of all
compounds and made signal assignment. All authors participated in interpreting the results and
preparing the final version of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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